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REVIEW / SYNTHÈSE The endothelium: influencing vascular smooth muscle in many ways Chris R. Triggle, Samson Mathews Samuel, Shalini Ravishankar, Isra Marei, Gnanapragasam Arunachalam, and Hong Ding Abstract: The endothelium, although only a single layer of cells lining the vascular and lymphatic systems, contributes in multiple ways to vascular homeostasis. Subsequent to the 1980 report by Robert Furchgott and John Zawadzki, there has been a phenomenal increase in our knowledge concerning the signalling molecules and pathways that regulate endothelial – vascular smooth muscle communication. It is now recognised that the endothelium is not only an important source of nitric oxide (NO), but also numerous other signalling molecules, including the putative endothelium-derived hyperpolarizing factor (EDHF), prostacyclin (PGI2), and hydrogen peroxide (H2O2), which have both vasodilator and vasoconstrictor properties. In addition, the endothelium, either via transferred chemical mediators, such as NO and PGI2, and (or) low-resistance electrical coupling through myoendothelial gap junctions, modulates flow-mediated vasodilatation as well as influencing mitogenic ac- tivity, platelet aggregation, and neutrophil adhesion. Disruption of endothelial function is an early indicator of the develop- ment of vascular disease, and thus an important area for further research and identification of potentially new therapeutic targets. This review focuses on the signalling pathways that regulate endothelial – vascular smooth muscle communication and the mechanisms that initiate endothelial dysfunction, particularly with respect to diabetic vascular disease. Key words: hyperglycaemia, endothelium, nitric oxide, EDHF, PGI2.. Résumé : L’endothélium, même s’il ne consiste qu’en une seule couche de cellules qui tapissent les systèmes vasculaire et lymphatique, contribue de multiples façons à l’homéostasie vasculaire. Depuis le rapport publié en 1980 par Robert Furch- gott et John Zawadzki, nos connaissances des molécules et des voies de signalisation qui régulent les communications entre l’endothélium et le muscle lisse vasculaire se sont accrues de façon phénoménale. On reconnaît maintenant que l’endothé- lium constitue non seulement une source importante d’oxyde nitrique, le NO, mais aussi de nombreuses autres molécules de signalisation, incluant le facteur d’hyperpolarisation présumé dérivé de l’endothélium (EDHF), la prostacycline (PGI2), et le peroxyde d’hydrogène (H2O2), qui possèdent des propriétés tant vasodilatatrices que vasoconstrictrices. De plus, l’endothé- lium, par l’intermédiaire du transfert de médiateurs chimiques comme le NO et la PGI2, et (ou) du couplage électrique de faible résistance à travers les jonctions communicantes myoendothéliales, module la vasodilatation dépendante de l’écoule- ment et influence l’activité mitogène, l’agrégation des plaquettes et l’adhésion des neutrophiles. La perturbation de la fonc- tion endothéliale est un indicateur précoce du développement de la maladie vasculaire et ainsi, constitue un domaine Received 23 October 2011. Accepted 12 April 2012. Published at www.nrcresearchpress.com/cjpp on 24 May 2012. Abbreviations: ACE, angiotensin converting enzyme; AMPK, AMP-activated protein kinase; Ang II, angiotensin II; BH4, tetrahydrobiopterin; BKCa (or BK), large conductance calcium-activated potassium channel; CaSR, calcium sensing receptor; CNP, C-type natriuretic peptide; COX, cyclo-oxygenase; Cx, connexins; CYP, cytochrome P450; ECs, endothelial cells; ECP, endothelial cell projection; EDCFs, endothelium dependent constricting factors; EDH, endothelium dependent hyperpolarization; EDHF, endothelium derived hyperpolarizing factor; EDRF(s), endothelium dependent relaxing factor(s); EDV, endothelium dependent vasodilatation; EETs, epoxy-eicosatrienoic acids; eNOS, endothelial nitric oxide synthase; FMD, flow mediated vasodilation; HNO, nitroxyl anion; H2O2, hydrogen peroxide; ICAM-1, intercellular adhesion molecule 1; IKCa (IK, or KCa3.1), intermediate conductance calcium-activated potassium channel; Kir, inward rectifying potassium channel; MEGJ, myo-endothelial gap junctions; NO, nitric oxide; NO•, nitric oxide free radical; PAI-1, plasminogen activator inhibitor-1; PGI2, prostacyclin; ROCK, Rho-associated protein kinase; ROS, reactive oxygen species; SKCa (SK, or KCa2.3), small conductance calcium-activated potassium channel; t-PA, tissue plasminogen activator; TXA2, thromboxane A2; VCAM-1, vascular cell adhesion molecule-1; VSMCs, vascular smooth muscle cells. C.R. Triggle and H. Ding. Department of Pharmacology, Weill Cornell Medical College in Qatar, P.O. Box 24144, Education City, Doha, Qatar; Department of Medical Education, Weill Cornell Medical College in Qatar, P.O. Box 24144, Education City, Doha, Qatar. S.M. Samuel, S. Ravishankar, I. Marei, and G. Arunachalam. Department of Pharmacology, Weill Cornell Medical College in Qatar, P.O. Box 24144, Education City, Doha, Qatar. Corresponding author: Chris R. Triggle (e-mail: firstname.lastname@example.org). This Invited Review is one of a number of papers published in the Special Issue on “Pharmacology, the Next 50 Years” commemorating the 50th Anniversary of the Department of Pharmacology at the University of Alberta. 713 Can. J. Physiol. Pharmacol. 90: 713–738 (2012) doi:10.1139/Y2012-073 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. important de recherche pour identifier de nouvelles cibles thérapeutiques potentielles. Cet article de revue se concentre sur les voies de signalisation qui régulent la communication entre l’endothélium et le muscle lisse vasculaire, et sur les mécanis- mes qui initient la dysfonction endothéliale, particulièrement dans le contexte de la maladie vasculaire diabétique. Mots‐clés : hyperglycémie, endothélium, oxyde nitrique, EDHF, PGI2. [Traduit par la Rédaction] Introduction The endothelium is a single layer of cells that provides the inner layer of all blood vessels and the lymphatic system, and in an adult human consists of approximately 10 trillion (1013) cells contributing close to 1.5% of the total body mass (Gal- ley and Webster 2004). Endothelial cells share a common origin from the mesoderm as hematopoietic cells, but despite a common origin, endothelial cells do show phenotype heter- ogeneity with respect to both structure and function (Choi et al. 1998; Aird 2007a, 2007b). In most vascular beds the en- dothelial cell layer appears continuous and nonfenestrated, but it can also be continuous fenestrated and discontinuous/ sinusoidal, reflecting differing functions within the body (Choi et al. 1998; Aird 2007a, 2007b). Endothelial cells also have the capacity to synthesize the proteins associated with the basal lamina, as well as matrix metalloproteinases, and thus play an important role in vascular remodelling (Kalebic et al. 1983). Despite their exposure to shear stress, endothe- lial cells have a relatively slow turnover rate that has been stated to be about 3 years (Brandes et al. 2005). Endothelial cell layer damage that can, for instance, result from athero- sclerosis, leads to replacement by regenerated endothelial cells that may not possess the same properties as normal cells (Brandes et al. 2005; Lee et al. 2007; Vanhoutte 2010). En- dothelial cell senescence is linked to telomere shortening, which is age-dependent in healthy humans, but accelerated by risk factors, such as atherosclerosis, hypertension, smok- ing, and the mechanical stress of a high heart rate. Such in- sults elevate oxidative stress, resulting in stress-induced senescence, growth arrest, a loss of repair capability, and ac- celerate a proatherogenic phenotype (Voghel et al. 2007; Thorin and Thorin-Trescases 2009; Thorin 2011). The statement “You are only as old as your endothelium” is attributed to Dr. Rudolf Altschul, former Head of the De- partment of Anatomy and Cell Biology at the University of Saskatchewan, Canada, but has been used by many others and reflects the important contributions of the endothelium to cardiovascular physiology and pathophysiology (Altschul 1955; Cooke and Zimmer 2002). Critical role of the endothelium in the regulation of blood flow The importance of the endothelium in the regulation of vascular function was first recognised as a result of the im- portant observation made by Robert Furchgott and his re- search assistant John Zawadzki that acetylcholine-mediated relaxation of rabbit aorta was endothelium-dependent (Furch- gott and Zawadzki 1980). Their study recognised that there was a diffusible factor, termed “endothelium-derived relaxing factor” (EDRF), that mediated relaxation (Furchgott and Za- wadzki 1980). In 1987, as a result of work by Moncada and colleagues, EDRF was convincingly identified as NO; how- ever, during a scientific meeting at the Mayo Clinic in 1986, both Ignarro and Furchgott independently presented evidence that also suggested that NO is an EDRF (Palmer et al. 1987; Furchgott 1996). Robert Furchgott, together with 2 other sci- entists in the field of nitric oxide research, Ferid Murad and Louis Ignarro, received the Nobel Prize in 1998 for their con- tributions to the discovery of the physiological importance of NO. We now know that NO has multiple cellular functions that are not restricted to the vascular system. Nonetheless, given the important functions of NO as a negative regulator of neutrophil adhesion, platelet aggregation, and smooth muscle proliferation, as well as by virtue of its antioxidant capability, a reduction in the bioavailability of NO has been clearly linked to the development of vascular disease (Félétou and Vanhoutte 2006a, 2006b; Tsutsui et al. 2010; Félétou 2011a). Thus, the endothelium can influence vascular smooth muscle and blood vessel function in many ways. Although the EDRF as first described by Furchgott and Zawadzki in 1980 was ultimately identified as NO, it was observed that other EDRFs also contribute to endothelium- dependent vasodilatation (EDV) in a vessel and species- dependent manner (Lundberg et al. 2005). The prostanoid prostacyclin (PGI2) is a contributor to EDV, as is evident from studies of flow-mediated (vaso)dilatation (FMD) in humans and rodents (Koller et al. 1993; Duffy et al. 1998). When nitric oxide synthase (NOS) activity is inhibited or impaired, as in disease states, the contribution of cyclooxy- genase (COX)-derived products, such as PGI2, to EDV may become more apparent (Corriu et al. 1996; Szerafin et al. 2006). The contribution of endothelial cell COX-2 to the synthesis of PGI2 potentially explains the deleterious effects of COX-2 inhibitors in terms of cardiovascular mortality, as COX-2 inhibition promotes a prothrombotic state that can be attributed, at least in part, to a reduction in PGI2 levels (Bulut et al. 2003; Antman et al. 2005, 2007; Andersohn et al. 2006). Nitrogen monoxide: is it just the free radical uncharged NO molecule that contributes to EDV? Although it is well known that NO can exist in 3 redox forms (the nitrosonium ion (NO+), the uncharged free radical (NO•), and the nitroxyl anion (NO– or HNO)), it is generally assumed, perhaps implicitly, that it is NO• that mediates the biological effects of NO (Stamler et al. 1992; Li et al. 1999; Kemp-Harper 2011). The relative importance of NO• to bio- logical signalling can be determined by the use of scavenging molecules such as carboxy PTIO (2-(4-carboxyphenyl)- 4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide) 714 Can. J. Physiol. Pharmacol. Vol. 90, 2012 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. (Akaike et al. 1993; Wanstall et al. 2001). The contribution of the one electron reduced nitroxyl form of NO, HNO, or, more accurately named, nitrosyl hydride or hydrogen oxyni- trate, to biological function has also been recognised (Li et al. 1999; Bullen et al. 2011; Fukuto and Carrington 2011; Kemp-Harper 2011; Tocchetti et al. 2011). HNO has phys- iological targets and actions that, at least partially, distin- guish it from NO•, including cardiac and vasoprotection as well as a role in nitrergic transmission (Li et al. 1999; Lundberg et al. 2005; Bullen et al. 2011; Fukuto and Car- rington 2011; Kemp-Harper 2011; Tocchetti et al. 2011). HNO can be generated via both NOS, as well as non-NOS sources such as S-nitrosothiols, and can contribute to the ef- fects mediated by NO• (Wong et al. 1998). HNO contrib- utes to EDV and spreading vasodilatation in small mesenteric arteries from the mouse and the rat, but with a somewhat different pharmacological profile than NO• (An- drews et al. 2009; Yuill et al. 2011). HNO may play a greater role than NO• when availability of tetrahydrobiop- terin (BH4), a key cofactor for maintaining endothelial nitric oxide synthase (eNOS) function to generate NO, is compro- mised (Fukuto et al. 1992). Endothelium-derived relaxing factor(s) and endothelium-derived contracting factors In addition to NO, PGI2, and other COX-products, multi- ple other EDRFs as well as endothelium-derived contracting factors (EDCFs) have now been identified. Thus, for the EDRFs, the list includes hydrogen peroxide (H2O2), carbon monoxide (CO), hydrogen sulphide (H2S), cytochrome P450 (CYP) products such as the epoxyeicosatrienoic acids (EETs) and, potentially, sulphur dioxide (SO2) and vasodilator pepti- des such as C-type natriuretic peptide (CNP), as well as small increases in extracellular K+ resulting from the opening of endothelial cell calcium-activated K+-channels. H2O2 may also function as an EDCF as, with considerable tissue and species variability, it has been shown to both contract and re- lax vascular smooth muscle from a variety of different spe- cies (see section on Endothelial-derived ROS as vasodilator factors). Additional EDCFs include the endothelin peptides, such as ET-1 and, via the activity of endothelial cell angio- tensin converting enzyme (ACE) that is located on the lumi- nal surface of endothelial cells, angiotensin II (Ang II). Endothelial cells also express thromboxane synthetase and can synthesize the vasoconstrictor thromboxane A2; further- more, PGI2 at high concentrations can also activate throm- boxane receptors and mediate vasoconstriction, and in a concentration-dependent fashion (Barton 2011; Corriu et al. 2001; Félétou 2011a; Williams et al. 1994). Further discus- sion of the contribution of EDCFs to endothelial function and dysfunction is presented later (Ingerman-Wojenski et al. 1981) (see section on Endothelial dysfunction). Myoendothelial gap junctions There is also very strong evidence that in addition to diffu- sible mediators, electrical continuity between adjacent endo- thelial cells as well as between the endothelial cell layer and the underlying vascular smooth muscle cells, plays a key role for endothelial – vascular smooth muscle cell communication and conducted vasodilatation. Myoendothelial gap junctions (MEGJs) are formed from connexin (Cx) proteins, notably, Cx37 and Cx40, but also Cx43 and Cx45. Adjacent endothe- lial cells are connected via low-resistance MEGJs, thus facilitating electrical continuity and the spread of hyper- polarization, which, in the microcirculation, is important for facilitating spreading vasodilatation (Takano et al. 2005). Conduction pathways may differ for different vasodilators, and in mouse cremaster arterioles, acetylcholine-mediated vasodilation is initiated via endothelial cell hyperpolariza- tion, whereas adenosine-initiated vasodilatation depends upon signalling along the vascular smooth muscle layer (de Wit 2010). Fenestrae are holes in the internal elastic lamina that are of particular importance in smaller blood vessels and allow MEGJs to provide low-resistance electrical cou- pling between endothelial and vascular smooth muscle cells, as well as the passage of small molecules of <1 kDa be- tween the cell layers (Figs. 1 and 2) (Takano et al. 2005). With smaller blood vessel diameter, the frequency of fenes- trae is increased and the continuity of the internal elastic lamina reduced and, possibly, absent (Sandow et al. 2009b). In disease states, such as pulmonary hypertension, there is evidence for a reduction in fenestrae as a result of an increase in elastin synthesis, and one can speculate that such morphological changes may contribute to the patho- physiology via a potential reduction in MEGJs (Aiello et al. 2003). The MEGJs are functionally of greater impor- tance in resistance arteries than in conduit vessels, and likely are primary contributors to non-NO/PGI2-mediated endothelium-dependent hyperpolarization (EDH) that has also been attributed to a putative chemical mediator, namely, endothelium-derived hyperpolarizing factor, or EDHF (see section on Importance of EDHF). The contribu- tion of MEGJs to the regulation of blood flow in humans is also suggested on the basis of the ability of carbenoxolone, albeit a nonspecific MEGJ inhibitor, to reduce FMD in the brachial artery of normal volunteers (Lan et al. 2011). Perivascular adipose tissue and the regulation of vascular smooth muscle Perivascular fat (perivascular adipose tissue, or PVAT) can also influence vascular smooth muscle and, potentially, endo- thelial function via the release of adipose-derived adipokines such as the antiatherogenic adiponectin, and also via nicoti- namide adenine dinucleotide phosphate (NADPH)-oxidase- generated reactive oxygen species (ROS) (Fig. 1). Thus, dif- fusible mediators from endothelial cells can affect vascular smooth muscle from the luminal side and diffusible media- tors from PVAT can modulate vascular function predomi- nantly via the adventitial layer. However, PVAT-derived transferable factors have been reported to have endothelium- independent as well as endothelium-dependent vasodilatation actions on rat thoracic aorta (Gao et al. 2007). NO, superox- ide (O 2 ), and H2O2 may all contribute to the regulation of vascular tone by PVAT via both endothelium-dependent and -independent mechanisms (Gao et al. 2007; Kassam et al. 2011; Li et al. 2011). Furthermore, adiponectin enhances the generation of NO via the synthesis of PI3K/AKT and AMPK signalling pathways, and offsets the effects of oxidative stress that are induced by hyperglycaemia, thus indicating that adi- Triggle et al. 715 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. pose tissue can be the source of pro- and anti-oxidative stress factors (Xiao et al. 2011). The endothelium as an “endocrine organ” Given the important functional role of the endothelium in the control of not only blood flow, but also angiogenesis, in- flammation, platelet aggregation, and vascular remodelling as well as metabolism, this tissue can be described as an endo- crine organ, despite being made up from only one cell type (Félétou 2011a). Figure 3 provides a summary of the multi- ple roles of the endothelium in the regulation of blood vessel function. The endothelium plays an important role in the regulation of VSMC growth and produces a number of growth factors such as platelet-derived growth factor (PDGF), basic fibro- blast growth factor (bFGF), insulin-like growth factor-1, and also growth inhibitory factors such as heparin (Peiró et al. 1995). NO and PGI2, in addition to their roles as EDRFs, have antiproliferative actions on VSMCs via cGMP- and cAMP-dependent mechanisms, respectively (Garg and Hassid 1989; Shirotani et al. 1991). Another endothelium-derived vasodilator factor that has cGMP-dependent antiproliferative activity on VSMCs is C-type natriuretic peptide (CNP) whose secretion is regulated by shear stress (Furuya et al. 1991; Zhang et al. 1999). Endothelium-derived endothelin 1 Fig. 1. Possible interactions between endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and perivascular adipose tissue. Intra- cellular Ca2+ release and extracellular Ca2+ entry into the EC is initiated by an endothelium-dependent vasodilator agonist resulting in (i) the generation of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS); (ii) the generation of prostacylcin (PGI2) by cyclooxygenase (COX); and (iii) the activation of endothelium derived hyperpolarizing factor (EDHF) pathway. NO-dependent activation of soluble guanylyl cyclase (sGC) results in the production of cyclic guanosine monophosphate (cGMP) and protein kinase G (PKG). Opening of the BKCa (BK) channel (Ca2+ activated large conductance K+ channel), hyperpolarisation, and closure of the voltage gated Ca2+ channel (VGCC) by cGMP and PKG results in the relaxation of VSMCs. Additionally activation of the IP1 receptor on the VSMC plasma membrane is activated by PGI2 and results in an increase in cyclic adenosine monophosphate (cAMP) levels, associated PKA activation, and relaxation of VSMC via inhibi- tion of myosin light chain kinase (MLCK). Hyperpolarisation of the VSMC is mediated by EDHF via direct EC–VSMC communication through the myoendothelial gap junctions (MEGJ), formation of cytochrome P450 (CYP) derived epoxy-eicosatrienoic acids (EETs), and their action on the BK channels in VSMC, and (or) efflux of K+ from EC via opening of small conductance (SK or SKCa, KCa2.3) and inter- mediate conductance (IK or IKCa, KCa3.1) calcium-activated K+ channels and subsequent hyperpolarisation of VSMC via activation of Na+– K+ ATPase and (or) opening of Kir (inward rectifying K+ channels). The EC–VSMC interactions may be modulated by perivascular adipose tissue derived factors such as adipokines, adipose-derived relaxing factor (ADRF), and superoxide generation from the NADPH oxidase system. 