Wai San Cheang

Uncoupling Protein-2 Protects Endothelial Function in Diet-Induced Obese Mice

Rationale: Previous studies indicate uncoupling protein-2 (UCP2) as an antioxidant defense against endothelial dysfunction in hypertension. UCP2 also regulates insulin secretion and action. However, the role of UCP2 in endothelial dysfunction associated with diabetes and obesity is unclear. Objective: UCP2 protects against endothelial dysfunction induced by high-fat diet through inhibition of reactive oxygen species (ROS) production, and subsequent increase of nitric oxide bioavailability. Methods and Results: Endothelium-dependent relaxation (EDR) in aortae and mesenteric arteries in response to acetylcholine was measured in wire myograph. Flow-mediated vasodilatation in 2nd-order mesenteric arteries was measured in pressure myograph. ROS production is measured by CM-H2DCFDA and DHE fluorescence. High-glucose exposure reduced EDR in mouse aortae, which was exaggerated in UCP2 knockout (KO) mice, whereas UCP2 overexpression by adenoviral infection (AdUCP2) restored the impaired EDR. Impairment of EDR and flow-mediated vasodilatation in aortae and mesenteric arteries from high-fat diet-induced obese mice (DIO) was exaggerated in UCP2KO DIO mice compared with wild-type DIO littermates, whereas AdUCP2 i.v. injection restored both EDR and flow-mediated vasodilatation in DIO mice. Improved EDR in mesenteric arteries was inhibited by nitric oxide synthase inhibitor. UCP2 overexpression also inhibited intracellular ROS production in the en face endothelium of aorta and mesenteric artery of DIO mice, whereas UCP2 deficiency enhanced ROS production. Conclusions: UCP2 preserves endothelial function through increasing nitric oxide bioavailability secondary to the inhibition of ROS production in the endothelium of obese diabetic mice.

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.

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.

Modifications of Dietary Flavonoids towards Improved Bioactivity: An Update on Structure-activity Relationship

ABSTRACT Over the past two decades, extensive studies have revealed that inflammation represents a major risk factor for various human diseases. Chronic inflammatory responses predispose to pathological progression of chronic illnesses featured with penetration of inflammatory cells, dysregulation of cellular signaling, excessive generation of cytokines, and loss of barrier function. Hence, the suppression of inflammation has the potential to delay, prevent, and to treat chronic diseases. Flavonoids, which are widely distributed in humans daily diet, such as vegetables, fruits, tea and cocoa, among others, are considered as bioactive compounds with anti-inflammatory potential. Modification of flavonoids including hydroxylation, o-methylation, and glycosylation, can alter their metabolic features and affect mechanisms of inflammation. Structure–activity relationships among naturally occurred flavonoids hence provide us with a preliminary insight into their anti-inflammatory potential, not only attributing to the antioxidant capacity, but also to modulate inflammatory mediators. The present review summarizes current knowledge and underlies mechanisms of anti-inflammatory activities of dietary flavonoids and their influences involved in the development of various inflammatory-related chronic diseases. In addition, the established structure–activity relationships of phenolic compounds in this review may give an insight for the screening of new anti-inflammatory agents from dietary materials.

Endothelial nitric oxide synthase enhancer reduces oxidative stress and restores endothelial function in db/db mice

AIMS Endothelial dysfunction is caused by reduced nitric oxide (NO) bioavailability and/or over-produced reactive oxygen species (ROS). The present study investigated a vascular benefit of AVE3085, an endothelial nitric oxide synthase (eNOS) enhancer, in preserving endothelial function in diabetic mice and the mechanisms involved. METHODS AND RESULTS Male db/db and db/m(+) mice were orally administered AVE3085 for 7 days (10 mg kg(-1) day(-1)). Vascular reactivity of arteries was studied via myography under both isometric and isobaric conditions. ROS levels in aortas were determined using dihydroethidium fluorescence dye and electron paramagnetic resonance spin trapping. Chronic treatment with AVE3085 reduced blood pressure, enhanced endothelium-dependent relaxations (EDR) to acetylcholine in aortas, mesenteric, and renal arteries, lowered oxidative stress, and augmented the attenuated flow-dependent dilatation in mesenteric resistance arteries from db/db mice. Incubation of aortas from C57BL/6J mice in high glucose (30 mmol L(-1)) culture medium for 48 h impaired EDR and elevated ROS generation, and these effects were reversed by co-treatment with AVE3085 (1 µmol L(-1)). Benefits of AVE3085 were abolished by the transcription inhibitor actinomycin D, the NOS inhibitor N(G)-nitro-L-arginine methyl ester, and in eNOS(-/-) mice. NO production in primary endothelial cells from mouse aortas was detected with a NO-sensitive fluorescence dye. Protein expression was assayed by western blotting. Treatment with AVE3085 enhanced NO production in endothelial cells and eNOS expression in aortas. CONCLUSION AVE3085 ameliorates endothelial dysfunction in db/db mice through increased NO bioavailability, which reduces oxidative stress in the vascular wall. Targeting eNOS and NO production may be a promising approach to combat diabetic vasculopathy.

