Zhongjian Cheng

Endothelial Dysfunction and Salt-Sensitive Hypertension in Spontaneously Diabetic Goto-Kakizaki Rats

Endothelial dysfunction is associated with hypertension, hypercholesterolemia, and heart failure. We tested the hypothesis that spontaneously diabetic Goto-Kakizaki (GK) rats, a model for type 2 diabetes, exhibit endothelial dysfunction. Rats also received a high-sodium diet (6% NaCl [wt/wt]) and chronic angiotensin type 1 (AT1) receptor blockade (10 mg/kg PO valsartan for 8 weeks). Compared with age-matched nondiabetic Wistar control rats, GK rats had higher blood glucose levels (9.3±0.5 versus 6.9±0.2 mmol/L for control rats), 2.7-fold higher serum insulin levels, and impaired glucose tolerance (all P <0.05). Telemetry-measured mean blood pressure was 15 mm Hg higher in GK rats (P <0.01) compared with control rats, whereas heart rates were not different. Heart weight– and kidney weight–to–body weight ratios were higher in GK rats (P <0.05), and 24-hour albuminuria was increased 50%. Endothelium-mediated relaxation of noradrenaline-precontracted mesenteric arterial rings by acetylcholine was impaired compared with the control condition (P <0.05), whereas the sodium nitroprusside–induced relaxation was similar. Preincubation of the arterial rings with the NO synthase inhibitor NG-nitro-l-arginine methyl ester and the cyclooxygenase inhibitor diclofenac inhibited relaxations to acetylcholine almost completely in GK rats but not in Wistar rats, suggesting that endothelial dysfunction can be in part attributed to reduced relaxation via arterial K+ channels. Perivascular monocyte/macrophage infiltration and intercellular adhesion molecule-1 overexpression were observed in GK rat kidneys. A high-sodium diet increased blood pressure by 24 mm Hg and 24-hour albuminuria by 350%, induced cardiac hypertrophy, impaired endothelium-dependent relaxation further, and aggravated inflammation (all P <0.05). The serum level of 8-isoprostaglandin F2&agr;, a vasoconstrictor and antinatriuretic arachidonic acid metabolite produced by oxidative stress, was increased 400% in GK rats on a high-sodium diet. Valsartan decreased blood pressure in rats fed a low-sodium diet and prevented the inflammatory response. In rats fed a high-sodium diet, valsartan did not decrease blood pressure or improve endothelial dysfunction but protected against albuminuria, inflammation, and oxidative stress. As measured by quantitative autoradiography, AT1 receptor expression in the medulla was decreased in GK compared with Wistar rats, whereas cortical AT1 receptor expression, medullary and cortical angiotensin type 2 (AT2) receptor expressions, and adrenal ACE and neutral endopeptidase expressions were unchanged. A high-sodium diet did not influence renal AT1, AT2, ACE, or neutral endopeptidase expressions. In valsartan-treated GK rats, the cortical and medullary AT1 receptor expressions were decreased in the presence and absence of a high-sodium diet. A high-sodium diet increased plasma brain natriuretic peptide concentrations in presence and absence of valsartan treatment. We conclude that hypertension in GK rats is salt sensitive and associated with endothelial dysfunction and perivascular inflammation. AT1 receptor blockade ameliorates inflammation during a low-sodium diet and partially protects against salt-induced vascular damage by blood pressure–independent mechanisms.

Endothelial Dysfunction and Xanthine Oxidoreductase Activity in Rats With Human Renin and Angiotensinogen Genes

We examined whether xanthine oxidoreductase (XOR), a hypoxia-inducible enzyme capable of generating reactive oxygen species, is involved in the onset of angiotensin (Ang) II–induced vascular dysfunction in double-transgenic rats (dTGR) harboring human renin and human angiotensinogen genes. In 7-week-old hypertensive dTGR, the endothelium-mediated relaxation of noradrenaline (NA)-precontracted renal arterial rings to acetylcholine (ACh) in vitro was markedly impaired compared with Sprague Dawley rats. Preincubation with superoxide dismutase (SOD) improved the endothelium-dependent vascular relaxation, indicating that in dTGR, endothelial dysfunction is associated with increased superoxide formation. Preincubation with the XOR inhibitor oxypurinol also improved endothelium-dependent vascular relaxation. The endothelium-independent relaxation to sodium nitroprusside was similar in both strains. In dTGR, serum 8-isoprostaglandin F2&agr;, a vasoconstrictor and antinatriuretic arachidonic acid metabolite produced by oxidative stress, was increased by 100%, and the activity of XOR in the kidney was increased by 40%. Urinary nitrate plus nitrite (NOx) excretion, a marker of total body NO generation, was decreased by 85%. Contractile responses of renal arteries to Ang II, endothelin-1 (ET-1), and NA were decreased in dTGR, suggesting hypertension-associated generalized changes in the vascular function rather than a receptor-specific desensitization. Valsartan (30 mg/kg PO for 3 weeks) normalized blood pressure, endothelial dysfunction, and the contractile responses to ET-1 and NA. Valsartan also normalized serum 8-isoprostaglandin F2&agr; levels, renal XOR activity, and, to a degree, NOx excretion. Thus, overproduction of Ang II in dTGR induces pronounced endothelial dysfunction, whereas the sensitivity of vascular smooth muscle cells to nitric oxide is unaltered. Ang II–induced endothelial dysfunction is associated with increased oxidative stress and vascular xanthine oxidase activity.