716 Can. J. Physiol. Pharmacol. Vol. 90, 2012 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. (ET-1) is not only a potent vasoconstrictor, but also demon- strates pro-proliferative actions on VSMCs, and its synthesis is inhibited by NO (Boulanger and Luscher 1990). Endothe- lial damage leads to dysregulation in the balance between anti- and pro-proliferative stimuli leading to intimal hyperpla- sia. Angiogenesis, a term broadly used in this review to reflect blood vessel growth, is the process of growing new blood vessels from pre-existing vessels and plays an important role in, for instance, wound healing as well as tumour growth. Angiogenesis involves the activation of signalling pathways that result in endothelial cells acquiring a proliferative and migratory phenotype, and involves the contribution of a num- ber of growth factors including fibroblast growth factor-1, FGF-1, bFGF, vascular endothelial growth factor (VEGF), and the angiopoietins as well as the matrix metalloprotei- nases (MMPs) (Adams and Alitalo 2007). Endothelial cells also have the capacity to synthesize the proteins associated with the basal lamina as well as MMPs (Kalebic et al. 1983). The proteolytic action of endothelial-cell-derived MMPs, particularly MMP-9, is essential for the release of en- dothelial cells into the interstitial matrix, as seen in sprouting angiogenesis and vascular remodelling (Kalebic et al. 1983; Rundhaug 2005). Although it is well established that angio- genesis is attenuated when the bioavailability of NO is re- duced, the mechanism(s) whereby NO regulates angiogenesis is(are) not clearly established, but include an up-regulation of VEGF, suppression of the angiogenesis inhibitor angiostatin, promotion of endothelial cell migration, as well as inhibition of apoptosis and extracellular matrix dissolution (Ziche et al. 1997; Murohara et al. 1999; Rössig et al. 1999; Dulak et al. 2000; Matsunaga et al. 2002). The endothelium also plays a key role in the regulation of immune responses, and the endothelial cell layer serves as Fig. 2. Vascular tone and endothelial cell (EC) – vascular smooth muscle cell (VSMC) communication. Close communication between ECs and VSMCs regulates the degree of vascular constriction (tone) and blood flow. (A) Factors such as shear stress cause endothelial dysfunction and platelet aggregation. (B) The endothelium-derived factors, notably endothelium-dependent hyperpolarizing factor (EDHF), NO, and PGI2 aid in the hyperpolarization of the VSMC and resultant vasodilation. Endothelial dysfunction caused by shear stress or other risk factors tend to reduce the levels of EDHF, NO, and PGI2, along with an increase in endothelium-derived constriction factors (EDCFs) leading to VSMC cell depolarization and vessel constriction. (C) The EC–VSMC contacts are made through holes (fenestrae) in the internal elastic lamina (IEL). The heterocellular contacts are made through myoendothelial gap junctions (MEGJ) made up of connexins 37 and 40. The MEGJs are closely associated to the Na+/K+ ATPase and Kir (inward rectifying K+ channel) on the VSMC membrane and KCa3.1 (IKCa), the calcium sensor receptor (CaSR) and Kir on EC membranes via “signalling microdomains”. Triggle et al. 717 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. the gateway for the entry of leukocytes into tissue in re- sponse to inflammatory stimuli by a transmigration process termed extravasation. In brief, transmigration of leukocytes initially requires the tethering and rolling of the leukocyte on the surface of the endothelial cells, involving members of the selectin superfamily, such as E-selectin, that are expressed by the leukocytes. The activated leukocytes then bind, via integ- rins, to endothelial cell adhesion molecules such as ICAM-1 and VCAM-1 and, finally, the leukocytes move through the endothelial cell layer via the gaps between adjacent endothe- lial cells — a process termed diapedesis (Johnson-Léger et al. 2000). The mechanisms whereby leukocytes are able to spe- cifically target sites of injury, rather than normal tissue, in- volves sensing mechanisms that allow the leukocytes to remain intravascular and localize to sites of injury through “necrotaxic signals” (McDonald et al. 2010). A reduction in the bioavailability of NO increases the recruitment of leuko- cytes to the endothelium, indicating that endothelial cell- derived NO plays an important anti-inflammatory role that, in part, is mediated by the inhibition of adhesion molecule expression (Kubes et al. 1991; Niu et al. 1994). The endothelium is an important site for the production of PGI2, a potent inhibitor of platelet aggregation, and it is also the source of molecules that affect fibrinolysis. Endothelial- cell-derived tissue-type plasminogen activator (t-PA) is re- leased from either stored pools or following de-novo synthesis, and is the key mediator of intravascular fibrinolysis converting plasminogen into plasmin (Kooistra et al. 1994). Acetylcholine and other endothelium-dependent vasodilators release t-PA by a calcium-dependent pathway similar to the signalling pathway that regulates the generation of NO, but is independent of NOS, or COX, activation (Tranquille and Emeis 1991, 1993; Brown et al. 2000). Among the endothelium-dependent vaso- dilators studied in rats, bradykinin is a particularly potent releaser of t-PA, suggesting an important role for bradykinin in the initiation of fibrinolysis (Smith et al. 1985). The physiological inhibitor of t-PA, the serine protease inhibitor plasminogen activator inhibitor-1, is also synthesized in en- dothelial cells as well as liver and adipose tissue (van Mourik et al. 1984; De Taeye et al. 2005). In conclusion, in addition to the important contributions of the endothelium to the regulation of blood flow, the endothe- lium has multiple roles that affect vascular function including the regulation of vascular growth, angiogenesis, inflamma- tion, and the coagulation pathway. Regulation of eNOS eNOS is tightly regulated via several distinct transcrip- tional and posttranscriptional mechanisms that are all poten- tially sites where changes in function may result in the development of vascular disease (Fig. 4). The key regulatory processes are briefly summarized and also schematically rep- resented in Fig. 4. Regulation of eNOS at the transcriptional and post- transcriptional (mRNA stability), as well as post-translational level is an important control step that can affect endothelial function. The catalytic activity of eNOS is influenced by Fig. 3. Evolving nonlumen functions of endothelial cells (ECs). Several endothelial-derived factors have a critical role in changing the vessel diameter, as per the requirement of the microenvironement. However, this single layer of ECs that line the lumen of vasculature, have come to be known as very dynamic entities that perform a wide range of functions. The endothelium is known to function as a selective “barrier,” regulating the solute flux and fluid permeability between blood and tissues. Apart from its “autocrine” functions, ECs exert significant para- crine and endocrine functions through their influence on the underlying smooth muscle cells and circulating blood elements such as the pla- telets and white blood cells. ECs play a critical role in maintaining blood homeostasis and fluidity/continuity while also initiating, when necessary, the blood coagulation pathway and subsequent fibrinolysis. Several of the EC-specific cell adhesion molecules are responsible for the capture and rolling, adhesion and arrest, and transendothelial migration (diapedesis) of leukocytes, as a part of the inflammatory or innate immune response. ECs are of prime importance in vessel formation during embryonic development of the circulatory system (vasculogenesis), vessel formation, and remodeling during the formation of new-blood vessels from pre-existing vasculature (angiogenesis), and vessel differ- entiation and maturation (arteriogenesis). CNP, C-type natriuretic peptide; ET-1, endothelin 1; ICAM-1, intercellular cell adhesion molecule 1; NO, nitric oxide; PAI-1, plasminogen activator inhibitor 1; PECAM-1, platelet endothelial cell adhesion molecule 1; PGI2, prostacyclin; tPA, tissue plasminogen activator; VCAM-1, vascular cell adhesion molecule 1. 718 Can. J. Physiol. Pharmacol. Vol. 90, 2012 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. regulatory proteins such as calmodulin and caveolin, and by a variety of post-translational modifications including phos- phorylation, acetylation, protein–protein interactions, and the availability of key substrates and cofactors for eNOS func- tion that include L-arginine, BH4, iron (Fe), FMN, FAD, NADPH, and ROS, the latter possibly via H2O2 (Drum- mond et al. 2000; Fleming and Busse 2003; Sessa 2004). Cotranslational myristoylation of eNOS on N-terminal gly- cine, and post-translational cysteine palmitoylation on C15 and C26 control the localization and subcellular targeting of eNOS to plasmalemmal caveolae. Myristoylation provides general membrane association, while palmitoylation specifi- cally directs proteins to the plasma membrane (García- Cardeña et al. 1996, Prabhakar et al. 2000; Sessa 2004; Dudzinski et al. 2006). Receptor-mediated agonist stimulation leads to rapid enzyme activation by the binding of calcium– calmodulin, depalmitoylation, displacement of caveolin-1, and release of eNOS from caveolae (Michel et al. 1997; Prabhakar et al. 1998). Physiological stimuli such as vascu- lar endothelial growth factor (VEGF), estrogen, sphingosine 1-phosphate (S-1-P), and bradykinin, and hemodynamic forces of blood, including laminar shear stress, activate phosphoinoside 3-kinase (PI3K) via different receptors. Stimulated PI3K further phosphorylates its downstream kin- ase AKT (protein kinase B). Different regulatory sites (Ser 617, Ser 635, Ser 1177, Ser 1179, Thr 495) on eNOS influ- ence its physiological and pathological activities. The cata- lytic activity of eNOS towards NO generation is increased when phosphorylated at Ser 1177 by numerous protein kin- ases including protein kinase A (PKA), AKT, AMPK, calmodulin-dependent protein kinase II, while phosphoryla- tion at Thr 495 and Ser 116 are inhibitory. Phosphorylation at Ser 617 by either PKA or AKT appears important for the recruitment of eNOS to calmodulin binding, whereas phos- phorylation at Thr 495 by PKC attenuates this association (Dimmeler et al. 1999; Fulton et al. 1999; Fleming et al. 2001; Fulton et al. 2001; Michell et al. 2002; Dudzinski and Michel 2007). Heat shock protein 90 (Hsp90), a molec- ular chaperone, associates with eNOS to accelerate its cata- lytic activity by creating a favorable biological environment for AKT-dependent phosphorylation. Thus, Hsp90/eNOS as- sociation influences the rate of AKT-dependent phosphory- lation, unmasking the phosphorylation sites on eNOS and maintaining the p-AKT levels by inhibition of proteasomal- mediated degradation of PI3K (García-Cardeña et al. 1998, Sato et al. 2000, Wei and Xia 2005; Takahashi et al. 2006). Despite the importance of eNOS phosphorylation, recent studies also suggest that post-translational modifications via nitrosylation, acetylation, and association with interacting proteins such as NOS interacting protein (NOSIP) and NOS trafficking inducer protein (NOSTRIN) are also simi- larly important for efficient catalytic activity. S-Nitrosylation of eNOS leads to enzyme inhibition, whereas denitrosyla- tion is associated with an increase in enzyme activity (Ravi et al. 2004; Erwin et al. 2006). Interaction between eNOS and NOSIP helps the translocation from plasma membrane to intra cellular membrane (Dedio et al. 2001), while NOS- TRIN serves as a common platform for the association of multiple proteins with eNOS (Icking et al. 2005). eNOS mRNA stability is also reduced by RhoA kinase activity and a number of studies have reported that inhibition of Rho-associated protein kinase, ROCK, improves endothelial function (Laufs and Liao 1998; Noma et al. 2006; Chan et al. 2009; El-Remessy et al. 2010). The enzyme sirtuin deacetylase (SIRT1) increases during calorie restriction in mammals, and may play a similar role as the nonmammalian homolog sitruin gene-product, Sir2, or silence information regulator, that determines longevity in Caenorhabditis elegans and other nonmammalian species (Chang and Min 2002; Bordone and Guarente 2005). Although the interpretation of the longevity data with C. ele- gans and Drosophila has recently been disputed, there is still an increasing level of interest in the role that the sirtuin fam- ily may play in mammalian homeostasis, and particularly as a sensor of calorie restriction (Burnett et al. 2011; Lombard et al. 2011). Attention has been placed on SIRT6, which also protects against the effects of a high-fat diet and is linked with longevity in mice (Kanfi et al. 2010, 2012). The protec- tive effects of SIRT1 on endothelial homeostasis via the reg- ulation of eNOS-mediated NO production may be a contributory factor to the benefits of SIRT1. SIRT1 has been shown to deacetylate Lys 496 and 506 in the calmodulin- binding domain of eNOS, which in turn regulates endothe- lial-dependent vasomotor tone (Mattagajasingh et al. 2007; Arunachalam et al. 2010). Mattagajasingh et al. (2007) re- ported that SIRT1-mediated increases in EDV result from an Fig. 4. Regulation of endothelial nitric oxide synthase (eNOS). In the plasma membrane, eNOS is mainly associated with caveolin-1 through a consensus site, which precludes calmodulin binding and activation of eNOS. An increase in free intracellular calcium, as well as receptor-mediated agonist stimulation, results in eNOS dissocia- tion from caveolae and binding of the calcium–calmodulin (CaM) complex. Recruitment of hsp90 to the complex further facilitates the strong binding of calcium-activated CaM and release of caveolin in- hibitory clamp. Based on the physiological stimuli, eNOS shuttles between the caveolae and subcellular compartments such as the cy- tosol and Golgi. Caveolae-free eNOS undergo various protein-kinase (PK)-mediated phosphorylation (red line) and SIRT1-mediated dea- cetylation (pink line). Phosphorylation at Ser 1177 by PKA, AKT (protein kinase B), PKC, and adenosine monophosphate kinase (AMPK) activate, while phosphorylation at Ser 116 and Thr 495 inhi- bit eNOS activity. Finally active eNOS catalyses the conversion of mo- lecular oxygen to NO, using a terminal guanidino group of L-arginine as the substrate and nitrogen donor (green line). Triggle et al. 719 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. eNOS-dependent, but not an eNOS-independent process, and this conclusion is supported by data indicating that overex- pression of SIRT1 in mice increases the conversion of L-[14C]arginine to L-[14C]citrulline. In calorie-restricted ani- mals, NO has been shown to activate the SIRT1 promoter, leading to an increase of SIRT1 mRNA and protein (Nisoli et al. 2005; Ota et al. 2008), indicating that a positive feed- back mechanism exists between SIRT1 and eNOS (Potente and Dimmeler 2008). Inhibition of SIRT1 in vascular endo- thelial cells decreases eNOS expression, resulting in an in- crease in cellular senescence, while over-expression of SIRT1 activates eNOS with subsequent generation of NO, thereby preventing endothelial cell senescence (Ota et al. 2007, 2008). SIRT1 also promotes endothelial cell prolifera- tion and prevents senescence via deacetylation of the up- stream regulator of AMPK, the serine/threonine kinase, LKB1, thus indicating that SIRT1 and LKB1/AMPK are key sensors with potentially opposing influences that are in- volved in fine-tuning endothelial cell survival (Zu et al. 2010). Deacetylation of eNOS by SIRT1 correlates with its phosphorylation/dephosphorylation status. Shear stress in- creases SIRT1 expression in endothelial cells, activates AMP-activated protein kinase, AMPK, and enhances eNOS phosphorylation at serine 633 and serine 1177 (Chen et al. 2010). In AMPKa2–/– mice, eNOS acetylation levels are higher, thus inferring that AMPK-mediated phosphorylation of eNOS is required for SIRT1 to deacetylate eNOS (Chen et al. 2010). In addition, cross-regulation by AMPK and SIRT1 results in activation/inactivation of many biological targets, including eNOS, and determines the beneficiary ef- fects of AMPK and SIRT1 in disorders such as diabetes and cardiovascular disease (Ruderman et al. 2010; Dolinsky and Dyck 2011). Thus, overexpression of SIRT1 in ApoE–/– mice reduces the expression of the adhesion molecules, in- tercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), by suppressing NF-kB sig- nalling and reducing ROS production (Stein et al. 2010). Despite the fact that eNOS phosphorylation sites have been extensively studied, the identification of SIRT1 and other protein kinase targeted sites in eNOS and their interplay with phosphorylation sites opens a new avenue for future research, particularly with respect to targeting endothelial dysfunction and vascular disease. Importance of EDHF There is extensive literature concerning the importance of EDHF in the regulation of vascular function, and much of that has been comprehensively reviewed by Edwards et al. (2010), Félétou and Vanhoutte (2006a), and Félétou (2011b). It is therefore not the intention of the current review to reiter- ate all that has already been reviewed by Edwards et al. (2010) and Félétou (2011b), but rather to highlight key as- pects concerning EDHF, and then focus on areas of contro- versy concerning putative chemical mediators of EDHF. Although both NO and PGI2 can hyperpolarize vascular smooth muscle, an EDHF-mediated EDV is usually, by defi- nition, considered to be both NO- and PGI2-independent. The classical EDHF-mediated EDV is therefore endothelium- dependent, insensitive to inhibition by a combination of NOS and COX inhibitors, results in hyperpolarization of VSMCs, and is inhibited by the presence of a combination of small (SKCa) and intermediate conductance calcium-activated K+-channel (IKCa) blockers such as apamin + charybdo- toxin, or apamin + TRAM-34 (Waldron and Garland 1994; Edwards et al. 2010; Félétou 2011a, 2011b). However, both IKCa and SKCa channels are also involved in the regulation of NO generation in the large mesenteric artery from the rat, and thus the designation of an EDV being mediated by EDHF requires verification that neither NO nor PGI2 is the mediator of the hyperpolarization (Félétou 2011b; Stankevi- cius et al. 2011). IKCa and SKCa channels are essential for the mediation of the EDHF response, and have been proven to be located on endothelial cells from several mammalian species, including humans (Edwards et al. 1998; Kohler et al. 2000; Burnham et al. 2002; Bychkov et al. 2002). Although activation of both IKCa and SKCa channels is required to initiate EDH and EDV, Crane et al. (2003) demonstrated that in a nonde- polarized state, EDH is apamin-sensitive and thus dependent on SKCa activation. In depolarized arteries there is a require- ment for both SKCa and IKCa activation, thus giving rise to the hypothesis that these KCa channels occupied different microdomains in endothelial cells (Crane