Palmitic Acid Methyl Ester and Its Relation to Control of Tone of Human Visceral Arteries and Rat Aortas by Perivascular Adipose Tissue.

Background: Perivascular adipose tissue (PVAT) exerts anti-contractile effects on visceral arteries by release of various perivascular relaxing factors (PVRFs) and opening voltage-gated K+ (Kv) channels in vascular smooth muscle cells (VSMCs). Palmitic acid methyl ester (PAME) has been proposed as transferable PVRF in rat aorta. Here, we studied PVAT regulation of arterial tone of human mesenteric arteries and clarified the contribution of Kv channels and PAME in the effects. Methods: Wire myography was used to measure vasocontractions of mesenteric artery rings from patients undergoing abdominal surgery. Isolated aortic rings from Sprague-Dawley rats were studied for comparison. PVAT was either left intact or removed from the arterial rings. Vasocontractions were induced by external high K+ (60 mM), serotonin (5-HT) or phenylephrine. PAME (10 nM-3 µM) was used as vasodilator. Kv channels were blocked by XE991, a Kv7 (KCNQ) channel inhibitor, or by 4-aminopyridine, a non-specific Kv channel inhibitor. PAME was measured in bathing solutions incubated with rat peri-aortic or human visceral adipose tissue. Results: We found that PVAT displayed anti-contractile effects in both human mesenteric arteries and rat aortas. The anti-contractile effects were inhibited by XE991 (30 µM). PAME (EC50 ~1.4 µM) was capable to produce relaxations of PVAT-removed rat aortas. These effects were abolished by XE991 (30 µM), but not 4-aminopyridine (2 mM) or NDGA (10 µM), a lipoxygenases inhibitor. The cytochrome P450 epoxygenase inhibitor 17-octadecynoic acid (ODYA 10 µM) and the soluble epoxide hydrolase inhibitor 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA 10 µM) slightly decreased PAME relaxations. PAME up to 10 µM failed to induce relaxations of PVAT-removed human mesenteric arteries. 5-HT induced endogenous PAME release from rat peri-aortic adipose tissue, but not from human visceral adipose tissue. Conclusions: Our data also suggest that Kv7 channels are involved in the anti-contractile effects of PVAT on arterial tone in both rat aorta and human mesenteric arteries. PAME could contribute to PVAT relaxations by activating Kv7 channels in rat aorta, but not in human mesenteric arteries.

Differential effects of cystathionine-γ-lyase-dependent vasodilatory H2S in periadventitial vasoregulation of rat and mouse aortas.

BACKGROUND: Hydrogen sulfide (H(2)S) is a potent vasodilator. However, the complex mechanisms of vasoregulation by H(2)S are not fully understood. We tested the hypotheses that (1) H(2)S exerts vasodilatory effects by opening KCNQ-type voltage-dependent (K(v)) K(+) channels and (2) that H(2)S-producing cystathionine-γ-lyase (CSE) in perivascular adipose tissue plays a major role in this pathway. METHODOLOGY/PRINCIPAL FINDINGS: Wire myography of rat and mouse aortas was used. NaHS and 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) were used as H(2)S donors. KCNQ-type K(v) channels were blocked by XE991. 4-Propargylglycine (PPG) and ß-cyano-l-alanine (BCA), or 2-(aminooxy)-acetic acid (AOAA) were used as inhibitors of CSE or cystathionine-ß-synthase (CBS), respectively. NaHS and ADTOH produced strong vasorelaxation in rat and mouse aortas, which were abolished by KCNQ channel inhibition with XE991. Perivascular adipose tissue (PVAT) exerted an anticontractile effect in these arteries. CSE inhibition by PPG and BCA reduced this effect in aortas from rats but not from mice. CBS inhibition with AOAA did not inhibit the anticontractile effects of PVAT. XE991, however, almost completely suppressed the anticontractile effects of PVAT in both species. Exogenous l-cysteine, substrate for the endogenous production of H(2)S, induced vasorelaxation only at concentrations >5 mmol/l, an effect unchanged by CSE inhibition. CONCLUSIONS/SIGNFICANCE: Our results demonstrate potent vasorelaxant effects of H(2)S donors in large arteries of both rats and mice, in which XE991-sensitive KCNQ-type channel opening play a pivotal role. CSE-H(2)S seems to modulate the effect of adipocyte-derived relaxing factor in rat but not in mouse aorta. The present study provides novel insight into the interaction of CSE-H(2)S and perivascular adipose tissue. Furthermore, with additional technical advances, a future clinical approach targeting vascular H(2)S/KCNQ pathways to influence states of vascular dysfunction may be possible.

