Chi Wai Lau

Cyclooxygenase-2–Derived Prostaglandin F2α Mediates Endothelium-Dependent Contractions in the Aortae of Hamsters With Increased Impact During Aging

Hypertension and vascular dysfunction result in the increased release of endothelium-derived contracting factors (EDCFs), whose identity is poorly defined. We tested the hypothesis that endothelial cyclooxygenase (COX)-2 can generate EDCFs and identified the possible EDCF candidate. Changes in isometric tension of aortae of young and aged hamsters were recorded on myograph. Real-time changes in intracellular calcium concentrations ([Ca2+]i) in native aortic endothelial cells were measured by imaging. Endothelium-dependent contractions were triggered by acetylcholine (ACh) after inhibition of nitric oxide production and they were abolished by COX-2 but not COX-1 inhibitors or by thromboxane–prostanoid receptor antagonists. 2-Aminoethoxydiphenyl borate (cation channel blocker) eliminated endothelium-dependent contractions and ACh-stimulated rises in endothelial cell [Ca2+]i. RT-PCR and Western blotting showed COX-2 expression mainly in the endothelium. Enzyme immunoassay and high-performance liquid chromatography-coupled mass spectrometry showed release of prostaglandin (PG)F2&agr; and prostacyclin (PGI2) increased by ACh; only PGF2&agr; caused contraction at relevant concentrations. COX-2 expression, ACh-stimulated contractions, and vascular sensitivity to PGF2&agr; were augmented in aortae from aged hamsters. Human renal arteries also showed thromboxane–prostanoid receptor–mediated ACh- or PGF2&agr;-induced contractions and COX-2–dependent release of PGF2&agr;. The present study demonstrates that PGF2&agr;, derived from COX-2, which is localized primarily in the endothelium, is the most likely EDCF underlying endothelium-dependent, thromboxane–prostanoid receptor–mediated contractions to ACh in hamster aortae. These contractions involved increases in endothelial cell [Ca2+]i. The results support a critical role of COX-2 in endothelium-dependent contractions in this species with an increased importance during aging and, possibly, a similar relevance in humans.

Dipeptidyl Peptidase 4 Inhibitor Sitagliptin Protects Endothelial Function in Hypertension Through a Glucagon–Like Peptide 1–Dependent Mechanism

Sitagliptin, a selective dipeptidyl peptidase 4 inhibitor, inhibits the inactivation and degradation of glucagon like peptide 1 (GLP-1), which is used for the treatment of type 2 diabetes mellitus. However, little is known about the role of GLP-1 in hypertension. This study investigated whether the activation of GLP-1 signaling protects endothelial function in hypertension. Two-week sitagliptin treatment (10 mg/kg per day, oral gavage) improved endothelium-dependent relaxation in renal arteries, restored renal blood flow, and reduced systolic blood pressure in spontaneously hypertensive rats. In vivo sitagliptin treatment elevated GLP-1 and GLP-1 receptor expressions, increased cAMP level, and subsequently activated protein kinase A, liver kinase B1, AMP-activated protein kinase-&agr; and endothelial NO synthase in spontaneously hypertensive rat renal arteries. Inhibition of GLP-1 receptor, adenylyl cyclase, protein kinase A, AMP-activated protein kinase-&agr;, or NO synthase reversed the protective effects of sitagliptin. We also demonstrate that GLP-1 receptor agonist exendin 4 in vitro treatment had similar vasoprotective effects in spontaneously hypertensive rat renal arteries and increased NO production in spontaneously hypertensive rat aortic endothelial cells. Studies using transient expressions of wild-type and dominant-negative AMP-activated protein kinase-&agr;2 support the critical role of AMP-activated protein kinase-&agr; in mediating the effect of GLP-1 in endothelial cells. Ex vivo exendin 4 treatment also improved endothelial function of renal arteries from hypertensive patients. Our results elucidate that upregulation of GLP-1 and related agents improve endothelial function in hypertension by restoring NO bioavailability, suggesting that GLP-1 signaling could be a therapeutic target in hypertension-related vascular events.