et al. 2003). This hypothesis is supported by the knowledge that typically IKCa channels are not located in caveolae, but colocated in microdomains in close association with other proteins in- volved in the initiation of the EDHF-mediated EDH, such as the calcium sensing receptor (CaSR), inositol trisphosphate (IP3) receptors, Kir (inward rectifying K+ channel), and Cx37/40 (Fig. 2) (Dora et al. 2008; Ledoux et al. 2008; Sandow et al. 2009a; Dora 2010). In contrast, SKCa channels are typically located in caveolin-rich areas of the cell together with, for instance, the bradykinin receptor and the shear- stress sensing TRPV4 channel, as well as with endothelial cell-to-endothelial cell gap junctions (Absi et al. 2007; Dora et al. 2008; Saliez et al. 2008; Sandow et al. 2009a; Dora 2010; Mendoza et al. 2010). These “signalling microdo- mains” are critical for the regulation of the conductance of a vasodilatory stimulus, and facilitate endothelial to vascular smooth muscle cell cross talk and the associated conducted vasomotor response and are potential sites for dysfunction in association with vascular disease (Sandow et al. 2006, 2009a, 2009b; de Wit and Griffith 2010; Dora 2010; Triggle and Ding 2011); see also Fig. 2C. What is EDHF? We have already stated that the classical EDHF-mediated EDV is endothelium-dependent and is insensitive to inhibi- tion by a combination of NOS and COX inhibitors. This def- inition is supported by data from eNOS/COX double knockout mice in which EDH has been shown to be unaf- fected (Scotland et al. 2005). A number of putative chemical mediators of EDHF have been identified including the gases CO and H2S, as well as H2O2, cytochrome P450 (CYP) prod- ucts such as the epoxyeicosatrienoic acids (EETs), the vaso- dilator peptide C-type natriuretic peptide, and small increases in extracellular K+ resulting from the opening of endothelial cell IKCa and SKCa channels. With the possible exception of K+, not all of the published data support these mediators as being EDHFs (McGuire et al. 2001; Ellis et al. 2003; Sandow and Tare 2007). The following 3 sections re- 720 Can. J. Physiol. Pharmacol. Vol. 90, 2012 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. view the evidence for the importance of NOS expression, the contribution of hydrogen sulphide (H2S), and the importance of ROS in the generation of an EDHF-mediated EDV. Is NOS expression required for EDHF-dependent EDV? An EDHF-like EDV persists in small mesenteric arteries from mice in which eNOS have been genetically ablated, albeit attenuated from that produced in vessels from eNOS- expressing mice (Waldron et al. 1999). In triple (“triply”) NOS knockout mice, wherein eNOS, iNOS, and nNOS have been genetically ablated, EDHF-mediated EDV was not de- tected in aortic ring preparations, inferring that a functional NO-generating pathway is essential for EDHF (Nakata et al. 2008; Tsutsui et al. 2010). However, since EDV in the mouse aorta is purely dependent on eNOS-derived NO, the interpre- tation of such data from the triply NOS–/– mice as inferring that a functional NO-generating system is an essential prereq- uisite for eliciting an EDHF-mediated EDV is clearly prob- lematic (Huang et al. 1995; Waldron et al. 1999). Is hydrogen sulphide an EDHF? The potential contribution of H2S to EDHF has been as- sessed in aortic and small mesenteric arteries from mice with intact NOS enzymes, but lacking the enzyme cystathionine g- lyase (CSE) for the synthesis of H2S (Mustafa et al. 2011). These data indicate a lack of EDV-and EDH-responses to acetylcholine in the aorta and mesenteric vessels from CSE- deficient animals and the mice also display pronounced hy- pertension (Mustafa et al. 2011). However, the H2S-mediated EDHF-like response described by Mustafa et al. (2011) re- sults primarily from the cysteine S-sulfhydration activation of glibenclamide-sensitive KATP channels, with only a mini- mal contribution (25%) of IKCa and SKCa — the latter chan- nels being those identified as mediating the “classic” EDHF- mediated EDV (Waldron and Garland 1994; Edwards et al. 2010). The interpretation of these observations as inferring CSE to be the putative “EDHF synthase,” or the previously dis- cussed data from triply NOS–/– mice that inferred a NOS re- quirement for EDHF-mediated EDV, requires caution as analysis of data from genetic knockout mice, such as for CSE and NOS knockouts, may be complicated as the result of compensatory changes that either mask or enhance the cel- lular mechanisms that mediate EDV. Is endothelial cell-derived ROS an EDHF? ROS include the paramagnetic free radical superoxide (O 2 ), and hydroxyl (HO•), as well as diamagnetic H2O2. ROS serve both cytotoxic functions as well as important physiological roles in cell signalling (Thannickal and Fan- burg 2000; Ellis and Triggle 2003). The Nox family (Nox 1–5 and DUOX1 and DUOX2) of NADPH oxidases are major cellular sources of ROS (Bedard and Krause 2007. Nox 1, 2, 4, and 5 are expressed in endo- thelial cells with Nox2 and Nox4, the major sources of ROS in endothelial cells; however, other sources include the COXs, xanthine oxidase, CYP, mitochondria, and the mono- meric “uncoupled” eNOS (Ellis and Triggle 2003; Touyz et al. 2011). H2O2, which is produced either by spontaneous dismuta- tion of superoxide (O 2 ) or dismutation of superoxide via superoxide dismutase (SOD), is of particular interest in terms of its role as both an EDCF and an EDRF and also as a pu- tative EDHF (see reviews by Ellis and Triggle 2003; Félétou 2011a). Low concentrations of H2O2, at least in blood vessels from the systemic circulation of rabbits, evoke contraction and higher concentrations (>0.3 mmol/L), or relaxation that may be preceded or followed by a contraction (Bény and von der Weid 1991; Bharadwaj and Prasad 1995; Gao et al. 2003; Ardanaz et al. 2008). It has been argued, based on the overexpression data of catalase, that the physiological func- tion of H2O2 is as a vasoconstrictor and, furthermore, H2O2 has been identified as a putative EDCF in rabbit arteries (Gao and Lee 2005; Suvorava and Kojda 2009). In the cere- bral circulation, however, ROS products, including H2O2, are prominent vasodilators, suggesting a different physiological function(s) in cerebral blood vessels for H2O2 than in the sys- temic circulation (Miller et al. 2006). The vascular actions and the role of the endothelium in the contribution of H2O2 to EDV are complex, and H2O2 may also serve as an EDHF, with some evidence indicating a redox-mediated activation mechanism involving thiol oxida- tion of voltage-gated potassium channels (KV) on vascular smooth muscle, whereas in some vascular beds its actions are either entirely eNOS and NO-dependent or endothelium- independent via the direct activation of soluble guanylyl cy- clase and, possibly, COX (Fujimoto et al. 2001; Thomas et al. 2002; Sato et al. 2003; Saitoh et al. 2007). Evidence for H2O2 as an EDHF in rodent, porcine, and human vessels has been presented by several laboratories and reflects a catalase- sensitive (inhibitable) response (Miura et al. 1999; Matoba et al. 2000, 2002, 2003; Shimokawa and Matoba 2004; Liu et al. 2011). However, in contrast to the report from Matoba et al. (2000), other studies with murine resistance vessels report that the EDHF-mediated EDV reflects a catalase-insensitive response (Ellis and Triggle 2003; Ellis et al. 2003). Similarly, data from Bény and von der Weid (1991) and Chadha et al. (2011) fail to confirm reports from Matoba et al. (2003) and Matoba et al. (2002) that H2O2 is an EDHF in, respectively, porcine coronary and human mesenteric arteries. Numerous other studies have also presented data that do not support a role for H2O2 as an EDHF, for instance Chaytor et al. (2003). Perhaps, as suggested by Edwards et al. (2008), the “EDHF-like” effect of H2O2 is not due to a direct hyperpola- rization, but results from a H2O2-mediated enhancement of calcium release in endothelial cells. However, the contribu- tion of H2O2 to EDH may depend on the stimulus for endo- thelial cell activation. In human coronary arteries, the transferable factor that mediates FMD has been identified as H2O2, and hyperpolarization of VSMC results from the open- ing of BKCa channels via a mechanism(s) requiring intact cell signalling pathways in VSMC (Liu et al. 2011). In summary, the vascular effects of H2O2 significantly dif- fer between vascular beds and are clearly concentration- dependent, and may also depend on the cellular source of H2O2, i.e., mitochondria, Nox1, or Nox2, versus Nox4. Thus, H2O2 generated from constitutively active Nox4, which has been associated with the endoplasmic reticulum rather than plasma membrane, may be the source of H2O2 as an EDHF and H2O2 from this source seems to serve a protective physiological function (Chen et al. 2008; Brandes et al. 2011; Ray et al. 2011). Furthermore, H2O2 can also Triggle et al. 721 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. regulate eNOS at both the transcriptional and posttranscrip- tional level, and may therefore play a role in helping to compensate for pathophysiological states wherein ROS pro- duction is elevated. (Drummond et al. 2000). Calcium signalling and the regulation of endothelial cell function An increase in free intracellular calcium, Ca2+, is a re- quirement for the generation of NO, PGI2, and also for the EDHF-mediated EDV (McSherry et al. 2005; Félétou and Vanhoutte 2006a; Liu et al. 2006). Hyperpolarization of en- dothelial cells in culture increases Ca2+ entry by increasing the electrochemical gradient for Ca2+ (Colden-Stanfield et al. 1987; Adams et al. 1989). The link between mechanosensi- tive shear stress activation of endothelial cells, Ca2+ entry, and EDV, at least in mouse blood vessels, is the transient re- ceptor potential vallinoid type 4 (TRPV4) channel (Mendoza et al. 2010). Endothelial cell Ca2+ and EDV are both en- hanced in mouse small mesenteric arteries from TRPV4 ex- pressing, but not in TRPV4(–/–) mice; furthermore, the TRPV4 channel blocker ruthenium red reduces Ca2+ levels and EDV in vessels from TRPV4 mice (Mendoza et al. 2010; Saliez et al. 2008). Calcium homeostasis is also dis- rupted in small mesenteric arteries from caveolin-1 knockout mice, with the resultant greater reduction in the EDHF- mediated versus the NO-mediated EDV (Saliez et al. 2008). There is also a lower expression of connexins 37, 40, and 43 in caveolin-1–/– mice, and disruption of EDHF-mediated EDV is thus linked to the loss of association between TRPV4, connexins, 37, 40, and 43 and caveolin-1 (Saliez et al. 2008). The greater sensitivity of EDHF versus NO- mediated EDV to the disruption of the caveolin-1 associated microdomain may also be linked to the higher threshold for intracellular calcium for the EDHF versus NO-mediated EDV that has been reported for rat cerebral arteries (Mar- relli 2001). Rather than global changes in free intracellular Ca2+, most likely it is the change in free intracellular Ca2+ within endo- thelial cell microdomains that is critical for the regulation of endothelial cell function. In pressurised small mesenteric ar- teries from rats, store depletion of intracellular Ca2+ rather than endothelial cell hyperpolarization drives Ca2+ influx, suggesting that discrete changes in free intracellular Ca2+ in spatially restricted “microdomains” is crucial for EDV (McSherry el al. 2005). For instance, activation of CaSR, a member of the G-protein-coupled receptor family, has been linked to the opening of the IKCa channel and the initiation of EDH (Fig. 2) (Weston et al. 2005). The activation and opening of IKCa channels are of particular importance in the generation of NO-independent EDH, whereas the opening of SKCa channels are of greater importance for facilitating NO- mediated EDH (McNeish et al. 2006). This separation of function for IKCa versus SKCa is reflected by their differing microdomain locations (Fig. 2) (Sandow et al. 2006, 2009a). The generation of “Ca2+ waves”, or“Ca2+ pulsars” in endo- thelial cells is also a critical event, and involves the release of inositol trisphosphate (IP3), which can enter endothelial cells via MEGJs from VSMCs and trigger the release of Ca2+ from the endoplasmic reticulum of endothelial cells (Kansui et al. 2008; Ledoux et al. 2008). In the microvasculature, the ECP microdomains depicted in Fig. 2 are thought to play an important role in the regula- tion of the non-NO/PGI2-mediated EDH and conducted vaso- dilatation (Ledoux et al. 2008; Dora 2010). The ECP microdomain functions as a repository for the key proteins involved in the regulation of the non-NO/PGI2-mediated EDH, including the connexins, CaSR, Kir, and IKCa (Dora et al. 2008; Sandow et al. 2009a, 2009b). Endothelial dysfunction Furchgott and Zawadzki first demonstrated the effects of physical damage to the endothelial cell layer on the vasoactive actions of acetylcholine in their 1980 publication in Nature (Furchgott and Zawadzki 1980). Using coronary angiography, it was subsequently reported that atherosclerotic human ves- sels relaxed in response to nitroglycerin but responded to ace- tylcholine with vasoconstriction, whereas vessels from angiographically normal patients relaxed to both agents, thus indicating that atherosclerosis was associated with endothelial dysfunction and not necessarily with vascular smooth muscle dysfunction (Ludmer et al. 1986). The existence and extent of endothelial dysfunction can be determined by assessing the EDV response to an endothelium-dependent vasodilator such as acetylcholine or bradykinin, but other changes are also evident. Thus, a broader definition includes not only a re- duced EDV, but also an elevation in the expression of adhe- sion molecules, enhanced VSMC proliferation, and the development of a hypercoagulatory state (Triggle and Ding 2010). Endothelial dysfunction, as is evident, for instance, in diabetic and hypertensive patients, also extends to im- pairment in the release of t-PA and (or) increased release of PAI-1, thus resulting in a procoagulatory state (Hrafn- kelsdóttir et al. 1998, 2004; Schneider et al. 1993). The chronic inhibition of NOS induces the expression of both PAI-1 and ACE in vascular tissues, and Ang II induces PAI-1 and PAI-2 expression in vascular endothelial cells, indicating that a reduction in the bioavailability of NO has multiple effects on blood vessel function beyond that affect- ing blood flow (Fig. 3) (Feener et al. 1995; Katoh et al. 2000). ROS and endothelial dysfunction ROS play important roles in the physiological regulation of vascular function; however, excessive ROS formation is associated with endothelial dysfunction, which, in part, is mediated by the oxidation of BH4 and the subsequent uncou- pling of eNOS from a dimeric to the monomeric state, and in the latter functions as an NADPH-oxidase and generates superoxide anions (Stroes et al. 1998; Xia et al. 1998; Cai et al. 2005; Ding and Triggle 2010). Heightened production of ROS is also associated with sepsis-induced endothelial dys- function (Aird 2003). Nonetheless, ROS do, as previously discussed, play important physiological roles in the vascula- ture, and thus a balance must be maintained between the gen- eration and elimination of ROS. The Nox family (Nox1–5) of NADPH oxidases are major sources for ROS production and, in the vasculature, in pathophysiological states have been as- sociated with the development of endothelial dysfunction and vascular disease primarily via Nox1 and Nox2 (Bedard and Krause 2007; Touyz et al. 2011). Nox4, unlike Nox1 and 722 Can. J. Physiol. Pharmacol. Vol. 90, 2012 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. Nox2, is constitutively active and continually produces H2O2 and is upregulated in response to a number of stimuli associ- ated with vascular disease, and thus may also be involved in the progression of cardiovascular diseases including heart failure and hypertension (Brandes et al. 2010; García- Redondo et al. 2009; Kuroda et al. 2010; see also section on Endothelial dysfunction: imbalance in EDRFs versus EDCFs). H2O2 can also up-regulate the expression of p47phox (a cytosolic subunit of NADPH oxidase) in endo- thelial cells, suggesting that in pathological situations such as sepsis, this H2O2-mediated increase in NADPH oxidase will further enhance the endotoxin and cytokine stimulated elevation in ROS production, and therefore accelerate the development of endothelial dysfunction (Wu et al. 2007). In contrast, endothelium-targeted overexpression of Nox4 has been shown to have beneficial effects on endothelial function and blood pressure inferring a beneficial role for Nox4, and inferring a detrimental role for Nox1 and Nox2 via the production of superoxide anions, to eNOS uncou- pling, and endothelial dysfunction (Ray et al. 2011). Thus, in terms of endothelial function, Nox4 may serve as “The Good NADPH Oxidase” (Brandes et al. 2011). Oxidative stress is an important contributor to endothelial dysfunction in cerebrovascular disease (Chrissobolis et al. 2011). Of interest is that the evidence for vessel-dependent compartmentalisation with respect to the subcellular sources that contribute to oxidative-induced endothelial dysfunction. Thus, in mouse basilar artery a lack of cytoloslic superoxide dismutase (CuZnSOD) impairs EDV, whereas in the cerebral arterioles, the lack of mitochondrial SOD (MnSOD) impairs EDV (Faraci et al. 2006; Modrick et al. 2009). Contribution of eNOS dyregulation to endothelial dysfunction eNOS–/– mice are insulin resistant and hypertensive, and triply NOS–/– mice exhibit a metabolic-syndrome-like pheno- type with visceral obesity, impaired glucose tolerance, ele- vated triglycerides, and hypertension (Duplain et al. 2001; Nakata et al. 2008). These observations suggest that a defi- ciency in NOS-derived NO contributes to cardiovascular and metabolic dysfunction, and supports additional data that indi- cates that there is an inverse relationship between eNOS ex- pression and the regulation of the genes that control adipogenesis (Razny et al. 2011). Based on plethysmography data, an increase in blood glucose, as occurs postprandially or following the ingestion of a 75 g oral glucose load for an impaired glucose tolerance test, results in an immediate re- duction of human forearm blood flow that can be offset by the administration of the SR, but not the inactive 6S (6S)- 5,6,7,8-tetrahydro-l-biopterin) isomer of BH4 (Kawano et al. 1999; Ihlemann et al. 2003). These data suggest that an early step in the development of diabetes-linked vascular disease is the dysregulation of eNOS that results from the generation of ROS from mitochondria following glucose metabolism (Giacco and Brownlee 2010). BH4 is an essential cofactor for eNOS, and facilitates equilibrium in favour of the dimeric form of the enzyme that optimizes the generation of NO; in the monomeric state, eNOS functions as an NADPH-oxidase (Stroes et al. 1998; Xia et al. 1998; Cai et al. 2005; Förster- mann 2010). This change in function of eNOS is referred to as “eNOS uncoupling” (Pannirselvam et al. 2002; Förster- mann and Münzel 2006). Based on data from the db/db lep- tin receptor mutant mouse model of type-2 diabetes, as well as studies with mouse microvascular endothelial cells in cul- ture, it is evident that BH4, particularly the ratio of BH4 to the oxidized biopterins, is important for maintaining endothe- lial cell function. Thus, a reduction in BH4 results in a re- duced ratio of eNOS dimer to eNOS monomer, as well as elevated oxidative stress, higher expression levels of pro- oxidant NADPH-oxidase, and reduced expression of antiox- idant SOD (Pannirselvam et al. 2002, 2003; Ding et al. 2007; Aljofan and Ding 2010). In the presence of elevated glucose and the heightened superoxide production by mito- chondria, NO and superoxide react to produce peroxynitrite, ONOO–, which, in turn, oxidizes BH4 and uncouples eNOS and reduces the dimer:monomer ratio, thus setting off the generation of more superoxide from the uncoupled eNOS — potentially this can result in an enhanced and an extended period of endothelial dysfunction (Ogonowski et al. 2000; Aljofan and Ding 2010). Treatment of db/db mice with se- piapterin, a precursor of BH4, improves the contribution of NO to EDV, but does not reduce plasma glucose levels (Pannirselvam et al. 