Hydrogen sulfide: potent regulator of vascular tone and stimulator of angiogenesis.

Hydrogen sulfide is the "third" gasotransmitter on the rise in cardiovascular research. Recent studies show that hydrogen sulfide has a great potential in the regulation of vascular tone of systemic arteries and many molecular targets are discussed. However, the complex mechanism of vascular tone regulation by hydrogen sulfide is only incompletely understood. It seems that a potent interaction of hydrogen sulfide with vascular endothelial growth factor (VEGF) becomes important in angiogenesis, in the process of wound healing, but also in tumor angiogenesis. Hydrogen sulfide exerts anti-inflammatory effects and it could be a pharmacological target in vascular dysfunction in association with obesity-related hypertension as well as in tumor development and progression. However, the underlying molecular pathways still need to be revealed. This review primarily focuses on the regulatory role of hydrogen sulfide in controlling vascular tone. We attempt to provide recent insights into mechanisms by which CSE-dependent hydrogen sulfide plays a role in the regulation of vascular tone by perivascular adipose tissue. The role of KCNQ channels and other ionic permeation pathways as key targets will be discussed. Recent findings which are summarized in this paper provide new insights into molecular mechanisms of hydrogen sulfide that are crucial for understanding vascular dysfunction in cardiovascular disease and possibly angiogenesis. Future research will be extended to investigate the therapeutic potential of hydrogen sulfide and their targets such as KCNQ channels in cardiovascular diseases, angiogenesis and tumor genesis.

Amplification of EDHF-type vasodilatations in TRPC1-deficient mice.

BACKGROUND AND PURPOSE: TRPC1 channels are expressed in the vasculature and are putative candidates for intracellular Ca(2+) handling. However, little is known about their role in endothelium-dependent vasodilatations including endothelium-derived hyperpolarizing factor (EDHF) vasodilatations, which require activation of Ca(2+) -activated K(+) channels (K(Ca)). To provide molecular information on the role of TRPC1 for K(Ca) function and the EDHF signalling complex, we examined endothelium-dependent and independent vasodilatations, K(Ca) currents and smooth muscle contractility in TRPC1-deficient mice (TRPC1-/-). EXPERIMENTAL APPROACH: Vascular responses were studied using pressure/wire myography and intravital microscopy. We performed electrophysiological measurements, and confocal Ca(2+) imaging for studying K(Ca) channel functions and Ca(2+) sparks. KEY RESULTS: TRPC1 deficiency in carotid arteries produced a twofold augmentation of TRAM-34- and UCL1684-sensitive EDHF-type vasodilatations and of endothelial hyperpolarization to acetylcholine. NO-mediated vasodilatations were unchanged. TRPC1-/- exhibited enhanced EDHF-type vasodilatations in resistance-sized arterioles in vivo associated with reduced spontaneous tone. Endothelial IK(Ca) /SK(Ca)-type K(Ca) currents, smooth muscle cell Ca(2+) sparks and associated BK(Ca)-mediated spontaneous transient outward currents were unchanged in TRPC1-/-. Smooth muscle contractility induced by receptor-operated Ca(2+) influx or Ca(2+) release and endothelium-independent vasodilatations were unaltered in TRPC1-/-. TRPC1-/- exhibited lower systolic blood pressure as determined by tail-cuff blood pressure measurements. CONCLUSIONS AND IMPLICATIONS: Our data demonstrate that TRPC1 acts as a negative regulator of endothelial K(Ca) channel-dependent EDHF-type vasodilatations and thereby contributes to blood pressure regulation. Thus, we propose a specific role of TRPC1 in the EDHF-K(Ca) signalling complex and suggest that pharmacological inhibition of TRPC1, by enhancing EDHF vasodilatations, may be a novel strategy for lowering blood pressure.