Berberine prevents hyperglycemia-induced endothelial injury and enhances vasodilatation via adenosine monophosphate-activated protein kinase and endothelial nitric oxide synthase

AIMS Endothelial dysfunction is a key event that links obesity, diabetes, hypertension, and cardiovascular diseases. The aim of the present study was to examine the protective effect of the alkaloid drug berberine against hyperglycemia-induced cellular injury and endothelial dysfunction. METHODS AND RESULTS In both cultured endothelial cells and blood vessels isolated from rat aorta, berberine concentration dependently enhanced phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177 and promoted the association of eNOS with heat shock protein 90 (HSP90), leading to an increased production of nitric oxide. Furthermore, berberine attenuated high glucose-induced generation of reactive oxygen species, cellular apoptosis, nuclear factor-kappaB activation, and expression of adhesion molecules, thus suppressing monocyte attachment to endothelial cells. In mouse aortic rings, berberine elicited endothelium-dependent vasodilatations and alleviated high glucose-mediated endothelial dysfunction. All these beneficial effects of berberine on the endothelium were abolished by either pharmacological inhibition of adenosine monophosphate-activated protein kinase (AMPK) or adenovirus-mediated overexpression of a dominant negative version of AMPK. CONCLUSION Berberine protects against endothelial injury and enhances the endothelium-dependent vasodilatation, which is mediated in part through activation of the AMPK signalling cascade. Berberine or its derivatives may be useful for the treatment and/or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease.

Calcitriol protects renovascular function in hypertension by down-regulating angiotensin II type 1 receptors and reducing oxidative stress

AIMS The present study investigated whether or not calcitriol, an active form of vitamin D, protects against renovascular dysfunction in hypertension and, if so, whether or not such protection alters the expression of key proteins involved in that dysfunction. METHODS AND RESULTS Changes in isometric tension showed that the impaired endothelium-dependent relaxations in renal arteries of hypertensive patients were enhanced by 12 h in vitro treatment with calcitriol. Dihydroethidium fluorescence revealed an elevated level of reactive oxygen species (ROS) in these arteries which was reduced by calcitriol. Immunofluorescence showed that calcitriol treatment reduced the expression of AT(1)R, NOX-2, NOX-4, and p67(phox) and increased that of superoxide dismutase (SOD)-1. Twelve-hour exposure to calcitriol prevented angiotensin (Ang) II-induced increases in ROS and the over-expression of NOX-2, NOX-4, and p67(phox) in renal arteries from normotensive patients. A specific antagonist of the human vitamin D receptor (VDR), TEI-9647, abolished these effects of calcitriol. Both in vitro exposure to and chronic in vivo administration of calcitriol enhanced relaxations to acetylcholine and abolished exaggerated endothelium-dependent contractions in renal arteries of normotensive rats pre-exposed to Ang II or harvested from spontaneously hypertensive rats (SHR). Reactive oxygen species levels and expressions of AT(1)R, NAD(P)H oxidase subunits, SOD-1, and SOD-2 in SHR arteries were normalized by the chronic treatment with calcitriol. CONCLUSION In vivo and in vitro activation of VDR with calcitriol improves endothelial function by normalizing the expressions of AT(1)R and radical generating and scavenging enzymes and thus preventing ROS over-production. The present findings suggest that calcitriol is effective in preserving endothelial function in hypertension.

Metformin Protects Endothelial Function in Diet-Induced Obese Mice by Inhibition of Endoplasmic Reticulum Stress Through 5′ Adenosine Monophosphate–Activated Protein Kinase–Peroxisome Proliferator–Activated Receptor δ Pathway

Objective—5′ Adenosine monophosphate–activated protein kinase (AMPK) interacts with peroxisome proliferator–activated receptor &dgr; (PPAR&dgr;) to induce gene expression synergistically, whereas the activation of AMPK inhibits endoplasmic reticulum (ER) stress. Whether the vascular benefits of antidiabetic drug metformin (AMPK activator) in diabetes mellitus and obesity is mediated by PPAR&dgr; remains unknown. We aim to investigate whether PPAR&dgr; is crucial for metformin in ameliorating ER stress and endothelial dysfunction induced by high-fat diet. Approach and Results—Acetylcholine-induced endothelium-dependent relaxation in aortae was measured on wire myograph. ER stress markers were determined by Western blotting. Superoxide production in mouse aortae and NO generation in mouse aortic endothelial cells were assessed by fluorescence imaging. Endothelium-dependent relaxation was impaired and ER stress markers and superoxide level were elevated in aortae from high-fat diet–induced obese mice compared with lean mice. These effects of high-fat diet were reversed by oral treatment with metformin in diet-induced obese PPAR&dgr; wild-type mice but not in diet-induced obese PPAR&dgr; knockout littermates. Metformin and PPAR&dgr; agonist GW1516 reversed tunicamycin (ER stress inducer)-induced ER stress, oxidative stress, and impairment of endothelium-dependent relaxation in mouse aortae as well as NO production in mouse aortic endothelial cells. Effects of metformin were abolished by cotreatment of GSK0660 (PPAR&dgr; antagonist), whereas effects of GW1516 were unaffected by compound C (AMPK inhibitor). Conclusions—Metformin restores endothelial function through inhibiting ER stress and oxidative stress and increasing NO bioavailability on activation of AMPK/PPAR&dgr; pathway in obese diabetic mice.