2003). In cell culture studies with mouse microvessel endothelial cells, sepiapterin reduces high glucose-induced oxidative stress and enhances the gen- eration of NO (Aljofan and Ding 2010). Collectively these data suggest that restoring eNOS function by providing se- piapterin, a precursor of BH4, enhances protection against glucose-induced endothelial dysfunction. Elevated glucose and the resulting increase in superoxide anion generation and ONOO– formation in streptozotocin-induced type 1 dia- betic rats decrease eNOS mRNA levels in aortic tissue, and this reduction is prevented by in-vivo treatment with the peroxynitrite inhibitor, FeTTPs (El-Remessy et al. 2010). Both the FeTTPs, and the RhoA kinase inhibitor, Y26732, prevent peroxynitrite-induced, or high-glucose-induced, changes in eNOS expression and RhoA kinase levels in bovine aortic endothelial cells in culture (El-Remessy et al. 2010). Similarly in rodent models of hypertension, oxidation of BH4 results in an uncoupling of eNOS, which then func- tions as an NADPH-oxidase (Landmesser et al. 2003). Epigenetic changes in endothelial function The persistence and continued development of diabetic complications, despite therapeutic interventions, support the concept that “hyperglycaemic, or metabolic, memory” is a key contributor to the high cardiovascular morbidity and mortality associated with diabetes (Pirola et al. 2010). Stud- ies to further elucidate the association between gene regula- tion and environmental factors in the pathophysiology of type-2 diabetes indicate an important contribution resulting from the persistent activation of the nuclear transcription fac- tor, NFkB (Bierhaus et al. 2001; Brownlee 2001; Cooper and El-Osta 2010). The sirtuins, such as SIRT1, have also been implicated in the epigenetic control of metabolic pathways via the deactylation of both histone and nonhistone proteins (Schwer and Verdin 2008; Siebel et al. 2010). The impor- tance of epigenetic changes induced by hyperglycaemia can be seen following a 6 hour exposure of mice to elevated glu- cose (20 mmol/L) equivalent to a prolonged postprandial ex- posure, which results in an increase in expression of the Triggle et al. 723 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. proinflammatory NFkB that persists in mice for at least 6 days, despite exposure to normal glucose (El-Osta et al. 2008). Comparable data have been obtained from cell culture studies with bovine aortic endothelial cells (El-Osta et al. 2008). NF-kB targets multiple genes involved in inflammation, in- cluding ICAM, VCAM, and IL-1, and can also be activated by ROS (Cogswell et al. 1994; Peng et al.1995; Barnes and Karin 1997; Li and Karin 1999; Ross 1999; Wilson et al. 2000; Kim et al. 2001). Activation of NF-kB is prevented by NO, and thus a reduction in the bioavailability of NO may result in epigenetic changes that promote vascular inflamma- tion and atherosclerosis (Peng et al. 1995). Restoring eNOS function in disease states Agents that target pathways associated with the regulation of eNOS are of potential benefit for the treatment of vascular disease (Fig. 5) and restoring endothelial progenitor cell function, which in turn will improve endothelial cell prolifer- ation (Ding and Triggle 2011; Hamed et al. 2011). In this re- gard, several studies have indicated that the plant polyphenol phytoalexin resveratrol, which is found, for instance, in grapes, and hence, notably, in red wine, not only prolongs life in fruit flies (Drosophila melanogaster) and nematodes (C. elegans), but also reverses the effects of cigarette smoke on eNOS acetylation in human umbilical vein endothelial cells, suggesting that resveratrol directly, or indirectly, targets Sir2 and SIRT1 (Bass et al. 2007; Arunachalam et al. 2010). The potential use of resveratrol for a number of cardiovascu- lar diseases has been reviewed (Dolinsky and Dyck 2011). Resveratrol also mediates an upregulation of the antioxidant enzymes SOD1, SOD2, SOD3, glutathione peroxidase, and catalase, as well as the rate-limiting enzyme for BH4 synthe- sis, and GTP cyclohydrolase, but it mediates the the downre- gulation of NADPH oxidase Nox2 and Nox4 in cardiac tissue from ApoE–/– mice, with comparable data for human endothe- lial cells in culture (Xia et al. 2010). Of additional interest was the observation that the SIRT1 inhibitor sirtinol inhibited some of the effects of resveratrol (Xia et al. 2010). Resvera- trol, however, is not a very potent activator of SIRT1, with a reported EC50 of approximately 50 µmol/L (Milne and Denu 2008). Metformin, the most frequently prescribed oral hypo- glycaemic agent for type-2 diabetes, in addition to having po- tentially multiple targets, also directly or indirectly activates SIRT1, possibly in part also involving AMPK, with subse- quent multiple target actions that include improved endothe- lial function and an inhibition of hepatic gluconeogenesis (Mather et al. 2001; Zhou et al. 2001; Detaille et al. 2005; Caton et al. 2010; Foretz et al. 2010). Enhanced expression of SIRT1 also protects against hyperglycaemia-induced endo- thelial senescence (Orimo et al. 2009). Agents that increase eNOS transcription, such as AVE9488, may prove to be of potential benefit for restoring Fig. 5. Potential therapeutic targets for endothelial protection. (A) In a diabetic milieu, there is a significant increase in Nox1/Nox2 derived pathological reactive oxygen species (ROS) and concomitant reduction in nitric oxide (NO) and prostacyclin (PGI2) levels. Additionally, the balance between endothelium dependent relaxing factors (EDRFs) and endothelium dependent constricting factors (EDCFs) is disrupted, leading to endothelial dysfunction and higher incidence of cardiovascular complications. (B) Physiological Nox4 activity is thought to be rather protective. Therapeutic strategies maybe used to restore Nox4 activity, reduce the level of pathological ROS, restore NO and PGI2 levels, and achieve a balance between the levels of EDRFs and EDCFs to protect the endothelium and thereby reduce associated cardiovas- cular complications, improve prognosis, and improve the quality of life in diabetic subjects. 724 Can. J. Physiol. Pharmacol. Vol. 90, 2012 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. endothelial function in disease states such as diabetes as, for example, treatment with AVE9488 has been shown to reverse eNOS uncoupling and restore endothelial function in aortae from db/db diabetic mice (Cheang et al. 2011). Furthermore, AVE9488 improves endothelial function and lowers blood pressure in the hypertensive rat (SHR) with comparable ef- fects on eNOS coupling in cell culture studies of mouse aortic endothelial cells (Yang et al. 2011). As previously noted, the RhoA – Rho kinase (ROCK) pathway may also contribute to endothelial dysfunction, as ROCK inhibitors have been shown to improve endothelial function in human patients with coronary artery disease, in- hibit EDCF-mediated vasoconstriction in hypertension, re- store NOS expression and Ser1177 phosphorylation, as well as protect eNOS mRNA (Eto et al. 2001; Nohria et al. 2006; Chan et al. 2009; Hassona et al. 2010; El-Remessy et al. 2010; Yao et al. 2010). Role of EDHF in disease states In the presence of disease states, such as in diabetes and hypertension, where ROS production is elevated and the bio- availability of NO is reduced, EDHF may function as a “back-up” to maintain FMD and blood flow. In diabetes, the contribution of EDHF seems to be maintained despite a re- duction in EDV as, for instance, is evident in small mesen- teric arteries and coronary arteries from db/db type 2 diabetic mice (Pannirselvam et al. 2002; Pannirselvam et al. 2006; Park et al. 2008). Of interest, however, is that the pharmaco- logical properties of the EDHF-mediated response appear to change, suggesting changes in the nature of the et mediators and (or) signalling pathways with an apparent greater role for a CYP-product(s) in the mediation of bradykinin-induced EDV in mesenteric arteries and for H2O2 in acetylcholine- mediated EDV in coronary arteries from db/db mice (Pan- nirselvam et al. 2002; Pannirselvam et al. 2006; Park et al. 2008). Results from a study of forearm vasodilator re- sponses to bradykinin in healthy humans versus those with hypercholesterolemia indicate a reduction in NO-mediated, but enhanced tetraethylammonium (TEA) and CYP-inhibitor sensitive, vasodilator responses, thus inferring an up-regula- tion of an EDHF contribution to EDV in disease states (Oz- kor et al. 2011). In pressurised small mesenteric arteries from ApoE–/– hypercholesterolaemic mice, the contribution of NO-independent, EDHF-mediated dilation is enhanced, thus supporting the argument that EDHF-mediated EDV may serve as a back-up pathway for the maintenance of EDV in disease states (Beleznai et al. 2011). A reduction in the bioavailability of NO as a result of the in- crease in oxidative stress that is associated with diabetes and hypertension may trigger changes in EDV that are vascular-bed specific. NO is a potent inhibitor of catalase and, in conse- quence, a reduction in NO will alter the vasoactive profile of H2O2 and catalase can also slowly consume NO (Mohazzab-H et al. 1996; Brunelli et al. 2001). Furthermore, a reduced bioavailabilty of NO may enhance the synthesis of EETs as NO binding to the haem enzyme CYP depresses CYP activ- ity (Khatsenko 1998). In human coronary arterioles taken from patients undergoing cardiopulmonary bypass proce- dures, both H2O2 and EETs mediate vasodilatation and are putative EDHFs; however, H2O2 inhibits CYP, and thus re- duces the synthesis of EETs suggesting an inhibitory role for H2O2 in EET-mediated EDH (Larsen et al. 2008). In mice with dyslipidaemia, a shift towards a greater contribu- tion of CYP metabolites to the EDHF-mediated EDV is evi- dent in small blood vessels from diabetic and dyslipidaemic mice; however, in these studies it is unclear whether the ac- tivation of CYP is associated with a physiological or patho- physiological compensation for the reduced contribution of NO to EDV (Krummen et al. 2005; Pannirselvam et al. 2006). In addition, Krummen et al. (2006) suggest that in mice with dyslipidaemia but normoglycaemia, there is an initial compensatory increase in the contribution of EETs to EDHF, but an age-related increase in the production of ROS results in the gradual loss of the contribution of EETs to EDV (Krummen et al. 2006). That the same pathophysi- ology may also develop in humans is suggested by the de- scription of a CYP2C9-derived, sulfaphenazole-sensitive EDHF as the mediator of bradykinin and acetylcholine- mediated forearm vasodilatation in hypertensive, but not normotensive, patients (Taddei et al. 2006). Thus, a CYP product provides a compensatory mechanism for partially maintaining EDV in disease states; however, as illustrated by the study from Larsen et al. (2008), elevated ROS, in the form of H2O2, can inhibit the synthesis of EETs and re- duce the contribution of EETs to EDH. Both NO and EDHF, and possibly a CYP product, are im- portant for the regulation of arterial stiffness, and with ageing there is a progressive increase in wall stiffness that is acceler- ated in cardiovascular disease (Bellien et al. 2010). Thus, ar- guably the maintanance of EDHF-mediated vascular regulation should be important in disease states to offset the negative effects of a reduced bioavailability of NO (Selemidis and Cocks 2002; Duprez 2010). However, ageing, as well as the progression of diseases such as hypertension, result in higher levels of ROS produc- tion that can compromise the contribution of these compensa- tory signalling pathways, and thus endothelial dysfunction continues to progress (Schulz et al. 2011). As was evident to the 16th century Swiss physician Paracelsus, the basic princi- ple of concentration (dose)-dependency prevails: “Alle Ding’ sind Gift, und nichts ohn’ Gift; allein die Dosis macht, daß ein Ding kein Gift ist”, or“All things are poison, and nothing is without poison; only the dose permits something not to be poisonous” (Deichmann et al. 1986; Langman and Kapur 2006). Thus, in conclusion, although ROS are important for the physiological regulation of vascular function excessive levels of ROS generation result in the development of endo- thelial dysfunction and vascular disease that is associated, for instance, with diabetes and hypertension (Ding and Triggle 2010; Triggle and Ding 2010; Schulz et al. 2011). Endothelial dysfunction: imbalance in EDRFs versus EDCFs Endothelium-dependent contractions are seen in vascular tissue under normal physiological conditions such as, in particular, in veins and result from diffusible endothelium- derived factors, EDCFs (Fig. 6) (De Mey and Vanhoutte 1982). In cardiovascular disease, as depicted in Fig. 5, the contribution of EDCFs to the influence of the endothelium Triggle et al. 725 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. on vascular tone is greatly enhanced (Moncada and Higgs 1986; Vanhoutte et al. 2005; Barton 2011). In the spontaneously hypertensive rat (SHR) model of hy- pertension, arachidonic-acid-derived EDCFs generated via enhanced expression and activity of constitutive COX-1 in- clude the elevated production of PGI2 that at high concentra- tions, mediates vasoconstriction via the activation of thromboxane receptors and a cellular pathway involving ROCK (Rapoport and Williams 1996; Traupe et al. 2002; Tang and Vanhoutte 2008; Vanhoutte and Tang 2008; Chan et al. 2009; Félétou et al. 2009). Parallel genomic changes are evident in the vasculature from SHR as well as ageing normotensive rats, and include enhanced expression of COX- 1, thromboxane synthase, and prostacyclin synthase (Tang and Vanhoutte 2008). The enhanced expression of COX-1, but not COX-2, results in elevated production of vasocon- strictor prostanoids whose vasoconstrictor effects are en- hanced as a result of increases in thromboxane synthase expression (Tang et al. 2005; Tang and Vanhoutte 2008). The enhanced expression of prostacyclin synthase results in the conversion of COX-1 derived endoperoxides into PGI2 at levels that result in the dominant effect of PGI2, being medi- ated by activation of thromboxane receptors with resultant vasoconstriction (Gluais et al. 2005, 2006; Tang and Van- houtte 2008). Mesenteric resistance arteries from SHR dem- onstrate a heightened contractile response to H2O2 compared with vessels from normotensive WKY rats, by a mechanism involving enhanced thromboxane production via COX-1 and a subsequent elevation of O2 production, perhaps via the ac- tivation of NADPH oxidase, and an elevation of intracellular Ca2+ (García-Redondo et al. 2009). Similarly, carotid arteries from SHR respond to H2O2 with a greater contraction than vessels from WKY, and this difference was abolished by in- hibition of COX-1 with the selective inhibitor SC-560, and also greatly reduced by thromboxane receptor blockade with SQ-29548, or ROCK inhibition by the selective antagonist Y- 27632 (Denniss et al. 2010). Thus, at least in the SHR model of hypertension, the heightened vasoconstrictor response to H2O2 involves COX-1. In contrast to the results from the SHR, a heightened ex- pression of COX-2 rather than COX-1 may be an important contributor to the development of endothelial, vascular, and pancreatic beta cell dysfunction in diabetes. Thus, COX-2 ex- pression has been shown to be enhanced following exposure of monocytes to advanced glycation end products (AGEs), and comparable data have been reported by endothelial cells from both macro- and micro-vascular sources following ex- posure to simulated hyperglycaemia (Cosentino et al. 2003; Shanmugam et al. 2003, 2006; Aljofan and Ding 2010). In human aortic endothelial cells, exposure to high levels of glucose (22.2 mmol/L) resulted in a shift in the balance from vasodilator to vasoconstrictor eicosanoids and, despite an increase in the expression of prostacyclin synthase, there was a decreased contribution of PGI2 that was attributed to tyrosine nitration and reduced activity of the enzyme (Cosen- tino et al. 2003). In addition, COX-2 derived vasoconstrictor prostanoids contribute to the elevated peripheral resistance in vessels from type-2 diabetic mice (Bagi, et al. 2005). In con- trast, an enhanced production of vasodilator prostanoids from COX-2 activity has been reported to help facilitate perfusion of human cardiac tissue from patients with diabetes (Szerafin et al. 2006). In conclusion, alterations in both COX-1 and COX-2 vaso- active products contribute to endothelial and vascular dys- function in diabetes and hypertension, but significant tissue, species, and disease-related differences are evident. The vari- able contributions of COX-1 and COX-2 in disease states probably reflects the differential way in which these two en- zymes are regulated, as well as the effects of other factors such as oxidative stress and dyslipidaemia (Davidge 2001). Similarities between the effects of ageing and disease states, such as diabetes and hypertension, on the expression of COX-1 and (or) COX-2 suggest that cardiovascular disease reflects an accelerated ageing process and a heightened con- tribution of COX-products to endothelial dysfunction and the development of vascular disease (Heymes et al. 2000). Ang II is an important vasoactive molecule that is involved in the etiology of endothelial dysfunction and vascular dis- ease. The generation of Ang II from Ang I is dependent upon endothelial cell expression of angiotensin-converting enzyme (ACE) activity that is predominantly associated with the luminal surface of endothelial cells, but Ang II generation can also occur within cells, thus implying both an autocrine and paracrine function (Félétou 2011a; Ryan et al. 1976). Ang II can, via the activation of the Ang II type 1 receptor (AT1R), mediate vasoconstriction or vasodilatation via acti- Fig. 6. Healthy versus unheatlhy endothelium. (A) The net balance of local endogenous healthy endothelium-derived vasodilator (EDRFs) and vasoconstrictor (EDCFs) factors maintains normal vascular tone. (B) In pathological conditions such as hypertension and diabetes, an increase in EDCFs, as well as a decrease in EDRFs, will favour vascular contraction and potentially pathophysiological changes. 