Systemic peripheral artery relaxation by KCNQ channel openers and hydrogen sulfide.

BACKGROUND: Perivascular adipose tissue secretes an adipocyte-derived relaxing factor (ADRF) that opens voltage-dependent K (Kv) channels in peripheral arteries. We studied the role of KCNQ-type Kv channels and tested the hypothesis that hydrogen sulfide (H2S) could be an ADRF. METHODS: We performed isometric contraction studies on systemic arteries of rats and mice. RESULTS: In mesenteric arteries and aortas without perivascular adipose tissue, the KCNQ channel openers retigabine, VRX0530727, VRX0621238, and VRX0621688 produced concentration-dependent vasorelaxation; VRX0621688 was the most potent vasodilator. The KCNQ inhibitor XE991 (30 micromol/l) blocked the effects of both the drugs and ADRF. Inhibitors of cystathionine gamma lyase (CSE) beta-cyano-L-alanine (BCA, 5 mmol/l) and 4-propargyl glycine (PPG, 10 mmol/l) also blocked the relaxations. CSE is expressed in perivascular adipose tissue and endogenously generates H2S. The H2S donor NaHS produced concentration-dependent vasorelaxation, which was also blocked by XE991. The vasodilatory capacities of retigabine, VRX0530727, VRX0621238, and VRX0621688 were preserved following inhibition of H2S generation in perivascular fat. CONCLUSION: We suggest that KCNQ channel opening is a powerful mechanism to produce vasorelaxation of systemic arteries in rats and mice. Furthermore, KCNQ channels play a major role in the paracrine control of vascular tone by perivascular adipose tissue, which is at least in part mediated or modulated by H2S. In conditions of reduced H2S release from perivascular adipose tissue, these paracrine effects can be mimicked by synthetic KCNQ channel openers.

A reduction in the amount and anti-contractile effect of periadventitial mesenteric adipose tissue precedes hypertension development in spontaneously hypertensive rats.

The aim of this study was to determine whether alterations in periadventitial adipose tissue and its anti-contractile effect precede hypertension development. We used 4-week-old male Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR), which were pre-hypertensive. Vascular function was studied in the perfused mesenteric bed (MB, 1.5 mL/min). MB weight was lower in SHR (8.0 +/- 0.3 mg/g body weight) than in WKY (9.0 +/- 0.3 mg/g body weight) rats. Concentration-response curves to KCI (6 to 75 mmol/L) and to acetylcholine (10(-9) to 10(-5) mol/L) were similar between groups. Contractile responses to serotonin (10(-9) to 10(-5) mol/L) were significantly higher in SHR compared to WKY. 4-Aminopyridine (4-AP, 2 mmol/L), a blocker of Kv channels, induced a similar increase in perfusion pressure in both strains. However, 4-AP (2 mmol/L) significantly increased the contractile response to serotonin (10(-9) to 10(-5) mol/L) only in WKY. The anti-contractile effect of fat was confirmed by a comparison of (+) fat and (-) fat mesenteric arteries, which revealed that 4-AP significantly enhanced contractions only in (+) fat rings from WKY. These results show that alterations in visceral periadventitial fat mass and function in SHR precede hypertension, suggesting a constitutive mechanism independent of age and the hypertensive state.

Glucocorticoid-related signaling effects in vascular smooth muscle cells.