NaHS relaxes rat cerebral artery in vitro via inhibition of l-type voltage-sensitive Ca2+ channel.

H(2)S, a gaseous signalling molecule, relaxes blood vessels partly through activation of ATP-sensitive K(+) channels. It is however unclear whether H(2)S or its donors could affect other ion transporting proteins. The present study examined the hypothesis that NaHS, a H(2)S donor inhibits voltage-sensitive Ca(2+) channels and thus relaxes vascular smooth muscle cells (VSMC) in the cerebral arteries. NaHS dilated cerebral arteries from Sprague-Dawley rats with the same potency against pre-contraction by 5-HT and 60 mmol/L KCl, which were unaffected by several K(+) channel blockers, N(G)-nitro-l-arginine methyl ester or indomethacin, as assessed in wire myograph under an isometric condition. Likewise, NaHS also dilated cerebral arteries against myogenic constriction in pressurized myograph under an isobaric condition. NaHS concentration-dependently inhibited CaCl(2)-induced contraction in Ca(2+)-free, 60mM K(+)-containing Krebs solution. Patch clamp recordings showed that NaHS reduced the amplitude of l-type Ca(2+) currents in single myocytes isolated enzymatically from the cerebral artery. Calcium fluorescent imaging using fluo-4 showed a reduced [Ca(2+)](i) in 60 mmol/L KCl-stimulated rat cerebral arteries in response to NaHS. H(2)S precursor l-cysteine-induced relaxation in cerebral arteries was inhibited by cystathionine γ-lyase (CSE) inhibitor dl-propargylglycine. CSE was expressed in cerebral arteries. In summary, NaHS dilates rat cerebral arteries by reducing l-type Ca(2+) currents and suppressing [Ca(2+)](i) of arterial myocyte, indicating that NaHS relaxes cerebral arteries primarily through inhibiting Ca(2+) influx via Ca(2+) channels.

PPARδ Activation Protects Endothelial Function in Diabetic Mice

Recent evidence highlights the therapeutic potential of peroxisome proliferator–activated receptor-δ (PPARδ) agonists to increase insulin sensitivity in diabetes. However, the role of PPARδ in regulating vascular function is incompletely characterized. We investigate whether PPARδ activation improves endothelial function in diabetic and obese mice. PPARδ knockout (KO) and wild-type (WT) mice fed with high-fat diet and db/db mice were used as diabetic mouse models, compared with PPARδ KO and WT mice on normal diet and db/m+ mice. Endothelium-dependent relaxation (EDR) was measured by wire myograph. Flow-mediated vasodilatation (FMD) was measured by pressure myograph. Nitric oxide (NO) production was examined in primary endothelial cells from mouse aortae. PPARδ agonist GW1516 restored EDRs in mouse aortae under high-glucose conditions or in db/db mouse aortae ex vivo. After oral treatment with GW1516, EDRs in aortae and FMDs in mesenteric resistance arteries were improved in obese mice in a PPARδ-specific manner. The effects of GW1516 on endothelial function were mediated through phosphatidylinositol 3-kinase (PI3K) and Akt with a subsequent increase of endothelial nitric oxide synthase (eNOS) activity and NO production. The current study demonstrates an endothelial-protective effect of PPARδ agonists in diabetic mice through PI3K/Akt/eNOS signaling, suggesting the therapeutic potential of PPARδ agonists for diabetic vasculopathy.