726 Can. J. Physiol. Pharmacol. Vol. 90, 2012 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. vation of the Ang II type 2 receptor (AT2R). Downstream signalling following AT1R activation is associated with the pathophysiological actions of Ang II, whereas activation of the AT2R is linked to antiproliferative and proapoptotic sig- naling pathways (Nguyen Dinh Cat and Touyz 2011). An im- portant contribution to the pathophysiological actions of Ang II results from the activation of NADPH oxidase that in- creases ROS generation including, via Nox4, H2O2 (Monte- zano et al. 2011). Endothelial dysfunction and the reduction in the bioavail- ability of NO results in elevated levels of oxidized lipopro- teins, activation of proinflammatory genes, leukocyte adhesion and adhesion molecule activity expression, prolifer- ation of VSMCs, and also the elevated synthesis of endothe- lin (Boulanger and Luscher 1990; Kubes et al. 1991; Marui, et al. 1993; Steinberg 1997; Griendling and Ushio-Fukai 1998; Vanhoutte 2000). Hickey et al. (1985) reported that porcine coronary artery cells in culture produced potent vaso- constrictor factor(s) (EDCF(s)), and a peptide (endothelin) was identified by Masaki and colleagues (Yanagisawa et al. 1988). We now know that the synthesis of endothelin is not restricted to endothelial cells, and that there is a family of 3 endothelins with their own precursor pro-endothelins and big-endothelins. The endothelins ET-1, ET-2, and ET-3, are 21 amino acid peptides that via the differential activation of ET-A and (or) ET-B receptors, mediate vasoconstriction or vasodilation (Taddei et al. 2001). Alterations in ET-mediated signalling, perhaps related to reduced NO, may contribute to vascular disease such as hypertension (Vanhoutte 2000; Tad- dei et al. 2001; Barton and Yanagisawa 2008). In conclusion, a reduced bioavailability of NO promotes endothelial and vascular dysfunction not only via profound effects on vascular tone and blood flow, but also via the pro- motion of cell proliferation and enhanced expression of adhe- sion molecules. In addition, a reduced bioavailability of NO and (or) PGI2 will also enhance the potential for platelet ag- gregation. Similarly, a reduction in the contribution of EDHF, possibly an EET, promotes platelet aggregation and CYP- products have also been reported to hyperpolarize the platelet membrane via the opening of large-conductance calcium- activated K+ channels (BKCa) on the platelet membrane (Krötz et al. 2004, 2010). See also Fig. 2. Endothelial dysfunction and alterations in the role of potassium channels and connexins Changes in the expression and (or) function of the ion channels associated with endothelial function have predict- able effects on the regulation of vascular homeostasis. Thus, mice deficient in SKCa channels are hypertensive, and those deficient in IKCa have defective EDHF-mediated EDV, and the loss of both SKCa and IKCa results in mice that have a higher blood pressure than that observed in mice where SKCa alone or IKCa alone are ablated (Taylor et al. 2003; Si et al. 2006; Brähler et al. 2009; Grgic et al. 2009; Kohler and Ruth 2010). EDHF-mediated EDV is impaired in blood vessels from a number of rat models of hypertension and, based on functional and molecular evidence, a component of this impairment can be attributed to a down-regulation of the SKCa and KIR channels with a putative link to a change in the dimer:monomer ratio for caveolin-1 (Weston et al. 2010). These data indicate the importance of the microdomains and the differential expression of the signalling molecules in- volved in the regulation of vascular tone by the endothelium (Garland 2010). Modulation of connexin expression and (or) function also affects both vascular development and function. Thus, Cx40- deficient mice are hypertensive, and the absence of both Cx37 and Cx40 results in lethal embryonic vascular abnor- malities, and the global ablation of either Cx43 or Cx45 is also lethal (Reaume et al. 1995; Kruger et al. 2000; de Wit et al. 2000; de Wit et al. 2003; Rummery et al. 2002, 2005). Changes in connexin expression and function in monocytes and platelets may also affect vascular health (Wong et al. 2006; Chanson and Kwak 2007). In addition to their impor- tant role in facilitating endothelial – vascular smooth muscle communication, connexins also regulate monocyte adhesion and platelet function. Cx37 expression has been reported to be important for limiting platelet aggregation, putatively by facilitating platelet-to-platelet transfer of cAMP, and the dele- tion of Cx37 enhances platelet reactivity; the Cx37 C1019T polymorphism in humans may also be a prognostic marker for coronary artery disease and atherosclerosis (Angelillo- Scherrer et al. 2011). In diabetic vascular disease where blood glucose levels are both elevated and not well controlled, advanced glycated end products (AGEs) may also impact on both the expression and function of potassium channels and connexins, and thus, con- tribute to the development of endothelial dysfunction and vascular disease (Wang et al. 2001, 2011). Summary The endothelium plays an essential role in maintaining healthy cardiovascular function, and early changes in endo- thelial function are indicators of future cardiovascular mor- bidity and mortality. Since the landmark report in 1980 by Professor Furchgott concerning the essential role that the en- dothelium plays in mediating the vasodilatation response to acetylcholine, our knowledge of the complexities of the endo- thelium has dramatically increased. The endothelium affects vascular function in many ways and, indeed, functions very much like an endocrine organ. NO and PGI2 are well estab- lished endothelium-derived vasoactive factors that have an important impact on vascular function and cardiovascular health and, in addition, the importance of the EDHF-mediated pathway(s) is an area of growing interest. Recognition of the key signalling pathways that regulate endothelial – vas- cular smooth cell communication and their contribution to the rapid and longer term regulation of cardiovascular ho- meostasis remains an important focus for further research and will aid in the identification of therapeutic targets (Fig. 4) for both the prevention and treatment of cardiovas- cular diseases. Acknowledgements The authors acknowledge the generous support of the Qa- tar Foundation in part via project grants through the National Priorities Research Program (NPRP: 08-165-3-054; 4-910-3- 244) as well as an establishment Biomedical Research Pro- gram grant. Triggle et al. 727 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. References Absi, M., Burnham, M.P., Weston, A.H., Harno, E., Rogers, M., and Edwards, G. 2007. Effects of methyl beta-cyclodextrin on EDHF responses in pig and rat arteries; association between SK(Ca) channels and caveolin-rich domains. Br. J. Pharmacol. 151(3): 332–340. doi:10.1038/sj.bjp.0707222. PMID:17450174. Adams, R.H., and Alitalo, K. 2007. Molecular regulation of angiogenesis and lymphangiogenesis. Nat. Rev. Mol. Cell Biol. 8(6): 464–478. doi:10.1038/nrm2183. PMID:17522591. Adams, D.J., Barakeh, J., Laskey, R., and Van Breemen, C. 1989. Ion channels and regulation of intracellular calcium in vascular endothelial cells. FASEB J. 3(12): 2389–2400. PMID:2477294. Aiello, V.D., Gutierrez, P.S., Chaves, M.J., Lopes, A.A., Higuchi, M.L., and Ramires, J.A. 2003. Morphology of the internal elastic lamina in arteries from pulmonary hypertensive patients: a confocal laser microscopy study. Mod. Pathol. 16(5): 411–416. doi:10.1097/01.MP.0000067685.57858.D7. PMID:12748246. Aird, W.C. 2003. The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood, 101(10): 3765– 3777. doi:10.1182/blood-2002-06-1887. PMID:12543869. Aird, W.C. 2007a. Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms. Circ. Res. 100(2): 158–173. doi:10.1161/01.RES.0000255691.76142.4a. PMID:17272818. Aird, W.C. 2007b. Phenotypic heterogeneity of the endothelium: II. Representative vascular beds. Circ. Res. 100(2): 174–190. doi:10. 1161/01.RES.0000255690.03436.ae. PMID:17272819. Akaike, T., Yoshida, M., Miyamoto, Y., Sato, K., Kohno, M., Sasamoto, K., et al. 1993. Antagonistic action of imidazolineoxyl N-oxides against endothelium-derived relaxing factor •NO (nitric oxide) through a radical reaction. Biochemistry, 32(3): 827–832. doi:10.1021/bi00054a013. PMID:8422387. Aljofan, M., and Ding, H. 2010. High glucose increases expression of cyclooxygenase-2, increases oxidative stress and decreases the generation of nitric oxide in mouse microvessel endothelial cells. J. Cell. Physiol. 222(3): 669–675. PMID:19950211. Altschul, R. 1955. Endothelium: Its Development, Morphology, Function, and Pathology. J. Am. Med. Assoc. 157(8): 691. doi:10. 1001/jama.1955.02950250065043. Andersohn, F., Suissa, S., and Garbe, E. 2006. Use of first- and second-generation cyclooxygenase-2-selective nonsteroidal antiin- flammatory drugs and risk of acute myocardial infarction. Circulation, 113(16): 1950–1957. doi:10.1161/ CIRCULATIONAHA.105.602425. PMID:16618816. Andrews, K.L., Irvine, J.C., Tare, M., Apostolopoulos, J., Favaloro, J.L., Triggle, C.R., and Kemp-Harper, B.K. 2009. A role for nitroxyl (HNO) as an endothelium-derived relaxing and hyperpolarizing factor in resistance arteries. Br. J. Pharmacol. 157(4): 540–550. doi:10.1111/j.1476-5381.2009.00150.x. PMID: 19338582. Angelillo-Scherrer, A., Fontana, P., Burnier, L., Roth, I., Sugamele, R., Brisset, A., et al. 2011. Connexin 37 limits thrombus propensity by downregulating platelet reactivity. Circulation, 124 (8): 930–939. doi:10.1161/CIRCULATIONAHA.110.015479. PMID:21810657. Antman, E.M., DeMets, D., and Loscalzo, J. 2005. Cyclooxygenase inhibition and cardiovascular risk. Circulation, 112(5): 759–770. doi:10.1161/CIRCULATIONAHA.105.568451. PMID:16061757. Antman, E.M., Bennett, J.S., Daugherty, A., Furberg, C., Roberts, H., and Taubert, K.A.American Heart Association. 2007. Use of nonsteroidal antiinflammatory drugs: an update for clinicians: a scientific statement from the American Heart Association. Circulation, 115(12): 1634–1642. doi:10.1161/ CIRCULATIONAHA.106.181424. PMID:17325246. Ardanaz, N., Beierwaltes, W.H., and Pagano, P.J. 2008. Distinct hydrogen peroxide-induced constriction in multiple mouse arteries: potential influence of vascular polarization. Pharmacol. Rep. 60(1): 61–67. PMID:18276986. Arunachalam, G., Yao, H., Sundar, I.K., Caito, S., and Rahman, I. 2010. SIRT1 regulates oxidant- and cigarette smoke-induced eNOS acetylation in endothelial cells: Role of resveratrol. Biochem. Biophys. Res. Commun. 393(1): 66–72. doi:10.1016/j. bbrc.2010.01.080. PMID:20102704. Bagi, Z., Erdei, N., Toth, A., Li, W., Hintze, T.H., Koller, A., and Kaley, G. 2005. Type 2 diabetic mice have increased arteriolar tone and blood pressure: enhanced release of COX-2-derived constrictor prostaglandins. Arterioscler. Thromb. Vasc. Biol. 25 (8): 1610–1616. doi:10.1161/01.ATV.0000172688.26838.9f. PMID:15947245. Barnes, P.J., and Karin, M. 1997. Nuclear factor-kB: a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med. 336(15): 1066–1071. doi:10.1056/NEJM199704103361506. PMID:9091804. Barton, M. 2011. The discovery of endothelium-dependent contrac- tion: the legacy of Paul M. Vanhoutte. Pharmacol. Res. 63(6): 455–462. doi:10.1016/j.phrs.2011.02.013. PMID:21385610. Barton, M., and Yanagisawa, M. 2008. Endothelin: 20 years from discovery to therapy. Can. J. Physiol. Pharmacol. 86(8): 485–498. doi:10.1139/Y08-059. PMID:18758495. Bass, T.M., Weinkove, D., Houthoofd, K., Gems, D., and Partridge, L. 2007. Effects of resveratrol on lifespan in Drosophila melanogaster and Caenorhabditis elegans. Mech. Ageing Dev. 128(10): 546–552. doi:10.1016/j.mad.2007.07.007. PMID: 17875315. Bedard, K., and Krause, K.H. 2007. The NOX family of ROS- generating NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87(1): 245–313. doi:10.1152/physrev.00044.2005. PMID:17237347. Beleznai, T., Takano, H., Hamill, C., Yarova, P., Douglas, G., Channon, K., and Dora, K. 2011. Enhanced K(+)-channel- mediated endothelium-dependent local and conducted dilation of small mesenteric arteries from ApoE(–/–) mice. Cardiovasc. Res. 92(2): 199–208. doi:10.1093/cvr/cvr181. PMID:21690174. Bellien, J., Favre, J., Iacob, M., Gao, J., Thuillez, C., Richard, V., et al. 2010. Arterial stiffness is regulated by nitric oxide and endothelium-derived hyperpolarizing factor during changes in blood flow in humans . Hypertension, 55(3): 674–680. doi:10. 1161/HYPERTENSIONAHA.109.142190. PMID:20083732. Bény, J.L., and von der Weid, P.Y. 1991. Hydrogen peroxide: an endogenous smooth muscle cell hyperpolarizing factor. Biochem. Biophys. Res. Commun. 176(1): 378–384. doi:10.1016/0006- 291X(91)90935-Z. PMID:1708249. Bharadwaj, L., and Prasad, K. 1995. Mediation of H2O2-induced vascular relaxation by endothelium-derived relaxing factor. Mol. Cell. Biochem. 149–150(1): 267–270. doi:10.1007/BF01076587. PMID:8569739. Bierhaus, A., Schiekofer, S., Schwaninger, M., Andrassy, M., Humpert, P.M., Chen, J., et al. 2001. Diabetes-associated sustained activation of the transcription factor nuclear factor-kappaB. Diabetes, 50(12): 2792–2808. doi:10.2337/diabetes.50.12.2792. PMID:11723063. Bordone, L., and Guarente, L. 2005. Calorie restriction, SIRT1 and metabolism: understanding longevity. Nat. Rev. Mol. Cell Biol. 6 (4): 298–305. doi:10.1038/nrm1616. PMID:15768047. Boulanger, C., and Luscher, T.F. 1990. Release of endothelin from the porcine aorta. Inhibition by endothelium-derived nitric oxide. J. Clin. Invest. 85(2): 587–590. doi:10.1172/JCI114477. PMID: 2153712. Brähler, S., Kaistha, A., Schmidt, V.J., Wolfle, S.E., Busch, C., 728 Can. J. Physiol. Pharmacol. Vol. 90, 2012 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. Kaistha, B.P., et al. 2009. Genetic deficit of SK3 and IK1 channels disrupts the endothelium-derived hyperpolarizing factor vasodi- lator pathway and causes hypertension. Circulation, 119(17): 2323–2332. doi:10.1161/CIRCULATIONAHA.108.846634. PMID:19380617. Brandes, R.P., Fleming, I., and Busse, R. 2005. Endothelial aging. Cardiovasc. Res. 66(2): 286–294. doi:10.1016/j.cardiores.2004.12. 027. PMID:15820197. Brandes, R.P., Weissmann, N., and Schroder, K. 2010. NADPH oxidases in cardiovascular disease. Free Radic. Biol. Med. 49(5): 687–706. doi:10.1016/j.freeradbiomed.2010.04.030. PMID: 20444433. Brandes, R.P., Takac, I., and Schroder, K. 2011. No superoxide–no stress?: Nox4, the good NADPH oxidase! Arterioscler. Thromb. Vasc. Biol. 31(6): 1255–1257. doi:10.1161/ATVBAHA.111. 226894. PMID:21593458. Brown, N.J., Gainer, J.V., Murphey, L.J., and Vaughan, D.E. 2000. Bradykinin stimulates tissue plasminogen activator release from human forearm vasculature through B(2) receptor-dependent, NO synthase-independent, and cyclooxygenase-independent pathway. Circulation, 102(18): 2190–2196. PMID:11056091. Brownlee, M. 2001. Biochemistry and molecular cell biology of diabetic complications. Nature, 414(6865): 813–820. doi:10.1038/ 414813a. PMID:11742414. Brunelli, L., Yermilov, V., and Beckman, J.S. 2001. Modulation of catalase peroxidatic and catalatic activity by nitric oxide. Free Radic. Biol. Med. 30(7): 709–714. doi:10.1016/S0891-5849(00) 00512-8. PMID:11275470. Bullen, M.L., Miller, A.A., Andrews, K.L., Irvine, J.C., Ritchie, R.H., Sobey, C.G., and Kemp-Harper, B.K. 2011. Nitroxyl (HNO) as a vasoprotective signaling molecule. Antioxid. Redox Signal. 14(9): 1675–1686. doi:10.1089/ars.2010.3327. PMID:20673125. Bulut, D., Liaghat, S., Hanefeld, C., Koll, R., Miebach, T., and Mügge, A. 2003. Selective cyclo-oxygenase-2 inhibition with parecoxib acutely impairs endothelium-dependent vasodilatation in patients with essential hypertension. J. Hypertens. 21(9): 1663– 1667. doi:10.1097/00004872-200309000-00015. PMID: 12923398. Burnett, C., Valentini, S., Cabreiro, F., Goss, M., Somogyvari, M., Piper, M.D., et al. 2011. Absence of effects of Sir2 overexpression on lifespan in C. elegans and Drosophila. Nature, 477(7365): 482– 485. doi:10.1038/nature10296. PMID:21938067. Burnham, M.P., Bychkov, R., Félétou, M., Richards, G.R., Vanhoutte, P.M., Weston, A.H., and Edwards, G. 2002. Characterization of an apamin-sensitive small-conductance Ca (2+)-activated K(+) channel in porcine coronary artery en- dothelium: relevance to EDHF. Br. J. Pharmacol. 135(5): 1133– 1143. doi:10.1038/sj.bjp.0704551. PMID:11877319. Bychkov, R., Burnham, M.P., Richards, G.R., Edwards, G., Weston, A.H., Félétou, M., and Vanhoutte, P.M. 2002. Characterization of a charybdotoxin-sensitive intermediate conductance Ca2+-activated K+ channel in porcine coronary endothelium: relevance to EDHF. Br. J. Pharmacol. 137(8): 1346–1354. doi:10.1038/sj.bjp. 0705057. PMID:12466245. Cai, S., Khoo, J., Mussa, S., Alp, N.J., and Channon, K.M. 2005. Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerization. Diabe- tologia, 48(9): 1933–1940. doi:10.1007/s00125-005-1857-5. PMID:16034613. Caton, P.W., Nayuni, N.K., Kieswich, J., Khan, N.Q., Yaqoob, M.M., and Corder, R. 2010. Metformin suppresses hepatic gluconeogen- esis through induction of SIRT1 and GCN5. J. Endocrinol. 205(1): 97–106. doi:10.1677/JOE-09-0345. PMID:20093281. Chadha, P.S., Liu, L., Rikard-Bell, M., Senadheera, S., Howitt, L., Bertrand, R.L., et al. 2011. Endothelium-dependent vasodilation in human mesenteric artery is primarily mediated by myoendothelial gap junctions intermediate conductance calcium-activated K+ channel and nitric oxide. J. Pharmacol. Exp. Ther. 336(3): 701– 708. doi:10.1124/jpet.110.165795. PMID:21172909. Chan, C.K., Mak, J.C., Man, R.Y., and Vanhoutte, P.M. 2009. Rho kinase inhibitors prevent endothelium-dependent contractions in the rat aorta. J. Pharmacol. Exp. Ther. 329(2): 820–826. doi:10. 1124/jpet.108.148247. PMID:19193928. Chang, K.T., and Min, K.T. 2002. Regulation of lifespan by histone deacetylase. Ageing Res. Rev. 1(3): 313–326. doi:10.1016/S1568- 1637(02)00003-X. PMID:12067588. Chanson, M., and Kwak, B.R. 2007. Connexin37: a potential modifier gene of inflammatory disease. J. Mol. Med. (Berl.), 85 (8): 787–795. doi:10.1007/s00109-007-0169-2. PMID:17318613. Chaytor, A.T., Edwards, D.H., Bakker, L.M., and Griffith, T.M. 2003. Distinct hyperpolarizing and relaxant roles for gap junctions and endothelium-derived H2O2 in NO-independent relaxations of rabbit arteries. Proc. Natl. Acad. Sci. U.S.A. 100(25): 15212– 15217. doi:10.1073/pnas.2435030100. PMID:14645719. Cheang, W.S., Wong, W.T., Tian, X.Y., Yang, Q., Lee, H.K., He, G.W., et al. 2011. Endothelial nitric oxide synthase enhancer reduces oxidative stress and restores endothelial function in db/ db mice. Cardiovasc. Res. 92(2): 267–275. doi:10.1093/cvr/ cvr233. PMID:21875904. Chen, K., Kirber, M.T., Xiao, H., Yang, Y., and Keaney, J.F., Jr. 2008. Regulation of ROS signal transduction by NADPH oxidase 4 localization. J. Cell Biol. 181(7): 1129–1139. doi:10.1083/jcb. 200709049. PMID:18573911. Chen, Z., Peng, I.C., Cui, X., Li, Y.S., Chien, S., and Shyy, J.Y. 2010. Shear stress, SIRT1, and vascular homeostasis. Proc. Natl. Acad. Sci. U.S.A. 107(22): 10268–10273. doi:10.1073/pnas. 1003833107. PMID:20479254. Choi, K., Kennedy, M., Kazarov, A., Papadimitriou, J.C., and Keller, G. 1998. A common precursor for hematopoietic and endothelial cells. Development, 125(4): 725–732. PMID:9435292. Chrissobolis, S., Miller, A.A., Drummond, G.R., Kemp-Harper, B.K., and Sobey, C.G. 2011. Oxidative stress and endothelial dysfunction in cerebrovascular disease. Front. Biosci. 16(1): 1733–1745. doi:10.2741/3816. PMID:21196259. Cogswell, J.P., Godlevski, M.M., Wisely, G.B., Clay, W.C., Leesnitzer, L.M., Ways, J.P., and Gray, J.G. 1994. NF-kB regulates IL-1b transcription through a consensus NF-kB binding site and a nonconsensus CRE-like site. J. Immunol. 153(2): 712– 723. PMID:8021507. Colden-Stanfield, M., Schilling, W.P., Ritchie, A.K., Eskin, S.G., Navarro, L.T., and Kunze, D.L. 1987. Bradykinin-induced increases in cytosolic calcium and ionic currents in cultured bovine aortic endothelial cells. Circ. Res. 61(5): 632–640. PMID:2444358. Cooke, J.P., Zimmer, J. 2002. The cardiovascular cure: How to strengthen your self-defense against heart attack and stroke. Broadway books, Portland, Ore. Cooper, M.E., and El-Osta, A. 2010. Epigenetics: mechanisms and implications for diabetic complications. Circ. Res. 107(12): 1403– 1413. doi:10.1161/CIRCRESAHA.110.223552. PMID:21148447. Corriu, C., Félétou, M., Canet, E., and Vanhoutte, P.M. 1996. Endothelium-derived factors and hyperpolarization of the carotid artery of the guinea-pig. Br. J. Pharmacol. 119(5): 959–964. PMID:8922746. Corriu, C., Félétou, M., Edwards, G., Weston, A.H., and Vanhoutte, P.M. 2001. Differential effects of prostacyclin and iloprost in the isolated carotid artery of the guinea-pig. Eur. J. Pharmacol. 426(1– 2): 89–94. doi:10.1016/S0014-2999(01)01203-1. PMID: 11525776. Triggle et al. 729 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. Cosentino, F., Eto, M., De Paolis, P., van der Loo, B., Bachschmid, M., Ullrich, V., et al. 2003. High glucose causes upregulation of cyclooxygenase-2 and alters prostanoid profile in human endothe- lial cells: role of protein kinase C and reactive oxygen species. Circulation, 107(7): 1017–1023. doi:10.1161/01.CIR. 0000051367.92927.07. PMID:12600916. Crane, G.J., Gallagher, N., Dora, K.A., and Garland, C.J. 2003. Small- and intermediate-conductance calcium-activated K+ chan- nels provide different facets of endothelium-dependent hyperpo- larization in rat mesenteric artery. J. Physiol. 553(1): 183–189. doi:10.1113/jphysiol.2003.051896. PMID:14555724. Davidge, S.T. 2001. Prostaglandin H synthase and vascular function. Circ. Res. 89(8): 650–660. doi:10.1161/hh2001.098351. PMID: 11597987. De Mey, J.G., and Vanhoutte, P.M. 1982. Heterogeneous behavior of the canine arterial and venous wall. Importance of the endothe- lium. Circ. Res. 51(4): 439–447. PMID:7127680. De Taeye, B., Smith, L.H., and Vaughan, D.E. 2005. Plasminogen activator inhibitor-1: a common denominator in obesity, diabetes and cardiovascular disease. Curr. Opin. Pharmacol. 