Mineralocorticoid receptor blockade protects from angiotensin II-induced target-organ damage. 11beta-Hydroxysteroid dehydrogenase type 2 protects the mineralocorticoid receptor from activation by glucocorticoids; however, high glucocorticoid concentrations and absent 11beta-hydroxysteroid dehydrogenase type 2 in some tissues make glucocorticoids highly relevant mineralocorticoid receptor ligands. We investigated the effects of corticosterone (10(-6) to 10(-12) mol/L) on early vascular mineralocorticoid receptor signaling by Western blotting, confocal microscopy, and myography. Corticosterone initiated extracellular signal-regulated kinase 1/2 phosphorylation in rat vascular smooth muscle cells at > or =10(-11) mol/L doses. Protein synthesis inhibitors had no effect, indicating a nongenomic action. Corticosterone also stimulated c-Jun N-terminal kinase, p38, Src, and Akt phosphorylation at 15 minutes and enhanced angiotensin II-induced signaling at 5 minutes. A specific epidermal growth factor receptor blocker, AG1478, as well as the Src inhibitor PP2, markedly reduced corticosterone-induced extracellular signal-regulated kinase 1/2 phosphorylation, as did preincubation of cells with the mineralocorticoid receptor antagonist spironolactone. Silencing mineralocorticoid receptor with small interfering RNA abolished corticosterone-induced effects. Corticosterone (10(-9) mol/L) enhanced phenylephrine-induced contraction of intact aortic rings. These effects were dependent on the intact endothelium, mineralocorticoid receptor, and mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinase signaling. We conclude that corticosterone induces rapid mineralocorticoid receptor signaling in vascular smooth muscle cells that involves mitogen-activated protein kinase kinase/extracellular signal-regulated kinase-dependent pathways. These new mineralocorticoid receptor-dependent signaling pathways suggest that glucocorticoids may contribute to vascular disease via mineralocorticoid receptor signaling, independent of circulating aldosterone.

Adiponectin is a novel humoral vasodilator.

OBJECTIVES: Perivascular adipose tissue secretes an adipocyte-derived relaxing factor(s) (ADRF) that opens K(v) channels in rat arteries. Visceral fat accumulation causes adipocyte dysfunction, including hyposecretion of adiponectin. We tested the hypothesis that ADRF might be adiponectin and that adiponectin plays a role in the paracrine control of vascular tone by perivascular adipose tissue. METHODS AND RESULTS: We studied Sprague-Dawley rats, wild-type and adiponectin gene-deficient (Apn 1-/-) mice, and New Zealand obese (NZO) mice. In rat aortas, recombinant adiponectin at serum levels (2-5 microg/ml) inhibited serotonin-induced contractions. The effects were abolished by K(v) channel inhibition with 4-aminopyridine (4-AP, 2 mM). Similar effects were observed in NZO mouse mesenteric arteries. To study vascular function in Apn 1-/- mice, the mesenteric vascular bed was isolated, cannulated, and perfused at a constant 4-5-ml/min flow in the absence and presence of serotonin. 4-AP (2 mM) induced a similar increase in perfusion pressure in the Apn 1-/- perfused isolated mesenteric vascular bed, compared to wild-type mice. Removal of perivascular fat increased the vasoconstrictor responses, but abolished the 4-AP effects. The anti-contractile effects of perivascular fat were similar in mesenteric artery and aortic rings from Apn 1-/- and wild-type mice. Despite high adiponectin levels, the anti-contractile effects of perivascular fat were diminished in mesenteric arteries of NZO mice with age. CONCLUSION: Adiponectin is a novel humoral vasodilator that relaxes aortic and mesenteric rings by opening K(v) channels. Similar to the rat, perivascular adipose tissue of the mouse harbors an ADRF, which is malfunctional in NZO mice and is not adiponectin.

Pressure-induced and store-operated cation influx in vascular smooth muscle cells is independent of TRPC1.