The peroxisome proliferator‐activated receptors in cardiovascular diseases: experimental benefits and clinical challenges

The peroxisome proliferator‐activated receptors, PPARα, PPARβ/δ and PPARγ, are ligand‐activated transcriptional factors belonging to the nuclear receptors superfamily and they are known to play important roles in glucose and lipid metabolism. Experimental studies in animal models of metabolic diseases have also revealed that activation of PPARs protects against the vascular complications of diabetes, hypertension, atherosclerosis, myocardial infarction and stroke, through exerting their anti‐inflammatory, anti‐atherogenic and antioxidant effects. In clinical trials and post‐market surveillance, agonists of PPARs have been shown to effectively prevent cardiovascular events. However, adverse effects, particularly for PPARγ agonists, are also observed with the use of investigational PPAR agonists and even some approved drugs. Further exploration of underlying mechanisms is needed to develop novel ways of PPAR activation without causing serious side effects. This article reviews the cardiovascular effects of PPARs, with emphasis on the therapeutic potential of PPAR agonists in combating metabolic vascular diseases.

Upregulation of Angiotensin (1-7)-Mediated Signaling Preserves Endothelial Function Through Reducing Oxidative Stress in Diabetes

AIMS Angiotensin-converting enzyme 2 (ACE2)-angiotensin (1-7) [Ang (1-7)]-Mas constitutes the vasoprotective axis and is demonstrated to antagonize the vascular pathophysiological effects of the classical renin-angiotensin system. We sought to study the hypothesis that upregulation of ACE2-Ang (1-7) signaling protects endothelial function through reducing oxidative stress that would result in beneficial outcome in diabetes. RESULTS Ex vivo treatment with Ang (1-7) enhanced endothelium-dependent relaxation (EDR) in renal arteries from diabetic patients. Both Ang (1-7) infusion via osmotic pump (500 ng/kg/min) for 2 weeks and exogenous ACE2 overexpression mediated by adenoviral ACE2 via tail vein injection (10(9) pfu/mouse) rescued the impaired EDR and flow-mediated dilatation (FMD) in db/db mice. Diminazene aceturate treatment (15 mg/kg/day) activated ACE2, increased the circulating Ang (1-7) level, and augmented EDR and FMD in db/db mouse arteries. In addition, activation of the ACE2-Ang (1-7) axis reduced reactive oxygen species (ROS) overproduction determined by dihydroethidium staining, CM-H2DCFDA fluorescence imaging, and chemiluminescence assay in db/db mouse aortas and also in high-glucose-treated endothelial cells. Pharmacological benefits of ACE2-Ang (1-7) upregulation on endothelial function were confirmed in ACE2 knockout (ACE2 KO) mice both ex vivo and in vitro. INNOVATION We elucidate that the ACE2-Ang (1-7)-Mas axis serves as an important signal pathway in endothelial cell protection in diabetic mice, especially in diabetic human arteries. CONCLUSION Endogenous ACE2-Ang (1-7) activation or ACE2 overexpression preserves endothelial function in diabetic mice through increasing nitric oxide bioavailability and inhibiting oxidative stress, suggesting the therapeutic potential of ACE2-Ang(1-7) axis activation against diabetic vasculopathy. Antioxid.

The peroxisome proliferator-activated receptors in cardiovascular diseases

The peroxisome proliferator‐activated receptors, PPARα, PPARβ/δ and PPARγ, are ligand‐activated transcriptional factors belonging to the nuclear receptors superfamily and they are known to play important roles in glucose and lipid metabolism. Experimental studies in animal models of metabolic diseases have also revealed that activation of PPARs protects against the vascular complications of diabetes, hypertension, atherosclerosis, myocardial infarction and stroke, through exerting their anti‐inflammatory, anti‐atherogenic and antioxidant effects. In clinical trials and post‐market surveillance, agonists of PPARs have been shown to effectively prevent cardiovascular events. However, adverse effects, particularly for PPARγ agonists, are also observed with the use of investigational PPAR agonists and even some approved drugs. Further exploration of underlying mechanisms is needed to develop novel ways of PPAR activation without causing serious side effects. This article reviews the cardiovascular effects of PPARs, with emphasis on the therapeutic potential of PPAR agonists in combating metabolic vascular diseases.