5(2): 149–154. doi:10.1016/j.coph.2005.01.007. PMID:15780823. de Wit, C. 2010. Different pathways with distinct properties conduct dilations in the microcirculation in vivo. Cardiovasc. Res. 85(3): 604–613. doi:10.1093/cvr/cvp340. PMID:19820254. de Wit, C., and Griffith, T.M. 2010. Connexins and gap junctions in the EDHF phenomenon and conducted vasomotor responses. Pflugers Arch. 459(6): 897–914. doi:10.1007/s00424-010-0830-4. PMID:20379740. de Wit, C., Roos, F., Bolz, S.S., Kirchhoff, S., Kruger, O., Willecke, K., and Pohl, U. 2000. Impaired conduction of vasodilation along arterioles in connexin40-deficient mice. Circ. Res. 86(6): 649– 655. PMID:10747000. de Wit, C., Roos, F., Bolz, S.S., and Pohl, U. 2003. Lack of vascular connexin 40 is associated with hypertension and irregular arteriolar vasomotion. Physiol. Genomics, 13(2): 169–177. PMID:12700362. Dedio, J., Konig, P., Wohlfart, P., Schroeder, C., Kummer, W., and Muller-Esterl, W. 2001. NOSIP, a novel modulator of endothelial nitric oxide synthase activity. FASEB J. 15(1): 79–89. doi:10. 1096/fj.00-0078com. PMID:11149895. Deichmann, W.B., Henschler, D., Holmsted, B., and Keil, G. 1986. What is there that is not poison? A study of the Third Defense by Paracelsus. Arch. Toxicol. 58(4): 207–213. doi:10.1007/ BF00297107. PMID:3521542. Denniss, S.G., Jeffery, A.J., and Rush, J.W. 2010. RhoA-Rho kinase signaling mediates endothelium- and endoperoxide-dependent contractile activities characteristic of hypertensive vascular dys- function. Am. J. Physiol. Heart Circ. Physiol. 298(5): H1391– H1405. doi:10.1152/ajpheart.01233.2009. PMID:20154258. Detaille, D., Guigas, B., Chauvin, C., Batandier, C., Fontaine, E., Wiernsperger, N., and Leverve, X. 2005. Metformin prevents high- glucose-induced endothelial cell death through a mitochondrial permeability transition-dependent process. Diabetes, 54(7): 2179– 2187. doi:10.2337/diabetes.54.7.2179. PMID:15983220. Dimmeler, S., Fleming, I., Fisslthaler, B., Hermann, C., Busse, R., and Zeiher, A.M. 1999. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature, 399 (6736): 601–605. doi:10.1038/21224. PMID:10376603. Ding, H., and Triggle, C.R. 2010. Endothelial dysfunction in diabetes: multiple targets for treatment. Pflugers Arch. 459(6): 977–994. doi:10.1007/s00424-010-0807-3. PMID:20238124. Ding, H., Triggle, C.R. 2011. Glycaemic control and protection ofthe vasculature from glucose toxicity. 87–108. Intech Publishers, Rijeka, Croatia. Ding, H., Aljofan, M., and Triggle, C.R. 2007. Oxidative stress and increased eNOS and NADPH oxidase expression in mouse microvessel endothelial cells. J. Cell. Physiol. 212(3): 682–689. doi:10.1002/jcp.21063. PMID:17443690. Dolinsky, V.W., and Dyck, J.R. 2011. Calorie restriction and resveratrol in cardiovascular health and disease. Biochim. Biophys. Acta, 1812(11): 1477–1489. PMID:21749920. Dora, K.A. 2010. Coordination of vasomotor responses by the endothelium. Circ. J. 74(2): 226–232. doi:10.1253/circj.CJ-09- 0879. PMID:20065608. Dora, K.A., Gallagher, N.T., McNeish, A., and Garland, C.J. 2008. Modulation of endothelial cell KCa3.1 channels during endothe- lium-derived hyperpolarizing factor signaling in mesenteric resistance arteries. Circ. Res. 102(10): 1247–1255. doi:10.1161/ CIRCRESAHA.108.172379. PMID:18403729. Drummond, G.R., Cai, H., Davis, M.E., Ramasamy, S., and Harrison, D.G. 2000. Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide. Circ. Res. 86(3): 347–354. PMID:10679488. Dudzinski, D.M., and Michel, T. 2007. Life history of eNOS: partners and pathways. Cardiovasc. Res. 75(2): 247–260. doi:10.1016/j. cardiores.2007.03.023. PMID:17466957. Dudzinski, D.M., Igarashi, J., Greif, D., and Michel, T. 2006. The regulation and pharmacology of endothelial nitric oxide synthase. Annu. Rev. Pharmacol. Toxicol. 46(1): 235–276. doi:10.1146/ annurev.pharmtox.44.101802.121844. PMID:16402905. Duffy, S.J., Tran, B.T., New, G., Tudball, R.N., Esler, M.D., Harper, R.W., and Meredith, I.T. 1998. Continuous release of vasodilator prostanoids contributes to regulation of resting forearm blood flow in humans. Am. J. Physiol. 274(4): H1174–H1183. PMID:9575920. Dulak, J., Jozkowicz, A., Dembinska-Kiec, A., Guevara, I., Zdzienicka, A., Zmudzinska-Grochot, D., et al. 2000. Nitric oxide induces the synthesis of vascular endothelial growth factor by rat vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 20 (3): 659–666. doi:10.1161/01.ATV.20.3.659. PMID:10712388. Duplain, H., Burcelin, R., Sartori, C., Cook, S., Egli, M., Lepori, M., et al. 2001. Insulin resistance, hyperlipidemia, and hypertension in mice lacking endothelial nitric oxide synthase. Circulation, 104(3): 342–345. PMID:11457755. Duprez, D.A. 2010. Arterial stiffness and endothelial function: key players in vascular health. Hypertension, 55(3): 612–613. doi:10. 1161/HYPERTENSIONAHA.109.144725. PMID:20083729. Edwards, G., Dora, K.A., Gardener, M.J., Garland, C.J., and Weston, A.H. 1998. K+ is an endothelium-derived hyperpolarizing factor in rat arteries. Nature, 396(6708): 269–272. doi:10.1038/24388. PMID:9834033. Edwards, D.H., Li, Y., and Griffith, T.M. 2008. Hydrogen peroxide potentiates the EDHF phenomenon by promoting endothelial Ca2+ mobilization.Arterioscler.Thromb. Vasc.Biol. 28(10):1774–1781. doi:10.1161/ATVBAHA.108.172692. PMID:18669883. Edwards, G., Félétou, M., and Weston, A.H. 2010. Endothelium- derived hyperpolarising factors and associated pathways: a synopsis. Pflugers Arch. 459(6): 863–879. doi:10.1007/s00424- 010-0817-1. PMID:20383718. El-Osta, A., Brasacchio, D., Yao, D., Pocai, A., Jones, P.L., Roeder, R.G., et al. 2008. Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia. J. Exp. Med. 205(10): 2409–2417. doi:10.1084/ jem.20081188. PMID:18809715. El-Remessy, A.B., Tawfik, H.E., Matragoon, S., Pillai, B., Caldwell, R.B., and Caldwell, R.W. 2010. Peroxynitrite mediates diabetes- induced endothelial dysfunction: possible role of Rho kinase activation. Exp. Diabetes Res. 2010: Article ID 247861. doi:10. 1155/2010/247861. PMID:21052489. 730 Can. J. Physiol. Pharmacol. Vol. 90, 2012 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. Ellis, A., and Triggle, C.R. 2003. Endothelium-derived reactive oxygen species: their relationship to endothelium-dependent hyperpolarization and vascular tone. Can. J. Physiol. Pharmacol. 81(11): 1013–1028. doi:10.1139/y03-106. PMID:14719036. Ellis, A., Pannirselvam, M., Anderson, T.J., and Triggle, C.R. 2003. Catalase has negligible inhibitory effects on endothelium-dependent relaxations in mouse isolated aorta and small mesenteric artery. Br. J. Pharmacol. 140(7): 1193–1200. doi:10.1038/sj.bjp. 0705549. PMID:14597598. Erwin, P.A., Mitchell, D.A., Sartoretto, J., Marletta, M.A., and Michel, T. 2006. Subcellular targeting and differential ·S- nitrosylation of endothelial nitric-oxide synthase. J. Biol. Chem. 281(1): 151–157. doi:10.1074/jbc.M510421200. PMID: 16286475. Eto, M., Barandier, C., Rathgeb, L., Kozai, T., Joch, H., Yang, Z., and Luscher, T.F. 2001. Thrombin suppresses endothelial nitric oxide synthase and upregulates endothelin-converting enzyme-1 expres- sion by distinct pathways: role of Rho/ROCK and mitogen- activated protein kinase. Circ. Res. 89(7): 583–590. doi:10.1161/ hh1901.097084. PMID:11577023. Faraci, F.M., Modrick, M.L., Lynch, C.M., Didion, L.A., Fegan, P.E., and Didion, S.P. 2006. Selective cerebral vascular dysfunction in Mn-SOD-deficient mice. J. Appl. Physiol. 100(6): 2089–2093. doi:10.1152/japplphysiol.00939.2005. PMID:16514005. Feener, E.P., Northrup, J.M., Aiello, L.P., and King, G.L. 1995. Angiotensin II induces plasminogen activator inhibitor-1 and -2 expression in vascular endothelial and smooth muscle cells. J. Clin. Invest. 95(3): 1353–1362. doi:10.1172/JCI117786. PMID: 7883982. Félétou, M. 2011a. The Endothelium. Part 1: Multiple functions of the endothelial cells — focus on endothelium-derived vasoactive mediators. Morgan & Claypool Life Sciences (www.morganclay- pool.com). Félétou, M. 2011b. The Endothelium. Part II: EDHF-mediated responses “The Classical Pathway”. Morgan & Claypool Life Sciences (www.morganclaypool.com). Félétou, M., and Vanhoutte, P.M. 2006a. Endothelium-derived hyperpolarizing factor: where are we now? Arterioscler. Thromb. Vasc. Biol. 26(6): 1215–1225. doi:10.1161/01.ATV.0000217611. 81085.c5. PMID:16543495. Félétou, M., and Vanhoutte, P.M. 2006b. Endothelial dysfunction: a multifaceted disorder (The Wiggers Award Lecture). Am. J. Physiol. Heart Circ. Physiol. 291(3): H985–H1002. doi:10.1152/ ajpheart.00292.2006. PMID:16632549. Félétou, M., Verbeuren, T.J., and Vanhoutte, P.M. 2009. Endothe- lium-dependent contractions in SHR: a tale of prostanoid TP and IP receptors. Br. J. Pharmacol. 156(4): 563–574. doi:10.1111/j. 1476-5381.2008.00060.x. PMID:19154435. Fleming, I., and Busse, R. 2003. Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am. J. Physiol. Regul. Integr. Comp. Physiol. 284(1): R1–R12. PMID: 12482742. Fleming, I., Fisslthaler, B., Dimmeler, S., Kemp, B.E., and Busse, R. 2001. Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin- dependent endothelial nitric oxide synthase activity. Circ. Res. 88 (11): e68–e75. doi:10.1161/hh1101.092677. PMID:11397791. Foretz, M., Hebrard, S., Leclerc, J., Zarrinpashneh, E., Soty, M., Mithieux, G., et al. 2010. Metformin inhibits hepatic gluconeo- genesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state. J. Clin. Invest. 120(7): 2355– 2369. doi:10.1172/JCI40671. PMID:20577053. Förstermann, U. 2010. Nitric oxide and oxidative stress in vascular disease. Pflugers Arch. 459(6): 923–939. doi:10.1007/s00424- 010-0808-2. PMID:20306272. Förstermann, U., and Münzel, T. 2006. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation, 113(13): 1708–1714. doi:10.1161/CIRCULATIONAHA.105. 602532. PMID:16585403. Fujimoto, S., Asano, T., Sakai, M., Sakurai, K., Takagi, D., Yoshimoto, N., and Itoh, T. 2001. Mechanisms of hydrogen peroxide-induced relaxation in rabbit mesenteric small artery. Eur. J. Pharmacol. 412(3): 291–300. doi:10.1016/S0014-2999(00) 00940-7. PMID:11166293. Fukuto, J.M., and Carrington, S.J. 2011. HNO signaling mechanisms. Antioxid. Redox Signal. 14(9): 1649–1657. doi:10.1089/ars.2010. 3855. PMID:21235348. Fukuto, J.M., Wallace, G.C., Hszieh, R., and Chaudhuri, G. 1992. Chemical oxidation of N-hydroxyguanidine compounds. Release of nitric oxide, nitroxyl and possible relationship to the mechanism of biological nitric oxide generation. Biochem. Pharmacol. 43(3): 607–613. doi:10.1016/0006-2952(92)90584-6. PMID:1540216. Fulton, D., Gratton, J.-P., McCabe, T.J., Fontana, J., Fujio, Y., Walsh, K., et al. 1999. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature, 399(6736): 597– 601. doi:10.1038/21218. PMID:10376602. Fulton, D., Gratton, J.P., and Sessa, W.C. 2001. Post-translational control of endothelial nitric oxide synthase: why isn't calcium/ calmodulin enough? J. Pharmacol. Exp. Ther. 299(3): 818–824. PMID:11714864. Furchgott, R.F. 1996. The 1996 Albert Lasker Medical Research Awards. The discovery of endothelium-derived relaxing factor and its importance in the identification of nitric oxide. JAMA, 276(14): 1186–1188. doi:10.1001/jama.1996.03540140074032. PMID: 8827976. Furchgott, R.F., and Zawadzki, J.V. 1980. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature, 288(5789): 373–376. doi:10.1038/ 288373a0. PMID:6253831. Furuya, M., Yoshida, M., Hayashi, Y., Ohnuma, N., Minamino, N., Kangawa, K., and Matsuo, H. 1991. C-type natriuretic peptide is a growth inhibitor of rat vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 177(3): 927–931. doi:10.1016/0006- 291X(91)90627-J. PMID:1647770. Galley, H.F., and Webster, N.R. 2004. Physiology of the endothe- lium. Br. J. Anaesth. 93(1): 105–113. doi:10.1093/bja/aeh163. PMID:15121728. Gao, Y.J., and Lee, R.M. 2005. Hydrogen peroxide is an endothelium-dependent contracting factor in rat renal artery. Br. J. Pharmacol. 146(8): 1061–1068. doi:10.1038/sj.bjp.0706423. PMID:16231001. Gao, Y.J., Hirota, S., Zhang, D.W., Janssen, L.J., and Lee, R.M. 2003. Mechanisms of hydrogen-peroxide-induced biphasic response in rat mesenteric artery. Br. J. Pharmacol. 138(6): 1085–1092. doi:10. 1038/sj.bjp.0705147. PMID:12684264. Gao, Y.J., Lu, C., Su, L.Y., Sharma, A.M., and Lee, R.M. 2007. Modulation of vascular function by perivascular adipose tissue: the role of endothelium and hydrogen peroxide. Br. J. Pharmacol. 151(3): 323–331. doi:10.1038/sj.bjp.0707228. PMID:17384669. García-Cardeña, G., Oh, P., Liu, J., Schnitzer, J.E., and Sessa, W.C. 1996. Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling. Proc. Natl. Acad. Sci. U.S.A. 93(13): 6448–6453. doi:10.1073/ pnas.93.13.6448. PMID:8692835. García-Cardeña, G., Fan, R., Shah, V., Sorrentino, R., Cirino, G., Papapetropoulos, A., and Sessa, W.C. 1998. Dynamic activation of endothelial nitric oxide synthase by Hsp90. Nature, 392(6678): 821–824. doi:10.1038/33934. PMID:9580552. García-Redondo, A.B., Briones, A.M., Beltrán, A.E., Alonso, M.J., Triggle et al. 731 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. Simonsen, U., and Salaices, M. 2009. Hypertension increases contractile responses to hydrogen peroxide in resistance arteries through increased thromboxane A2, Ca2+, and superoxide anion levels. J. Pharmacol. Exp. Ther. 328(1): 19–27. doi:10.1124/jpet. 108.144295. PMID:18818375. Garg, U.C., and Hassid, A. 1989. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J. Clin. Invest. 83(5): 1774–1777. doi:10.1172/JCI114081. PMID: 2540223. Garland, C.J. 2010. Compromised vascular endothelial cell SK(Ca) activity: a fundamental aspect of hypertension? Br. J. Pharmacol. 160(4): 833–835. doi:10.1111/j.1476-5381.2010.00692.x. PMID: 20590582. Giacco, F., and Brownlee, M. 2010. Oxidative stress and diabetic complications. Circ. Res. 107(9): 1058–1070. doi:10.1161/ CIRCRESAHA.110.223545. PMID:21030723. Gluais, P., Lonchampt, M., Morrow, J.D., Vanhoutte, P.M., and Félétou, M. 2005. Acetylcholine-induced endothelium-dependent contractions in the SHR aorta: the Janus face of prostacyclin. Br. J. Pharmacol. 146(6): 834–845. doi:10.1038/sj.bjp.0706390. PMID: 16158068. Gluais, P., Paysant, J., Badier-Commander, C., Verbeuren, T., Vanhoutte, P.M., and Félétou, M. 2006. SHR aorta, calcium ionophore A-23187 releases prostacyclin and thromboxane A2 as endothelium-derived contracting factors. Am. J. Physiol. Heart Circ. Physiol. 291(5): H2255–H2264. doi:10.1152/ajpheart.01115. 2005. PMID:16798820. Grgic, I., Kaistha, B.P., Hoyer, J., and Kohler, R. 2009. Endothelial Ca+-activated K+ channels in normal and impaired EDHF-dilator responses — relevance to cardiovascular pathologies and drug discovery. Br. J. Pharmacol. 157(4): 509–526. doi:10.1111/j.1476- 5381.2009.00132.x. PMID:19302590. Griendling, K.K., and Ushio-Fukai, M. 1998. Redox control of vascular smooth muscle proliferation. J. Lab. Clin. Med. 132(1): 9–15. doi:10.1016/S0022-2143(98)90019-1. PMID:9665366. Hamed, S., Brenner, B., and Roguin, A. 2011. Nitric oxide: a key factor behind the dysfunctionality of endothelial progenitor cells in diabetes mellitus type-2. Cardiovasc. Res. 91(1): 9–15. doi:10. 1093/cvr/cvq412. PMID:21186243. Hassona, M.D., Abouelnaga, Z.A., Elnakish, M.T., Awad, M.M., Alhaj, M., Goldschmidt-Clermont, P.J., and Hassanain, H. 2010. Vascular hypertrophy-associated hypertension of profilin1 trans- genic mouse model leads to functional remodeling of peripheral arteries. Am. J. Physiol. Heart Circ. Physiol. 298(6): H2112– H2120. doi:10.1152/ajpheart.00016.2010. PMID:20400688. Heymes, C., Habib, A., Yang, D., Mathieu, E., Marotte, F., Samuel, J., and Boulanger, C.M. 2000. Cyclo-oxygenase-1 and -2 contribution to endothelial dysfunction in ageing. Br. J. Pharma- col. 131(4): 804–810. doi:10.1038/sj.bjp.0703632. PMID: 11030731. Hickey, K.A., Rubanyi, G., Paul, R.J., and Highsmith, R.F. 1985. Characterization of a coronary vasoconstrictor produced by cultured endothelial cells. Am. J. Physiol. 248(5): C550–C556. PMID:3993773. Hrafnkelsdóttir, T., Wall, U., Jern, C., and Jern, S. 1998. Impaired capacity for endogenous fibrinolysis in essential hypertension. Lancet, 352(9140): 1597–1598. doi:10.1016/S0140-6736(05) 61044-6. PMID:9843110. Hrafnkelsdottir, T., Ottosson, P., Gudnason, T., Samuelsson, O., and Jern, S. 2004. Impaired endothelial release of tissue-type plasminogen activator in patients with chronic kidney disease and hypertension. Hypertension, 44(3): 300–304. doi:10.1161/01. HYP.0000137380.91476.fb. PMID:15249548. Huang, P.L., Huang, Z., Mashimo, H., Bloch, K.D., Moskowitz, M. A., Bevan, J.A., and Fishman, M.C. 1995. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature, 377 (6546): 239–242. doi:10.1038/377239a0. PMID:7545787. Icking, A., Matt, S., Opitz, N., Wiesenthal, A., Muller-Esterl, W., and Schilling, K. 2005. NOSTRIN functions as a homotrimeric adaptor protein facilitating internalization of eNOS. J. Cell Sci. 118(21): 5059–5069. doi:10.1242/jcs.02620. PMID:16234328. Ihlemann, N., Rask-Madsen, C., Perner, A., Dominguez, H., Hermann, T., Kober, L., and Torp-Pedersen, C. 2003. Tetrahy- drobiopterin restores endothelial dysfunction induced by an oral glucose challenge in healthy subjects. Am. J. Physiol. Heart Circ. Physiol. 285(2): H875–H882. PMID:12730050. Ingerman-Wojenski, C., Silver, M.J., Smith, J.B., and Macarak, E. 1981. Bovine endothelial cells in culture produce thromboxane as well as prostacyclin. J. Clin. Invest. 67(5): 1292–1296. doi:10. 1172/JCI110157. PMID:7014633. Johnson-Léger, C., Aurrand-Lions, M., and Imhof, B.A. 2000. The parting of the endothelium: miracle, or simply a junctional affair? J. Cell Sci. 113(6): 921–933. PMID:10683141. Kalebic, T., Garbisa, S., Glaser, B., and Liotta, L.A. 1983. Basement membrane collagen: degradation by migrating endothelial cells. Science, 221(4607): 281–283. doi:10.1126/science.6190230. PMID:6190230. Kanfi, Y., Peshti, V., Gil, R., Naiman, S., Nahum, L., Levin, E., et al. 2010. SIRT6 protects against pathological damage caused by diet- induced obesity. Aging Cell, 9(2): 162–173. doi:10.1111/j.1474- 9726.2009.00544.x. PMID:20047575. Kanfi, Y., Naiman, S., Amir, G., Peshti, V., Zinman, G., Nahum, L., et al. 2012. The sirtuin SIRT6 regulates lifespan in male mice. Nature, 483(7388): 218–221. doi:10.1038/nature10815. PMID: 22367546. Kansui, Y., Garland, C.J., and Dora, K.A. 2008. Enhanced spontaneous Ca2+ events in endothelial cells reflect signalling through myoendothelial gap junctions in pressurized mesenteric arteries. Cell Calcium, 44(2): 135–146. doi:10.1016/j.ceca.2007. 11.012. PMID:18191200. Kassam, S.I., Lu, C., Buckley, N., and Lee, R.M. 2011. The mechanisms of propofol-induced vascular relaxation and modula- tion by perivascular adipose tissue and endothelium. Anesth. Analg. 112(6): 1339–1345. doi:10.1213/ANE. 0b013e318215e094. PMID:21543785. Katoh, M., Egashira, K., Mitsui, T., Chishima, S., Takeshita, A., and Narita, H. 2000. Angiotensin-converting enzyme inhibitor pre- vents plasminogen activator inhibitor-1 expression in a rat model with cardiovascular remodeling induced by chronic inhibition of nitric oxide synthesis. J. Mol. Cell. Cardiol. 32(1): 73–83. doi:10. 1006/jmcc.1999.1053. PMID:10652192. Kawano, H., Motoyama, T., Hirashima, O., Hirai, N., Miyao, Y., Sakamoto, T., et al. 1999. Hyperglycemia rapidly suppresses flow- mediated endothelium-dependent vasodilation of brachial artery. J. Am. Coll. Cardiol. 34(1): 146–154. doi:10.1016/S0735-1097(99) 00168-0. PMID:10400004. Kemp-Harper, B.K. 2011. Nitroxyl (HNO): a novel redox signaling molecule. Antioxid. Redox Signal. 14(9): 1609–1613. doi:10. 1089/ars.2011.3937. PMID:21299468. Khatsenko, O. 1998. Interactions between nitric oxide and cyto- chrome P-450 in the liver. Biochemistry (Mosc.), 63(7): 833–839. PMID:9721336. Kim, I., Moon, S.O., Kim, S.H., Kim, H.J., Koh, Y.S., and Koh, G.Y. 2001. Vascular endothelial growth factor expression of inter- cellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor- 732 Can. J. Physiol. Pharmacol. Vol. 90, 2012 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. kappa B activation in endothelial cells. J. Biol. Chem. 276(10): 7614–7620. doi:10.1074/jbc.M009705200. PMID:11108718. Köhler, R., and Ruth, P. 2010. Endothelial dysfunction and blood pressure alterations in K+-channel transgenic mice. Pflugers Arch. 459(6): 969–976. doi:10.1007/s00424-010-0819-z. PMID: 20349244. Köhler, R.,Degenhardt,C.,Kuhn, M., Runkel, N., Paul,M.,andHoyer, J. 2000. Expression and function of endothelial Ca(2+)-activated K(+) channels in human mesenteric artery: A single-cell reverse transcriptase-polymerase chain reaction and electrophysiological study in situ. Circ. Res. 87(6): 496–503. PMID:10988242. Koller, A., Sun, D., and Kaley, G. 1993. Role of shear stress and endothelial prostaglandins in flow- and viscosity-induced dilation of arterioles in vitro. Circ. Res. 72(6): 1276–1284. PMID: 8495555. Kooistra, T., Schrauwen, Y., Arts, J., and Emeis, J.J. 1994. Regulation of endothelial cell t-PA synthesis and release. Int. J. Hematol. 