Vasorelaxant and antiproliferative effects of berberine.

The present study was intended to examine the relaxant effects of berberine in rat isolated mesenteric arteries. Berberine produced a rightward shift of the concentration-response curve to phenylephrine and significantly reduced the maximal contractile response to phenylephrine. Berberine (10(-7)-3x10(-5) M) also relaxed the phenylephrine- and 9,11-dideoxy-11alpha, 9alpha-epoxy-methanoprostaglandin F(2alpha)-precontracted arteries with respective IC(50) values of 1.48+/-0.16x10(-6) and 2.23+/-0. 22x10(-6) M. Removal of a functional endothelium significantly attenuated the berberine-induced relaxation (IC(50): 4.73+/-0. 32x10(-6) M) without affecting the maximum relaxant response. Pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME) or methylene blue reduced the relaxant effect of berberine, and L-arginine (10(-3) M) partially antagonized the effect of L-NAME. In contrast, pretreatment with 10(-6) M glibenclamide or 10(-5) M indomethacin had no effect. Berberine (10(-5) M) reduced over by 50% the transient contraction induced by caffeine or phenylephrine in endothelium-denuded rings bathed in Ca(2+)-free Krebs solution. Pretreatment with putative K(+) channel blockers, such as tetrapentylammonium ions (1-3x10(-6) M), 4-aminopyridine (10(-3) M), or Ba(2+) (3x10(-4) M), significantly attenuated the berberine-induced relaxation in endothelium-denuded arteries. In contrast, tetraethylammonium ions (3x10(-3) M), charybdotoxin (10(-7) M) or glibenclamide (10(-6) M) were without effect. Berberine reduced the high-K(+)-induced sustained contraction and the relaxant response to berberine was greater in rings with endothelium (IC(50): 4.41+/-0.47x10(-6) M) than in those without endothelium (IC(50): 8.73+/-0.74x10(-6) M). However, berberine (10(-6)-10(-4) M) did not affect the high-K(+)-induced increase of intracellular [Ca(2+)] in cultured aortic smooth muscle cells. Berberine did not affect active phorbol ester-induced contraction in Ca(2+)-free Krebs solution. In addition, berberine inhibited proliferation of cultured rat aortic smooth muscle cells with an IC(50) of 2.3+/-0.43x10(-5) M. These findings suggest that berberine could act at both endothelium and the underlying vascular smooth muscle to induce relaxation. Nitric oxide from endothelium may account primarily for the berberine-induced endothelium-dependent relaxation, while activation of tetrapentylammonium-, 4-aminopyridine- and Ba(2+)-sensitive K(+) channels, inhibition of intracellular Ca(2+) release from caffeine-sensitive pools, or a direct relaxant effect, is likely responsible for the berberine-induced endothelium-independent relaxation. Mechanisms related to either Ca(2+) influx or protein kinase C activation may not be involved. Both vasorelaxant and antiproliferative effects may contribute to a long-term benefit of berberine in the vascular system.

4-Aminopyridine-sensitive K+ channels contributes to NaHS-induced membrane hyperpolarization and relaxation in the rat coronary artery

The present study aimed at examining the role of potassium channels and endothelium in relaxations induced by sodium hydrogen sulphide (NaHS), which is the donor of gaseous hydrogen sulphide (H(2)S) and the effect of NaHS on endothelium-dependent relaxations in rat coronary arteries. Rat coronary arteries were suspended in a myograph for force measurement and changes of the membrane potential in arteries were determined by membrane potential-sensitive fluorescence dye. NaHS relaxed coronary arteries pre-contracted by U46619 and the relaxation was significantly less in high KCl-contracted rings. NaHS-induced relaxations were reduced by 4-aminopyridine (4-AP) but unaffected by glibenclamide, iberiotoxin, N(G)-nitro-L-arginine methyl ester, ODQ, indomethacin or by endothelium removal. The inhibitory effect of 4-AP was absent in NaHS-induced relaxations in high KCl-contracted rings. Addition of NaHS caused membrane hyperpolarization and this effect was inhibited by 4-AP but not by glibenclamide. NaHS causes endothelium-independent relaxations in rat coronary arteries partially through activation of 4-AP-sensitive potassium channel and ensuring hyperpolarization. Other potassium channels, Na(+)-K(+) pump or endothelium-derived relaxing factors play little role.