Among the classical transient receptor potential (TRPC) subfamily, TRPC1 is described as a mechanosensitive and store-operated channel proposed to be activated by hypoosmotic cell swelling and positive pipette pressure as well as regulated by the filling status of intracellular Ca(2+) stores. However, evidence for a physiological role of TRPC1 may most compellingly be obtained by the analysis of a TRPC1-deficient mouse model. Therefore, we have developed and analyzed TRPC1(-/-) mice. Pressure-induced constriction of cerebral arteries was not impaired in TRPC1(-/-) mice. Smooth muscle cells from cerebral arteries activated by hypoosmotic swelling and positive pipette pressure showed no significant differences in cation currents compared to wild-type cells. Moreover, smooth muscle cells of TRPC1(-/-) mice isolated from thoracic aortas and cerebral arteries showed no change in store-operated cation influx induced by thapsigargin, inositol-1,4,5 trisphosphate, and cyclopiazonic acid compared to cells from wild-type mice. In contrast to these results, small interference RNAs decreasing the expression of stromal interaction molecule 1 (STIM1) inhibited thapsigargin-induced store-operated cation influx, demonstrating that STIM1 and TRPC1 are mutually independent. These findings also imply that, as opposed to current concepts, TRPC1 is not an obligatory component of store-operated and stretch-activated ion channel complexes in vascular smooth muscle cells.

Perivascular adipose tissue and mesenteric vascular function in spontaneously hypertensive rats.

OBJECTIVE: Perivascular adipose tissue of normotensive rats releases a transferable factor that induces relaxation by opening voltage-dependent K+ (Kv) channels. The relevance of these observations to hypertension is unknown. METHODS AND RESULTS: We characterized mesenteric perivascular adipose tissue from 3-month-old Wistar Kyoto rats (WKY) and aged-matched spontaneously hypertensive rats (SHR). Mesenteric bed (MB) weight and MB total lipid content were lower in SHR than in WKY. Freshly isolated MB adipocytes were smaller in SHR. Plasma triglycerides, glycerol, nonesterified free-fatty acids, and cholesterol were also lower in SHR. Plasma and mesenteric leptin were correlated with the quantity of mesenteric fat. To study vascular function, the MB was cannulated and perfused at a constant 2 mL/min flow. The Kv channel blocker 4-aminopyridine (4-AP; 2 mmol/L) increased perfusion pressure less in SHR MB than WKY and was directly correlated with the mesenteric fat amount. In isolated mesenteric artery rings, 4-AP (2 mmol/L) induced a contractile effect that was attenuated in SHR compared with WKY. The anticontractile effects of perivascular fat were reduced in SHR mesenteric artery rings compared with WKY. CONCLUSIONS: Differences in visceral perivascular adipose tissue mass and function may contribute to the increased vascular resistance observed in SHR.

Increased vascular smooth muscle contractility in TRPC6-/- mice.

Among the TRPC subfamily of TRP (classical transient receptor potential) channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, we have developed a TRPC6-deficient mouse model. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, was completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6(-)(/)(-) smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells resulted in increased basal activity, while TRPC6 expression in TRPC6(-/-) smooth muscle cells reduced basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone.

Paracrine role for periadventitial adipose tissue in the regulation of arterial tone.

Recent studies propose a paracrine role for periadventitial adipose tissue in the regulation of vascular tone. This regulation depends on the anatomical integrity of the periadventitial adipose tissue and involves adipocyte-derived relaxing factor (ADRF). Although the nature of ADRF is largely unknown, it is released by periadventitial adipocytes and induces vasorelaxation by opening K+ channels in the plasma membrane of smooth muscle cells. Alterations in the paracrine role of periadventitial adipose tissue might have a role in vascular dysfunction in hypertension and metabolic disease. Therefore, understanding alterations in ADRF release and the K+ channels involved will help further our understanding of the increased cardiovascular risk and development of chronic vascular disease in obesity. Furthermore, ADRF and perhaps its putative targets might represent exciting new targets for the development of drugs to treat cardiovascular disorders.

Visceral periadventitial adipose tissue regulates arterial tone of mesenteric arteries.