59 (4): 233–255. PMID:8086618. Krötz, F., Riexinger, T., Buerkle, M.A., Nithipatikom, K., Gloe, T., Sohn, H.Y., et al. 2004. Membrane-potential-dependent inhibition of platelet adhesion to endothelial cells by epoxyeicosatrienoic acids. Arterioscler. Thromb. Vasc. Biol. 24(3): 595–600. doi:10. 1161/01.ATV.0000116219.09040.8c. PMID:14715644. Krötz, F., Hellwig, N., Burkle, M.A., Lehrer, S., Riexinger, T., Mannell, H., et al. 2010. A sulfaphenazole-sensitive EDHF opposes platelet-endothelium interactions in vitro and in the hamster microcirculation in vivo. Cardiovasc. Res. 85(3): 542– 550. doi:10.1093/cvr/cvp301. PMID:19717402. Krüger, O., Plum, A., Kim, J.S., Winterhager, E., Maxeiner, S., Hallas, G., et al. 2000. Defective vascular development in connexin 45-deficient mice. Development, 127(19): 4179–4193. PMID:10976050. Krummen, S., Falck, J.R., and Thorin, E. 2005. Two distinct pathways account for EDHF-dependent dilatation in the gracilis artery of dyslipidaemic hApoB+/+ mice. Br. J. Pharmacol. 145(2): 264–270. doi:10.1038/sj.bjp.0706194. PMID:15765099. Krummen, S., Drouin, A., Gendron, M.E., Falck, J.R., and Thorin, E. 2006. ROS-sensitive cytochrome P450 activity maintains en- dothelial dilatation in ageing but is transitory in dyslipidaemic mice. Br. J. Pharmacol. 147(8): 897–904. doi:10.1038/sj.bjp. 0706679. PMID:16474414. Kubes, P., Suzuki, M., and Granger, D.N. 1991. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc. Natl. Acad. Sci. U.S.A. 88(11): 4651–4655. doi:10.1073/pnas.88.11.4651. PMID:1675786. Kuroda, J., Ago, T., Matsushima, S., Zhai, P., Schneider, M.D., and Sadoshima, J. 2010. NADPH oxidase 4 (Nox4) is a major source of oxidative stress in the failing heart. Proc. Natl. Acad. Sci. U.S. A. 107(35): 15565–15570. doi:10.1073/pnas.1002178107. PMID: 20713697. Lan, W.R., Hou, C.J., Yen, C.H., Shih, B.F., Wang, A.M., Lee, T.Y., et al. 2011. Effects of carbenoxolone on flow-mediated vasodilata- tion in healthy adults. Am. J. Physiol. Heart Circ. Physiol. 301(3): H1166–H1172. doi:10.1152/ajpheart.00967.2010. PMID: 21622817. Landmesser, U., Dikalov, S., Price, S.R., McCann, L., Fukai, T., Holland, S.M., et al. 2003. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J. Clin. Invest. 111(8): 1201–1209. PMID: 12697739. Langman, L.J., and Kapur, B.M. 2006. Toxicology: then and now. Clin. Biochem. 39(5): 498–510. doi:10.1016/j.clinbiochem.2006. 03.004. PMID:16730254. Larsen, B.T., Gutterman, D.D., Sato, A., Toyama, K., Campbell, W.B., Zeldin, D.C., et al. 2008. Hydrogen peroxide inhibits cytochrome p450 epoxygenases: interaction between two en- dothelium-derived hyperpolarizing factors. Circ. Res. 102(1): 59–67. doi:10.1161/CIRCRESAHA.107.159129. PMID:17975109. Laufs, U., and Liao, J.K. 1998. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J. Biol. Chem. 273(37): 24266–24271. doi:10.1074/jbc.273.37. 24266. PMID:9727051. Ledoux, J., Taylor, M.S., Bonev, A.D., Hannah, R.M., Solodushko, V., Shui, B., et al. 2008. Functional architecture of inositol 1,4,5- trisphosphate signaling in restricted spaces of myoendothelial projections. Proc. Natl. Acad. Sci. U.S.A. 105(28): 9627–9632. doi:10.1073/pnas.0801963105. PMID:18621682. Lee, M.Y., Tse, H.F., Siu, C.W., Zhu, S.G., Man, R.Y., and Vanhoutte, P.M. 2007. Genomic changes in regenerated porcine coronary arterial endothelial cells. Arterioscler. Thromb. Vasc. Biol. 27(11): 2443–2449. doi:10.1161/ATVBAHA.107.141705. PMID:17942849. Li, N., and Karin, M. 1999. Is NF-kappaB the sensor of oxidative stress? FASEB J. 13(10): 1137–1143. PMID:10385605. Li, C.G., Karagiannis, J., and Rand, M.J. 1999. Comparison of the redox forms of nitrogen monoxide with the nitrergic transmitter in the rat anococcygeus muscle. Br. J. Pharmacol. 127(4): 826–834. doi:10.1038/sj.bjp.0702540. PMID:10433488. Li, Y., Mihara, K., Saifeddine, M., Krawetz, A., Lau, D.C., Li, H., et al. 2011. Perivascular adipose tissue-derived relaxing factors: release by peptide agonists via proteinase-activated receptor-2 (PAR2) and non-PAR2 mechanisms. Br. J. Pharmacol. 164(8): 1990–2002. doi:10.1111/j.1476-5381.2011.01501.x. PMID: 21615723. Liu, C., Ngai, C.Y., Huang, Y., Ko, W.H., Wu, M., He, G.W., et al. 2006. Depletion of intracellular Ca2+ stores enhances flow- induced vascular dilatation in rat small mesenteric artery. Br. J. Pharmacol. 147(5): 506–515. doi:10.1038/sj.bjp.0706639. PMID: 16415911. Liu, Y., Bubolz, A.H., Mendoza, S., Zhang, D.X., and Gutterman, D.D. 2011. H2O2 is the transferrable factor mediating flow-induced dilation in human coronary arterioles. Circ. Res. 108(5): 566–573. doi:10.1161/CIRCRESAHA.110.237636. PMID:21233456. Lombard, D.B., Pletcher, S.D., Canto, C., and Auwerx, J. 2011. Ageing: longevity hits a roadblock. Nature, 477(7365): 410–411. doi:10.1038/477410a. PMID:21938058. Ludmer, P.L., Selwyn, A.P., Shook, T.L., Wayne, R.R., Mudge, G.H., Alexander, R.W., and Ganz, P. 1986. Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic coronary arteries. N. Engl. J. Med. 315(17): 1046–1051. doi:10.1056/ NEJM198610233151702. PMID:3093861. Lundberg, J.O., and Weitzberg, E. 2005. NO generation from nitrite and its role in vascular control. Arterioscler. Thromb. Vasc. Biol. 25(5): 915–922. doi:10.1161/01.ATV.0000161048.72004.c2. PMID:15746440. Marrelli, S.P. 2001. Mechanisms of endothelial P2Y1- and P2Y2- mediated vasodilatation involve differential [Ca2+]i responses. Am. J. Physiol. Heart Circ. Physiol. 281(4): H1759–H1766. PMID: 11557568. Marui, N., Offermann, M.K., Swerlick, R., Kunsch, C., Rosen, C.A., Ahmad, M., et al. 1993. Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells. J. Clin. Invest. 92(4): 1866–1874. doi:10.1172/ JCI116778. PMID:7691889. Mather, K.J., Verma, S., and Anderson, T.J. 2001. Improved endothelial function with metformin in type 2 diabetes mellitus. Triggle et al. 733 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. J. Am. Coll. Cardiol. 37(5): 1344–1350. doi:10.1016/S0735-1097 (01)01129-9. PMID:11300445. Matoba, T., Shimokawa, H., Nakashima, M., Hirakawa, Y., Mukai, Y., Hirano, K., et al. 2000. Hydrogen peroxide is an endothelium- derived hyperpolarizing factor in mice. J. Clin. Invest. 106(12): 1521–1530. doi:10.1172/JCI10506. PMID:11120759. Matoba, T., Shimokawa, H., Kubota, H., Morikawa, K., Fujiki, T., Kunihiro, I., et al. 2002. Hydrogen peroxide is an endothelium- derived hyperpolarizing factor in human mesenteric arteries. Biochem. Biophys. Res. Commun. 290(3): 909–913. doi:10. 1006/bbrc.2001.6278. PMID:11798159. Matoba, T., Shimokawa, H., Morikawa, K., Kubota, H., Kunihiro, I., Urakami-Harasawa, L., et al. 2003. Electron spin resonance detection of hydrogen peroxide as an endothelium-derived hyperpolarizing factor in porcine coronary microvessels. Arter- ioscler. Thromb. Vasc. Biol. 23(7): 1224–1230. doi:10.1161/01. ATV.0000078601.79536.6C. PMID:12763764. Matsunaga, T., Weihrauch, D.W., Moniz, M.C., Tessmer, J., Warltier, D.C., and Chilian, W.M. 2002. Angiostatin inhibits coronary angiogenesis during impaired production of nitric oxide. Circula- tion, 105(18): 2185–2191. doi:10.1161/01.CIR.0000015856. 84385.E9. PMID:11994253. Mattagajasingh, I., Kim, C.S., Naqvi, A., Yamamori, T., Hoffman, T. A., Jung, S.B., et al. 2007. SIRT1 promotes endothelium- dependent vascular relaxation by activating endothelial nitric oxide synthase. Proc. Natl. Acad. Sci. U.S.A. 104(37): 14855– 14860. doi:10.1073/pnas.0704329104. PMID:17785417. McDonald, B., Pittman, K., Menezes, G.B., Hirota, S.A., Slaba, I., Waterhouse, C.C., et al.2010. Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science, 330(6002): 362–366. doi:10.1126/science.1195491. PMID:20947763. McGuire, J.J., Ding, H., and Triggle, C.R. 2001. Endothelium- derived relaxing factors: a focus on endothelium-derived hyper- polarizing factor(s). Can. J. Physiol. Pharmacol. 79(6): 443–470. doi:10.1139/y01-025. PMID:11430583. McNeish, A.J., Sandow, S.L., Neylon, C.B., Chen, M.X., Dora, K.A., and Garland, C.J. 2006. Evidence for involvement of both IKCa and SKCa channels in hyperpolarizing responses of the rat middle cerebral artery. Stroke, 37(5): 1277–1282. doi:10.1161/01.STR. 0000217307.71231.43. PMID:16556879. McSherry, I.N., Spitaler, M.M., Takano, H., and Dora, K.A. 2005. Endothelial cell Ca2+ increases are independent of membrane potential in pressurized rat mesenteric arteries. Cell Calcium, 38 (1): 23–33. doi:10.1016/j.ceca.2005.03.007. PMID:15907999. Mendoza, S.A., Fang, J., Gutterman, D.D., Wilcox, D.A., Bubolz, A.H., Li, R., et al. 2010. TRPV4-mediated endothelial Ca2+ influx and vasodilation in response to shear stress. Am. J. Physiol. Heart Circ. Physiol. 298(2): H466–H476. doi:10.1152/ ajpheart.00854.2009. PMID:19966050. Michel, J.B., Feron, O., Sacks, D., and Michel, T. 1997. Reciprocal regulation of endothelial nitric-oxide synthase by Ca2+-calmodulin and caveolin. J. Biol. Chem. 272(25): 15583–15586. doi:10.1074/ jbc.272.25.15583. PMID:9188442. Michell, B.J., Harris, M.B., Chen, Z.P., Ju, H., Venema, V.J., Blackstone, M.A., et al. 2002. Identification of regulatory sites of phosphorylation of the bovine endothelial nitric-oxide synthase at serine 617 and serine 635. J. Biol. Chem. 277(44): 42344–42351. doi:10.1074/jbc.M205144200. PMID:12171920. Miller, A.A., Drummond, G.R., and Sobey, C.G. 2006. Reactive oxygen species in the cerebral circulation: are they all bad? Antioxid. Redox Signal. 8(7–8): 1113–1120. doi:10.1089/ars. 2006.8.1113. PMID:16910759. Milne, J.C., and Denu, J.M. 2008. The Sirtuin family: therapeutic targets to treat diseases of aging. Curr. Opin. Chem. Biol. 12(1): 11–17. doi:10.1016/j.cbpa.2008.01.019. PMID:18282481. Miura, H., Liu, Y., and Gutterman, D.D. 1999. Human coronary arteriolar dilation to bradykinin depends on membrane hyperpo- larization: contribution of nitric oxide and Ca2+-activated K+ channels. Circulation, 99(24): 3132–3138. PMID:10377076. Modrick, M.L., Didion, S.P., Lynch, C.M., Dayal, S., Lentz, S.R., and Faraci, F.M. 2009. Role of hydrogen peroxide and the impact of glutathione peroxidase-1 in regulation of cerebral vascular tone. J. Cereb. Blood Flow Metab. 29(6): 1130–1137. doi:10.1038/jcbfm. 2009.37. PMID:19352401. Mohazzab-H, K.M., Fayngersh, R.P., and Wolin, M.S. 1996. Nitric oxide inhibits pulmonary artery catalase and H2O2-associated relaxation. Am. J. Physiol. 271(5): H1900–H1906. PMID: 8945907. Moncada, S., and Higgs, E.A. 1986. Arachidonate metabolism in blood cells and the vessel wall. Clin. Haematol. 15(2): 273–292. PMID:3015465. Montezano, A.C., Burger, D., Ceravolo, G.S., Yusuf, H., Montero, M., and Touyz, R.M. 2011. Novel Nox homologues in the vasculature: focusing on Nox4 and Nox5. Clin. Sci. (Lond.), 120 (4): 131–141. doi:10.1042/CS20100384. PMID:21039341. Murohara, T., Witzenbichler, B., Spyridopoulos, I., Asahara, T., Ding, B., Sullivan, A., et al. 1999. Role of endothelial nitric oxide synthase in endothelial cell migration. Arterioscler. Thromb. Vasc. Biol. 19(5): 1156–1161. doi:10.1161/01.ATV.19.5.1156. PMID: 10323764. Mustafa, A.K., Sikka, G., Gazi, S.K., Steppan, J., Jung, S.M., Bhunia, A.K., et al. 2011. Hydrogen sulfide as endothelium-derived hyperpolarizing factor sulfhydrates potassium channels. Circ. Res. 109(11): 1259–1268. doi:10.1161/CIRCRESAHA.111.240242. PMID:21980127. Nakata, S., Tsutsui, M., Shimokawa, H., Suda, O., Morishita, T., Shibata, K., et al. 2008. Spontaneous myocardial infarction in mice lacking all nitric oxide synthase isoforms. Circulation, 117(17): 2211–2223. doi:10.1161/CIRCULATIONAHA.107.742692. PMID:18413498. Nguyen Dinh Cat, A., and Touyz, R.M. 2011. Cell signaling of angiotensin II on vascular tone: novel mechanisms. Curr. Hypertens. Rep. 13(2): 122–128. doi:10.1007/s11906-011-0187- x. PMID:21274755. Nisoli, E., Tonello, C., Cardile, A., Cozzi, V., Bracale, R., Tedesco, L., et al. 2005. Calorie restriction promotes mitochondrial biogenesis by inducing the expression of eNOS. Science, 310 (5746): 314–317. doi:10.1126/science.1117728. PMID:16224023. Niu, X.F., Smith, C.W., and Kubes, P. 1994. Intracellular oxidative stress induced by nitric oxide synthesis inhibition increases endothelial cell adhesion to neutrophils. Circ. Res. 74(6): 1133– 1140. PMID:7910528. Nohria, A., Grunert, M.E., Rikitake, Y., Noma, K., Prsic, A., Ganz, P., et al. 2006. Rho kinase inhibition improves endothelial function in human subjects with coronary artery disease. Circ. Res. 99(12): 1426–1432. doi:10.1161/01.RES.0000251668.39526.c7. PMID: 17095725. Noma, K., Oyama, N., and Liao, J.K. 2006. Physiological role of ROCKs in the cardiovascular system. Am. J. Physiol. Cell Physiol. 290(3): C661–C668. doi:10.1152/ajpcell.00459.2005. PMID: 16469861. Ogonowski, A.A., Kaesemeyer, W.H., Jin, L., Ganapathy, V., Leibach, F.H., and Caldwell, R.W. 2000. Effects of NO donors and synthase agonists on endothelial cell uptake of L-Arg and superoxide production. Am. J. Physiol. Cell Physiol. 278(1): C136–C143. PMID:10644521. Orimo, M., Minamino, T., Miyauchi, H., Tateno, K., Okada, S., 734 Can. J. Physiol. Pharmacol. Vol. 90, 2012 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. Moriya, J., and Komuro, I. 2009. Protective role of SIRT1 in diabetic vascular dysfunction. Arterioscler. Thromb. Vasc. Biol. 29 (6): 889–894. doi:10.1161/ATVBAHA.109.185694. PMID: 19286634. Ota, H., Akishita, M., Eto, M., Iijima, K., Kaneki, M., and Ouchi, Y. 2007. Sirt1 modulates premature senescence-like phenotype in human endothelial cells. J. Mol. Cell. Cardiol. 43(5): 571–579. doi:10.1016/j.yjmcc.2007.08.008. PMID:17916362. Ota, H., Eto, M., Kano, M.R., Ogawa, S., Iijima, K., Akishita, M., and Ouchi, Y. 2008. Cilostazol inhibits oxidative stress-induced premature senescence via upregulation of Sirt1 in human endothelial cells. Arterioscler. Thromb. Vasc. Biol. 28(9): 1634– 1639. doi:10.1161/ATVBAHA.108.164368. PMID:18556572. Ozkor, M.A., Murrow, J.R., Rahman, A.M., Kavtaradze, N., Lin, J., Manatunga, A., and Quyyumi, A.A. 2011. Endothelium-derived hyperpolarizing factor determines resting and stimulated forearm vasodilator tone in health and in disease. Circulation, 123(20): 2244–2253. doi:10.1161/CIRCULATIONAHA.110.990317. PMID:21555712. Palmer, R.M., Ferrige, A.G., and Moncada, S. 1987. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature, 327(6122): 524–526. doi:10.1038/ 327524a0. PMID:3495737. Pannirselvam, M., Verma, S., Anderson, T.J., and Triggle, C.R. 2002. Cellular basis of endothelial dysfunction in small mesenteric arteries from spontaneously diabetic (db/db–/–) mice: role of decreased tetrahydrobiopterin bioavailability. Br. J. Pharmacol. 136(2): 255–263. doi:10.1038/sj.bjp.0704683. PMID:12010774. Pannirselvam, M., Simon, V., Verma, S., Anderson, T., and Triggle, C.R. 2003. Chronic oral supplementation with sepiapterin prevents endothelial dysfunction and oxidative stress in small mesenteric arteries from diabetic (db/db) mice. Br. J. Pharmacol. 140(4): 701– 706. doi:10.1038/sj.bjp.0705476. PMID:14534153. Pannirselvam, M., Ding, H., Anderson, T.J., and Triggle, C.R. 2006. Pharmacological characteristics of endothelium-derived hyperpo- larizing factor-mediated relaxation of small mesenteric arteries from db/db mice. Eur. J. Pharmacol. 551(1–3): 98–107. doi:10. 1016/j.ejphar.2006.08.086. PMID:17027963. Park, Y., Capobianco, S., Gao, X., Falck, J.R., Dellsperger, K.C., and Zhang, C. 2008. Role of EDHF in type 2 diabetes-induced endothelial dysfunction. Am. J. Physiol. Heart Circ. Physiol. 295 (5): H1982–H1988. doi:10.1152/ajpheart.01261.2007. PMID: 18790831. Peiró, C., Redondo, J., Rodríguez-Martínez, M.A., Angulo, J., Marín, J., and Sánchez-Ferrer, C.F. 1995. Influence of endothelium on cultured vascular smooth muscle cell proliferation. Hypertension, 25(4): 748–751. PMID:7721427. Peng, H.B., Libby, P., and Liao, J.K. 1995. Induction and stabilization of Ik Ba by nitric oxide mediates inhibition of NF- kB. J. Biol. Chem. 270(23): 14214–14219. PMID:7775482. Pirola, L., Balcerczyk, A., Okabe, J., and El-Osta, A. 2010. Epigenetic phenomena linked to diabetic complications. Nat. Rev. Endocrinol. 6(12): 665–675. doi:10.1038/nrendo.2010.188. PMID:21045787. Potente, M., and Dimmeler, S. 2008. NO targets SIRT1: a novel signaling network in endothelial senescence. Arterioscler. Thromb. Vasc. Biol. 28(9): 1577–1579. doi:10.1161/ATVBAHA.108. 173682. PMID:18716320. Prabhakar, P., Thatte, H.S., Goetz, R.M., Cho, M.R., Golan, D.E., and Michel, T. 1998. Receptor-regulated translocation of endothe- lial nitric-oxide synthase. J. Biol. Chem. 273(42): 27383–27388. doi:10.1074/jbc.273.42.27383. PMID:9765266. Prabhakar, P., Cheng, V., and Michel, T. 2000. A chimeric transmembrane domain directs endothelial nitric-oxide synthase palmitoylation and targeting to plasmalemmal caveolae. J. Biol. Chem. 275(25): 19416–19421. doi:10.1074/jbc.M001952200. PMID:10787410. Rapoport, R.M., and Williams, S.P. 1996. Role of prostaglandins in acetylcholine-induced contraction of aorta from spontaneously hypertensive and Wistar–Kyoto rats. Hypertension, 28(1): 64–75. PMID:8675266. Ravi, K., Brennan, L.A., Levic, S., Ross, P.A., and Black, S.M. 2004. S-Nitrosylation of endothelial nitric oxide synthase is associated with monomerization and decreased enzyme activity. Proc. Natl. Acad. Sci. U.S.A.101(8): 2619–2624. doi:10.1073/pnas. 0300464101. PMID:14983058. Ray, R., Murdoch, C.E., Wang, M., Santos, C.X., Zhang, M., Alom- Ruiz, S., et al. 2011. Endothelial Nox4 NADPH oxidase enhances vasodilatation and reduces blood pressure in vivo. Arterioscler. Thromb. Vasc. Biol. 31(6): 1368–1376. doi:10.1161/ATVBAHA. 110.219238. PMID:21415386. Razny, U., Kiec-Wilk, B., Wator, L., Polus, A., Dyduch, G., Solnica, B., et al. 2011. Increased nitric oxide availability attenuates high fat diet metabolic alterations and gene expression associated with insulin resistance. Cardiovasc. Diabetol. 10(1): 68–81. doi:10. 1186/1475-2840-10-68. PMID:21781316. Reaume, A.G., de Sousa, P.A., Kulkarni, S., Langille, B.L., Zhu, D., Davies, T.C., et al. 1995. Cardiac malformation in neonatal mice lacking connexin43. Science, 267(5205): 1831–1834. doi:10. 1126/science.7892609. PMID:7892609. Ross, R. 1999. Atherosclerosis is an inflammatory disease. Am. Heart J. 138(5): S419–S420. doi:10.1016/S0002-8703(99)70266-8. PMID:10539839. Rössig, L., Fichtlscherer, B., Breitschopf, K., Haendeler, J., Zeiher, A.M., Mülsch, A., and Dimmeler, S. 1999. Nitric oxide inhibits caspase-3 by S-nitrosation in vivo. J. Biol. Chem. 274(11): 6823– 6826. doi:10.1074/jbc.274.11.6823. PMID:10066732. Ruderman, N.B., Xu, X.J., Nelson, L., Cacicedo, J.M., Saha, A.K., Lan, F., and Ido, Y. 2010. AMPK and SIRT1: a long-standing partnership? Am. J. Physiol. Endocrinol. Metab. 298(4): E751– E760. doi:10.1152/ajpendo.00745.2009. PMID:20103737. Rummery, N.M., McKenzie, K.U., Whitworth, J.A., and Hill, C.E. 2002. Decreased endothelial size and connexin expression in rat caudal arteries during hypertension. J. Hypertens. 20(2): 247–253. doi:10.1097/00004872-200202000-00014. PMID:11821709. Rummery, N.M., Grayson, T.H., and Hill, C.E. 2005. Angiotensin- converting enzyme inhibition restores endothelial but not medial connexin expression in hypertensive rats. J. Hypertens. 23(2): 317–328. doi:10.1097/00004872-200502000-00014. PMID: 15662220. Rundhaug, J.E. 2005. Matrix metalloproteinases and angiogenesis. J. Cell. Mol. Med. 9(2): 267–285. doi:10.1111/j.1582-4934.2005. tb00355.x. PMID:15963249. Ryan, U.S., Ryan, J.W., Whitaker, C., and Chiu, A. 1976. Localization of angiotensin converting enzyme (kininase II). II. Immunocytochemistry and immunofluorescence. Tissue Cell, 8 (1): 125–145. doi:10.1016/0040-8166(76)90025-2. PMID:178068. Saitoh, S., Kiyooka, T., Rocic, P., Rogers, P.A., Zhang, C., Swafford, A., et al. 2007. Redox-dependent coronary metabolic dilation. Am. J. Physiol. Heart Circ. Physiol. 293(6): H3720–H3725. doi:10. 