Urocortin‐induced endothelium‐dependent relaxation of rat coronary artery: role of nitric oxide and K+ channels

The mechanisms underlying the vasodilator response to urocortin are incompletely understood. The present study was designed to examine the role of endothelial nitric oxide and Ba2+‐sensitive K+ channels in the endothelium‐dependent component of urocortin‐induced relaxation in the rat left anterior descending coronary artery. Urocortin induced both endothelium‐dependent and ‐independent relaxation with respective pD2 of 8.64±0.03 and 7.90±0.10. Removal of endothelium reduced the relaxing potency of urocortin. In rings pretreated with 10−4 M NG‐nitro‐L‐arginine methyl ester, 10−5 M methylene blue or 10−5 M ODQ, the urocortin‐induced relaxation was similar to that observed in endothelium‐denuded rings. L‐Arginine (5×10−4 M) antagonized the effect of NG‐nitro‐L‐arginine methyl ester. The relaxant response to urocortin was reduced in endothelium‐intact rings preconstricted by 3.5×10−2 M K+ and abolished when extracellular K+ was raised to 5×10−2 M. Pretreatment with 10−4 M BaCl2 significantly inhibited urocortin‐induced relaxation. Combined treatment with 10−4 M BaCl2 plus 10−4 M NG‐nitro‐L‐arginine methyl ester did not cause further inhibition. In urocortin (10−8 M)‐relaxed rings, BaCl2 induced concentration‐dependent reversal in vessel tone. Tertiapin‐Q (10−6 M) also attenuated urocortin‐induced relaxation. In contrast, BaCl2 did not alter urocortin‐induced relaxation in endothelium‐denuded rings. In endothelium‐denuded rings, hydroxylamine‐ and nitroprusside‐induced relaxation was inhibited by 10−4 M BaCl2, but not by 10−6 M tertiapin‐Q. The endothelium of the coronary artery was moderately stained with the antiserum against urocortin. Taken together, the present results indicate that the urocortin‐induced endothelium‐dependent relaxation of rat coronary arteries is likely attributable to endothelial nitric oxide and subsequent activation of Ba2+‐ or tertiapin‐Q‐sensitive K+ channels. The urocortin‐induced endothelium‐dependent relaxation appears to be mediated by cyclic GMP‐dependent mechanisms.

Vasorelaxant effects of purified green tea epicatechin derivatives in rat mesenteric artery

The effects of four epicatechin derivatives, (-) epicatechin, (-) epicatechin gallate, (-) epigallocatechin and (-) epigallocatechin gallate, isolated from jasmine green tea, on the contractions were studied in mesenteric arteries isolated from male Sprague-Dawley rats. All four derivatives (30-500 microM) non-competitively reduced the contractile response to phenylephrine in a concentration-dependent manner with epigallocatechin gallate being the most potent. The relaxant effects of epicatechin derivatives were unaffected by the ATP-sensitive K+ channel blocker glibenclamide (3 microM) or the Ca2+-activated K+ channel blocker charybdotoxin (100 nM). Four epicatechin derivatives also reduced the sustained contractions induced by phenylephrine (1 microM) and endothelin I (5 nM) in normal Krebs solution, whilst they did not relax the phorbol 12-myristate 13-acetate (TPA, 2 microM)-contracted arteries in the absence of extracellular Ca2+. In arteries contracted with 60 mM K+, each of epicatechins caused a relaxation. However, epicatechin derivatives did not affect the transient contraction induced by 100 microM caffeine in Ca2+-free solution. The present results suggest that epicatechin derivatives from green tea leaves relaxed rat mesenteric arteries probably by inhibiting Ca2+ influx. The protein kinase C-dependent contractile pathway and intracellular Ca2+ release may not be involved.

Adiponectin Is Required for PPARγ-Mediated Improvement of Endothelial Function in Diabetic Mice

Rosiglitazone is a PPARγ agonist commonly used to treat diabetes. In addition to improving insulin sensitivity, rosiglitazone restores normal vascular function by a mechanism that remains poorly understood. Here we show that adiponectin is required to mediate the PPARγ effect on vascular endothelium of diabetic mice. In db/db and diet-induced obese mice, PPARγ activation by rosiglitazone restores endothelium-dependent relaxation of aortae, whereas diabetic mice lacking adiponectin or treated with an anti-adiponectin antibody do not respond. Rosiglitazone stimulates adiponectin release from fat explants, and subcutaneous fat transplantation from rosiglitazone-treated mice recapitulates vasodilatation in untreated db/db recipients. Mechanistically, adiponectin activates AMPK/eNOS and cAMP/PKA signaling pathways in aortae, which increase NO bioavailability and reduce oxidative stress. Taken together, these results demonstrate that adipocyte-derived adiponectin is required for PPARγ-mediated improvement of endothelial function in diabetes. Thus, the adipose tissue represents a promising target for treating diabetic vasculopathy.