Periadventitial adipose tissue produces vasoactive substances that influence vascular contraction. Earlier studies addressed this issue in aorta, a vessel that does not contribute to peripheral vascular resistance. We tested the hypothesis that periadventitial adipose tissue modulates contraction of smaller arteries more relevant to blood pressure regulation. We studied mesenteric artery rings surrounded by periadventitial adipose tissue from adult male Sprague-Dawley rats. The contractile response to serotonin, phenylephrine, and endothelin I was markedly reduced in intact vessels compared with vessels without periadventitial fat. The contractile response to U46619 or depolarizing high K+-containing solutions (60 mmol/L) was similar in vessels with and without periadventitial fat. The K+ channel opener cromakalim induced relaxation of vessels precontracted by serotonin but not by U46619 or high K+-containing solutions (60 mmol/L), suggesting that K+ channels are involved. The intracellular membrane potential of smooth muscle cells was more hyperpolarized in intact vessels than in vessels without periadventitial fat. Both the anticontractile effect and membrane hyperpolarization of periadventitial fat were abolished by inhibition of delayed-rectifier K+ (K(v)) channels with 4-aminopyridine (2 mmol/L) or 3,4-diaminopyridine (1 mmol/L). Blocking other K+ channels with glibenclamide (3 micromol/L), apamin (1 micromol/L), iberiotoxin (100 nmol/L), tetraethylammonium ions (1 mmol/L), tetrapentylammonium ions (10 micromol/L), or Ba2+ (3 micromol/L) had no effect. Longitudinal removal of half the perivascular tissue reduced the anticontractile effect of fat by almost 50%, whereas removal of the endothelium had no effect. We suggest that visceral periadventitial adipose tissue controls mesenteric arterial tone by inducing vasorelaxation via K(v) channel activation in vascular smooth muscle cells.

Mechanisms of ADRF release from rat aortic adventitial adipose tissue.

Blood vessels are surrounded by variable amounts of adipose tissue. We showed earlier that adventitial adipose tissue inhibits rat aortic contraction by release of a transferable factor, adventitium-derived relaxing factor (ADRF), which activates smooth muscle K(+) channels. However, little is known about the mechanisms of ADRF release. Using isolated rat aortic rings and isometric contraction measurements, we show that ADRF release depends on extracellular [Ca(2+)] (EC(50) approximately 4.7 mM). ADRF effects do not involve neuronal presynaptic N-type Ca(2+) and Na(+) channels or vanilloid, cannabinoid, and CGRP receptors. ADRF release is strongly inhibited by the protein tyrosine kinase inhibitors genistein and tyrphostin A25. In contrast, daidzein, an inactive genistein analog, and the protein tyrosine kinase inhibitor ST638 had no effect. Protein kinase A inhibition by H89 also inhibited ADRF release, whereas the protein kinase G inhibitor KT-5823 had no effect. We propose that ADRF release is Ca(2+) dependent and is regulated by intracellular signaling pathways involving tyrosine kinase and protein kinase A. Furthermore, ADRF release does not depend on perivascular nerve endings.

Periadventitial fat releases a vascular relaxing factor.

Virtually all blood vessels are surrounded by adventitial fat. Adipocytes produce a host of vasoactive substances that may influence vascular contraction. We tested whether or not perivascular adipose tissue modulates contraction of aortic ring preparations. We studied aortic rings surrounded by periadventitial adipose tissue from adult Sprague-Dawley rats. At a maximum concentration of 300 nM angiotensin II, 6.5 microM serotonin, and 5 microM phenylephrine, the contractile response of intact rings was 95%, 80%, and 30% lower than that of vessels without periadventitial fat. The anticontractile effect of periadventitial fat was reduced by inhibition of ATP-dependent K+ channels with glibenclamide (3 microM) and by the tyrosine kinase inhibitor genistein (10 microM). Blocking NOS, cyclo-oxygenase, cytochrome P450, or adenosine receptors did not restore the vascular response in intact vessels. The anticontractile effect of perivascular fat was present in Zucker fa/fa rats, suggesting that leptin receptors were not responsible. Transferring the bath solution from intact vessels, isolated periadventitial tissue, and cultured rat adipocytes to precontracted vessels lacking periadventitial fat resulted in a rapid relaxation. We suggest that perivascular adventitial adipose tissue releases a transferable adventitium-derived relaxing factor that acts by tyrosine kinase-dependent activation of K+ channels in vascular smooth muscle cells.