1152/ajpheart.00436.2007. PMID:17965288. Saliez, J., Bouzin, C., Rath, G., Ghisdal, P., Desjardins, F., Rezzani, R., et al. 2008. Role of caveolar compartmentation in endothelium- derived hyperpolarizing factor-mediated relaxation: Ca2+ signals and gap junction function are regulated by caveolin in endothelial cells. Circulation, 117(8): 1065–1074. doi:10.1161/ CIRCULATIONAHA.107.731679. PMID:18268148. Sandow, S.L., and Tare, M. 2007. C-type natriuretic peptide: a new Triggle et al. 735 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. endothelium-derived hyperpolarizing factor? Trends Pharmacol. Sci. 28(2): 61–67. doi:10.1016/j.tips.2006.12.007. PMID: 17208309. Sandow, S.L., Neylon, C.B., Chen, M.X., and Garland, C.J. 2006. Spatial separation of endothelial small- and intermediate-con- ductance calcium-activated potassium channels (K(Ca)) and connexins: possible relationship to vasodilator function? J. Anat. 209(5): 689–698. doi:10.1111/j.1469-7580.2006.00647.x. PMID: 17062025. Sandow, S.L., Haddock, R.E., Hill, C.E., Chadha, P.S., Kerr, P.M., Welsh, D.G., and Plane, F. 2009a. What's where and why at a vascular myoendothelial microdomain signalling complex. Clin. Exp. Pharmacol. Physiol. 36(1): 67–76. doi:10.1111/j.1440-1681. 2008.05076.x. PMID:19018806. Sandow, S.L., Gzik, D.J., and Lee, R.M. 2009b. Arterial internal elastic lamina holes: relationship to function? J. Anat. 214(2): 258–266. doi:10.1111/j.1469-7580.2008.01020.x. PMID: 19207987. Sato, S., Fujita, N., and Tsuruo, T. 2000. Modulation of Akt kinase activity by binding to Hsp90. Proc. Natl. Acad. Sci. U.S.A. 97(20): 10832–10837. doi:10.1073/pnas.170276797. PMID:10995457. Sato, A., Sakuma, I., and Gutterman, D.D. 2003. Mechanism of dilation to reactive oxygen species in human coronary arterioles. Am. J. Physiol. Heart Circ. Physiol. 285(6): H2345–H2354. PMID:14613909. Schneider, D.J., Nordt, T.K., and Sobel, B.E. 1993. Attenuated fibrinolysis and accelerated atherogenesis in type II diabetic patients. Diabetes, 42(1): 1–7. doi:10.2337/diabetes.42.1.1. PMID: 8420806. Schulz, E., Gori, T., and Munzel, T. 2011. Oxidative stress and endothelial dysfunction in hypertension. Hypertens. Res. 34(6): 665–673. doi:10.1038/hr.2011.39. PMID:21512515. Schwer, B., and Verdin, E. 2008. Conserved metabolic regulatory functions of sirtuins. Cell Metab. 7(2): 104–112. doi:10.1016/j. cmet.2007.11.006. PMID:18249170. Scotland, R.S., Madhani, M., Chauhan, S., Moncada, S., Andresen, J., Nilsson, H., et al. 2005. Investigation of vascular responses in endothelial nitric oxide synthase/cyclooxygenase-1 double-knock- out mice: key role for endothelium-derived hyperpolarizing factor in the regulation of blood pressure in vivo. Circulation, 111(6): 796–803. doi:10.1161/01.CIR.0000155238.70797.4E. PMID: 15699263. Selemidis, S., and Cocks, T.M. 2002. Endothelium-dependent hyperpolarization as a remote anti-atherogenic mechanism. Trends Pharmacol. Sci. 23(5): 213–220. doi:10.1016/S0165-6147(02) 01998-3. PMID:12007998. Sessa, W.C. 2004. eNOS at a glance. J. Cell Sci. 117(12): 2427–2429. doi:10.1242/jcs.01165. PMID:15159447. Shanmugam, N., Kim, Y.S., Lanting, L., and Natarajan, R. 2003. Regulation of cyclooxygenase-2 expression in monocytes by ligation of the receptor for advanced glycation end products. J. Biol. Chem. 278(37): 34834–34844. doi:10.1074/jbc. M302828200. PMID:12837757. Shanmugam, N., Todorov, I.T., Nair, I., Omori, K., Reddy, M.A., and Natarajan, R. 2006. Increased expression of cyclooxygenase-2 in human pancreatic islets treated with high glucose or ligands of the advanced glycation endproduct-specific receptor (AGER), and in islets from diabetic mice. Diabetologia, 49(1): 100–107. doi:10. 1007/s00125-005-0065-7. PMID:16341840. Shimokawa, H., and Matoba, T. 2004. Hydrogen peroxide as an endothelium-derived hyperpolarizing factor. Pharmacol. Res. 49 (6): 543–549. doi:10.1016/j.phrs.2003.10.016. PMID:15026032. Shirotani, M., Yui, Y., Hattori, R., and Kawai, C. 1991. U-61,431F, a stable prostacyclin analogue, inhibits the proliferation of bovine vascular smooth muscle cells with little antiproliferative effect on endothelial cells. Prostaglandins, 41(2): 97–110. doi:10.1016/ 0090-6980(91)90023-9. PMID:1708156. Si, H., Heyken, W.T., Wolfle, S.E., Tysiac, M., Schubert, R., Grgic, I., et al. 2006. Impaired endothelium-derived hyperpolarizing factor-mediated dilations and increased blood pressure in mice deficient of the intermediate-conductance Ca2+-activated K+ channel. Circ. Res. 99(5): 537–544. doi:10.1161/01.RES. 0000238377.08219.0c. PMID:16873714. Siebel, A.L., Fernandez, A.Z., and El-Osta, A. 2010. Glycemic memory associated epigenetic changes. Biochem. Pharmacol. 80 (12): 1853–1859. doi:10.1016/j.bcp.2010.06.005. PMID: 20599797. Smith, D.S., Harmon, J., and Owen, W.G. 1985. A sensitive and specific assay for plasminogen activators. Thromb. Res. 37(4): 533–541. doi:10.1016/0049-3848(85)90099-4. PMID:4039078. Stamler, J.S., Singel, D.J., and Loscalzo, J. 1992. Biochemistry of nitric oxide and its redox-activated forms. Science, 258(5090): 1898–1902. doi:10.1126/science.1281928. PMID:1281928. Stankevicius, E., Dalsgaard, T., Kroigaard, C., Beck, L., Boedtkjer, E., Misfeldt, M.W., et al. 2011. Opening of small and intermediate calcium-activated potassium channels induces relaxation mainly mediated by nitric-oxide release in large arteries and endothelium- derived hyperpolarizing factor in small arteries from rat. J. Pharmacol. Exp. Ther. 339(3): 842–850. doi:10.1124/jpet.111. 179242. PMID:21880870. Stein, S., Schafer, N., Breitenstein, A., Besler, C., Winnik, S., Lohmann, C., et al. 2010. SIRT1 reduces endothelial activation without affecting vascular function in ApoE–/– mice. Aging (Albany NY), 2(6): 353–360. PMID:20606253. Steinberg, D. 1997. Lewis A. Conner Memorial Lecture. Oxidative modification of LDL and atherogenesis. Circulation, 95(4): 1062– 1071. PMID:9054771. Stroes, E., Hijmering, M., van Zandvoort, M., Wever, R., Rabelink, T. J., and van Faassen, E.E. 1998. Origin of superoxide production by endothelial nitric oxide synthase. FEBS Lett. 438(3): 161–164. doi:10.1016/S0014-5793(98)01292-7. PMID:9827538. Suvorava, T., and Kojda, G. 2009. Reactive oxygen species as cardiovascular mediators: lessons from endothelial-specific protein overexpression mouse models. Biochim. Biophys. Acta, 1787(7): 802–810. doi:10.1016/j.bbabio.2009.04.005. PMID:19393613. Szerafin, T., Erdei, N., Fulop, T., Pasztor, E.T., Edes, I., Koller, A., and Bagi, Z. 2006. Increased cyclooxygenase-2 expression and prostaglandin-mediated dilation in coronary arterioles of patients with diabetes mellitus. Circ. Res. 99(5): e12–e17. doi:10.1161/01. RES.0000241051.83067.62. PMID:16917094. Taddei, S., Virdis, A., Ghiadoni, L., Sudano, I., Magagna, A., and Salvetti, A. 2001. Role of endothelin in the control of peripheral vascular tone in human hypertension. Heart Fail. Rev. 6(4): 277– 285. doi:10.1023/A:1011400124060. PMID:11447302. Taddei, S., Versari, D., Cipriano, A., Ghiadoni, L., Galetta, F., Franzoni, F., et al. 2006. Identification of a cytochrome P450 2C9- derived endothelium-derived hyperpolarizing factor in essential hypertensive patients. J. Am. Coll. Cardiol. 48(3): 508–515. doi:10.1016/j.jacc.2006.04.074. PMID:16875977. Takahashi, S., Harigae, H., Yokoyama, H., Ishikawa, I., Abe, S., Imaizumi, M., et al. 2006. Synergistic effect of arsenic trioxide and flt3 inhibition on cells with flt3 internal tandem duplication. Int. J. Hematol. 84(3): 256–261. doi:10.1532/IJH97.06076. PMID: 17050201. Takano, H., Dora, K.A., and Garland, C.J. 2005. Spreading vasodilatation in resistance arteries. J. Smooth Muscle Res. 41 (6): 303–311. doi:10.1540/jsmr.41.303. PMID:16557004. Tang, E.H., and Vanhoutte, P.M. 2008. Gene expression changes of 736 Can. J. Physiol. Pharmacol. Vol. 90, 2012 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. prostanoid synthases in endothelial cells and prostanoid receptors in vascular smooth muscle cells caused by aging and hypertension. Physiol. Genomics, 32(3): 409–418. PMID:18056786. Tang, E.H., Ku, D.D., Tipoe, G.L., Félétou, M., Man, R.Y., and Vanhoutte, P.M. 2005. Endothelium-dependent contractions occur in the aorta of wild-type and COX2–/– knockout but not COX1–/– knockout mice. J. Cardiovasc. Pharmacol. 46(6): 761–765. doi:10. 1097/01.fjc.0000187174.67661.67. PMID:16306799. Taylor, M.S., Bonev, A.D., Gross, T.P., Eckman, D.M., Brayden, J.E., Bond, C.T., et al. 2003. Altered expression of small-conductance Ca2+-activated K+ (SK3) channels modulates arterial tone and blood pressure. Circ. Res. 93(2): 124–131. doi:10.1161/01.RES. 0000081980.63146.69. PMID:12805243. Thannickal, V.J., and Fanburg, B.L. 2000. Reactive oxygen species in cell signaling. Am. J. Physiol. Lung Cell. Mol. Physiol. 279(6): L1005–L1028. PMID:11076791. Thomas, S.R., Chen, K., and Keaney, J.F., Jr. 2002. Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phos- phoinositide 3-kinase-dependent signaling pathway. J. Biol. Chem. 277(8): 6017–6024. doi:10.1074/jbc.M109107200. PMID: 11744698. Thorin, E. 2011. Vascular disease risk in patients with hypertrigly- ceridemia: endothelial progenitor cells, oxidative stress, acceler- ated senescence, and impaired vascular repair. Can. J. Cardiol. 27 (5): 538–540. doi:10.1016/j.cjca.2011.03.014. PMID:21764253. Thorin, E., and Thorin-Trescases, N. 2009. Vascular endothelial ageing, heartbeat after heartbeat. Cardiovasc. Res. 84(1): 24–32. doi:10.1093/cvr/cvp236. PMID:19586943. Tocchetti, C.G., Stanley, B.A., Murray, C.I., Sivakumaran, V., Donzelli, S., Mancardi, D., et al. 2011. Playing with cardiac “redox switches”: the “HNO way” to modulate cardiac function. Antioxid. Redox Signal. 14(9): 1687–1698. doi:10.1089/ars.2010. 3859. PMID:21235349. Touyz, R.M., Briones, A.M., Sedeek, M., Burger, D., and Montezano, A.C. 2011. NOX isoforms and reactive oxygen species in vascular health. Mol. Interv. 11(1): 27–35. doi:10.1124/ mi.11.1.5. PMID:21441119. Tranquille, N., and Emeis, J.J. 1991. On the role of calcium in the acute release of tissue-type plasminogen activator and von Willebrand factor from the rat perfused hindleg region. Thromb. Haemost. 66(4): 479–483. PMID:1724576. Tranquille, N., and Emeis, J.J. 1993. The role of cyclic nucleotides in the release of tissue-type plasminogen activator and von Will- ebrand factor. Thromb. Haemost. 69(3): 259–261. PMID:8097063. Traupe, T., Lang, M., Goettsch, W., Munter, K., Morawietz, H., Vetter, W., and Barton, M. 2002. Obesity increases prostanoid- mediated vasoconstriction and vascular thromboxane receptor gene expression. J. Hypertens. 20(11): 2239–2245. doi:10.1097/ 00004872-200211000-00024. PMID:12409963. Triggle, C.R., and Ding, H. 2010. A review of endothelial dysfunction in diabetes: a focus on the contribution of a dysfunctional eNOS. J. Am. Soc. Hypertens. 4(3): 102–115. doi:10.1016/j.jash.2010.02.004. PMID:20470995. Triggle, C.R., and Ding, H. 2011. The endothelium in compliance and resistance vessels. Front. Biosci. (Schol. Ed.) 3: 730–744. PMID: 21196408. Tsutsui, M., Shimokawa, H., Otsuji, Y., and Yanagihara, N. 2010. Pathophysiological relevance of NO signaling in the cardiovas- cular system: novel insight from mice lacking all NO synthases. Pharmacol. Ther. 128(3): 499–508. doi:10.1016/j.pharmthera. 2010.08.010. PMID:20826180. van Mourik, J.A., Lawrence, D.A., and Loskutoff, D.J. 1984. Purification of an inhibitor of plasminogen activator (antiactivator) synthesized by endothelial cells. J. Biol. Chem. 259(23): 14914– 14921. PMID:6438106. Vanhoutte, P.M. 2000. Say NO to ET. J. Auton. Nerv. Syst. 81(1–3): 271–277. doi:10.1016/S0165-1838(00)00126-0. PMID:10869731. Vanhoutte, P.M. 2010. Regeneration of the endothelium in vascular injury. Cardiovasc. Drugs Ther. 24(4): 299–303. doi:10.1007/ s10557-010-6257-5. PMID:20689986. Vanhoutte, P.M., and Tang, E.H. 2008. Endothelium-dependent contractions: when a good guy turns bad! J. Physiol. 586(22): 5295–5304. doi:10.1113/jphysiol.2008.161430. PMID:18818246. Vanhoutte, P.M., Félétou, M., and Taddei, S. 2005. Endothelium- dependent contractions in hypertension. Br. J. Pharmacol. 144(4): 449–458. doi:10.1038/sj.bjp.0706042. PMID:15655530. Voghel, G., Thorin-Trescases, N., Farhat, N., Nguyen, A., Villeneuve, L., Mamarbachi, A.M., et al. 2007. Cellular senescence in endothelial cells from atherosclerotic patients is accelerated by oxidative stress associated with cardiovascular risk factors. Mech. Ageing Dev. 128(11–12): 662–671. doi:10.1016/j.mad.2007.09. 006. PMID:18022214. Waldron, G.J., and Garland, C.J. 1994. Effect of potassium channel blockers on L-NAME insensitive relaxations in rat small mesenteric artery. Can. J. Physiol. Pharmacol. 72(Suppl. 1): 115. Waldron, G.J., Ding, H., Lovren, F., Kubes, P., and Triggle, C.R. 1999. Acetylcholine-induced relaxation of peripheral arteries isolated from mice lacking endothelial nitric oxide synthase. Br. J. Pharmacol. 128(3): 653–658. doi:10.1038/sj.bjp.0702858. PMID:10516645. Wang, R., Wang, Z., Wu, L., Hanna, S.T., and Peterson-Wakeman, R. 2001. Reduced vasorelaxant effect of carbon monoxide in diabetes and the underlying mechanisms. Diabetes, 50(1): 166–174. doi:10. 2337/diabetes.50.1.166. PMID:11147783. Wang, C.Y., Liu, H.J., Chen, H.J., Lin, Y.C., Wang, H.H., Hung, T. C., and Yeh, H.-I. 2011. AGE-BSA down-regulates endothelial connexin43 gap junctions. BMC Cell Biol. 12(1): 19–30. doi:10. 1186/1471-2121-12-19. PMID:21575204. Wanstall, J.C., Jeffery, T.K., Gambino, A., Lovren, F., and Triggle, C. R. 2001. Vascular smooth muscle relaxation mediated by nitric oxide donors: a comparison with acetylcholine, nitric oxide and nitroxyl ion. Br. J. Pharmacol. 134(3): 463–472. doi:10.1038/sj. bjp.0704269. PMID:11588100. Wei, Q., and Xia, Y. 2005. Roles of 3-phosphoinositide-dependent kinase 1 in the regulation of endothelial nitric-oxide synthase phosphorylation and function by heat shock protein 90. J. Biol. Chem. 280(18): 18081–18086. doi:10.1074/jbc.M413607200. PMID:15737995. Weston, A.H., Absi, M., Ward, D.T., Ohanian, J., Dodd, R.H., Dauban, P., et al. 2005. Evidence in favor of a calcium-sensing receptor in arterial endothelial cells: studies with calindol and Calhex 231. Circ. Res. 97(4): 391–398. doi:10.1161/01.RES. 0000178787.59594.a0. PMID:16037572. Weston, A.H., Porter, E.L., Harno, E., and Edwards, G. 2010. Impairment of endothelial SK(Ca) channels and of downstream hyperpolarizing pathways in mesenteric arteries from sponta- neously hypertensive rats. Br. J. Pharmacol. 160(4): 836–843. doi:10.1111/j.1476-5381.2010.00657.x. PMID:20233221. Williams, S.P., Dorn, G.W., II, and Rapoport, R.M. 1994. Prostaglandin I2 mediates contraction and relaxation of vascular smooth muscle. Am. J. Physiol. 267(2): H796–H803. PMID: 8067435. Wilson, S.H., Caplice, N.M., Simari, R.D., Holmes, D.R., Jr, Carlson, P.J., and Lerman, A. 2000. Activated nuclear factor-kB is present in the coronary vasculature in experimental hypercholesterolemia. Atherosclerosis, 148(1): 23–30. doi:10.1016/S0021-9150(99) 00211-7. PMID:10580167. Triggle et al. 737 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. Wong, P.S., Hyun, J., Fukuto, J.M., Shirota, F.N., DeMaster, E.G., Shoeman, D.W., and Nagasawa, H.T. 1998. Reaction between S- nitrosothiols and thiols: generation of nitroxyl (HNO) and subsequent chemistry. Biochemistry, 37(16): 5362–5371. doi:10. 1021/bi973153g. PMID:9548918. Wong, C.W., Christen, T., Roth, I., Chadjichristos, C.E., Derouette, J. P., Foglia, B.F., et al. 2006. Connexin37 protects against atherosclerosis by regulating monocyte adhesion. Nat. Med. 12 (8): 950–954. doi:10.1038/nm1441. PMID:16862155. Wu, F., Schuster, D.P., Tyml, K., and Wilson, J.X. 2007. Ascorbate inhibits NADPH oxidase subunit p47phox expression in micro- vascular endothelial cells. Free Radic. Biol. Med. 42(1): 124–131. doi:10.1016/j.freeradbiomed.2006.10.033. PMID:17157199. Xia, Y., Tsai, A.L., Berka, V., and Zweier, J.L. 1998. Superoxide generationfromendothelialnitric-oxidesynthase.ACa2+/calmodulin- dependent and tetrahydrobiopterin regulatory process. J. Biol. Chem. 273(40): 25804–25808. doi:10.1074/jbc.273.40.25804. PMID:9748253. Xia, N., Daiber, A., Habermeier, A., Closs, E.I., Thum, T., Spanier, G., et al. 2010. Resveratrol reverses endothelial nitric-oxide synthase uncoupling in apolipoprotein E knockout mice. J. Pharmacol. Exp. Ther. 335(1): 149–154. doi:10.1124/jpet.110. 168724. PMID:20610621. Xiao, X., Dong, Y., Zhong, J., Cao, R., Zhao, X., Wen, G., and Liu, J. 2011. Adiponectin protects endothelial cells from the damages induced by the intermittent high level of glucose. Endocrine, 40 (3): 386–393. doi:10.1007/s12020-011-9531-9. PMID:21948177. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., et al. 1988. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature, 332(6163): 411– 415. doi:10.1038/332411a0. PMID:2451132. Yang, Q., Xue, H.M., Wong, W.T., Tian, X.Y., Huang, Y., Tsui, S.K., et al. 2011. AVE3085, an enhancer of endothelial nitric oxide synthase, restores endothelial function and reduces blood pressure in spontaneously hypertensive rats. Br. J. Pharmacol. 163(5): 1078–1085. doi:10.1111/j.1476-5381.2011.01308.x. PMID: 21385179. Yao, L., Romero, M.J., Toque, H.A., Yang, G., Caldwell, R.B., and Caldwell, R.W. 2010. The role of RhoA/Rho kinase pathway in endothelial dysfunction. J. Cardiovasc. Dis. Res. 1(4): 165–170. doi:10.4103/0975-3583.74258. PMID:21264179. Yuill, K.H., Yarova, P., Kemp-Harper, B.K., Garland, C.J., and Dora, K.A. 2011. A novel role for HNO in local and spreading vasodilatation in rat mesenteric resistance arteries. Antioxid. Redox Signal. 14(9): 1625–1635. doi:10.1089/ars.2010.3279. PMID:20615121. Zhang, Z., Xiao, Z., and Diamond, S.L. 1999. Shear stress induction of C-type natriuretic peptide (CNP) in endothelial cells is independent of NO autocrine signaling. Ann. Biomed. Eng. 27 (4): 419–426. doi:10.1114/1.203. PMID:10468226. Zhou, G., Myers, R., Li, Y., Chen, Y., Shen, X., Fenyk-Melody, J., et al. 2001. Role of AMP-activated protein kinase in mechanism of metformin action. J. Clin. Invest. 108(8): 1167–1174. PMID: 11602624. Ziche, M., Parenti, A., Ledda, F., Dell'Era, P., Granger, H.J., Maggi, C.A., and Presta, M. 1997. Nitric oxide promotes proliferation and plasminogen activator production by coronary venular endothe- lium through endogenous bFGF. Circ. Res. 80(6): 845–852. PMID:9168787. Zu, Y., Liu, L., Lee, M.Y., Xu, C., Liang, Y., Man, R.Y., et al. 2010. SIRT1 promotes proliferation and prevents senescence through targeting LKB1 in primary porcine aortic endothelial cells. Circ. Res. 106(8): 1384–1393. doi:10.1161/CIRCRESAHA.109. 215483. PMID:20203304. 738 Can. J. Physiol. Pharmacol. Vol. 90, 2012 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. ia. Atherosclerosis, 148(1): 23–30. doi:10.1016/S0021-9150(99) 00211-7. PMID:10580167. Triggle et al. 737 Published by NRC Research Press Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NATIONAL INST OF HEALTH LIB on 08/04/16 For personal use only. Wong, P.S., Hyun, J., Fukuto, J.M., Shirota, F.N., DeMaster, E.G., Shoeman, D.W., and Nagasawa, H.T. 1998. Reaction between S- nitrosothiols and thiols: generation of nitroxyl (HNO) and subsequent chemistry. Biochemistry, 37(16): 5362–5371. doi:10. 1021/bi973153g. PMID:9548918. Wong, C.W., Christen, T., Roth, I., Chadjichristos, C.E., Derouette, J. P., Foglia, B.F., et al. 2006. Connexin37 protects against atherosclerosis by regulating monocyte adhesion. Nat. Med. 12 (8): 950–954. doi:10.1038/nm1441. PMID:16862155. Wu, F., Schuster, D.P., Tyml, K., and Wilson, J.X. 2007. Ascorbate inhibits NADPH oxidase subunit p47phox expression in micro- vascular endothelial cells. Free Radic. Biol. Med. 42(1): 124–131. doi:10.1016/j.freeradbiomed.2006.10.033. PMID:17157199. Xia, Y., Tsai, A.L., Berka, V., and Zweier, J.L. 1998. Superoxide generationfromendothelialnitric-oxidesynthase.ACa2+/calmodulin- dependent and tetrahydrobiopterin regulatory process. J. Biol. Chem. 273(40): 25804–25808. doi:10.1074/jbc.273.40.25804. PMID:9748253. Xia, N., Daiber, A., Habermeier, A., Closs, E.I., Thum, T., Spanier, G., et al. 2010. Resveratrol reverses endothelial nitric-oxide synthase uncoupling in apolipoprotein E knockout mice. J. Pharmacol. Exp. Ther. 335(1): 149–154. doi:10.1124/jpet.110. 168724. PMID:20610621. Xiao, X., Dong, Y., Zhong, J., Cao, R., Zhao, X., Wen, G., and Liu, J. 2011. Adiponectin protects endothelial cells from the damages induced by the intermittent high level of glucose. Endocrine, 40 (3): 386–393. doi:10.1007/s12020-011-9531-9. PMID:21948177. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., et al. 1988. A novel potent vasocon