Hyperhomocysteinemia and Endothelial Dysfunction

Hyperhomocysteinemia (HHcy) is a significant and independent risk factor for cardiovascular diseases. Endothelial dysfunction (ED) is the earliest indicator of atherosclerosis and vascular diseases. We and others have shown that HHcy induced ED in human and in animal models of HHcy induced by either high-methionine load or genetic deficiency. Six mechanisms have been suggested explaining HHcy-induced ED. These include 1) nitric oxide inhibition, 2) prostanoids regulation, 3) endothelium-derived hyperpolarizing factors suppression, 4) angiotensin II receptor-1 activation, 5) endothelin-1 induction, and 6) oxidative stress. The goal of this review is to elaborate these mechanisms and to discuss biological and molecular events related to HHcy-induced ED.

Hyperhomocysteinemia impairs endothelium-derived hyperpolarizing factor–mediated vasorelaxation in transgenic cystathionine beta synthase–deficient mice

Hyperhomocysteinemia (HHcy) is associated with endothelial dysfunction (ED), but the mechanism is largely unknown. In this study, we investigated the role and mechanism of HHcy-induced ED in microvasculature in our newly established mouse model of severe HHcy (plasma total homocysteine, 169.5 μM). We found that severe HHcy impaired nitric oxide (NO)- and endothelium-derived hyperpolarizing factor (EDHF)-mediated, endothelium-dependent relaxations of small mesenteric arteries (SMAs). Endothelium-independent and prostacyclin-mediated endothelium-dependent relaxations were not changed. A nonselective Ca(2+)-activated potassium channel (K(Ca)) inhibitor completely blocked EDHF-mediated relaxation. Selective blockers for small-conductance K(Ca) (SK) or intermediate-conductance K(Ca) (IK) failed to inhibit EDHF-mediated relaxation in HHcy mice. HHcy increased the levels of SK3 and IK1 protein, superoxide (O(2)(-)), and 3-nitrotyrosine in the endothelium of SMAs. Preincubation with antioxidants and peroxynitrite (ONOO(-)) inhibitors improved endothelium-dependent and EDHF-mediated relaxations and decreased O(2)(-) production in SMAs from HHcy mice. Further, EDHF-mediated relaxation was inhibited by ONOO(-) and prevented by catalase in the control mice. Finally, L-homocysteine stimulated O(2)(-) production, which was reversed by antioxidants, and increased SK/IK protein levels and tyrosine nitration in cultured human cardiac microvascular endothelial cells. Our results suggest that HHcy impairs EDHF relaxation in SMAs by inhibiting SK/IK activities via oxidation- and tyrosine nitration-related mechanisms.

Targeted STIM deletion impairs calcium homeostasis, NFAT activation, and growth of smooth muscle

The Ca2+‐sensing stromal interaction molecule (STIM) proteins are crucial Ca2+ signal coordinators. Cre‐lox technology was used to generate smooth muscle (sm)‐targeted STIM1‐, STIM2‐, and double STIM1/STIM2‐knockout (KO) mouse models, which reveal the essential role of STIM proteins in Ca2+ homeostasis and their crucial role in controlling function, growth, and development of smooth muscle cells (SMCs). Compared to Cre+/– littermates, sm‐STIM1‐KO mice showed high mortality (50% by 30 d) and reduced bodyweight. While sm‐STIM2‐KO was without detectable phenotype, the STIM1/STIM double‐KO was perinatally lethal, revealing an essential role of STIM1 partially rescued by STIM2. Vascular and intestinal smooth muscle tissues from sm‐STIM1‐KO mice developed abnormally with distended, thinned morphology. While depolarization‐induced aortic contraction was unchanged in sm‐STIM1‐KO mice, α1‐adrenergic‐mediated contraction was 26% reduced, and store‐dependent contraction almost eliminated. Neointimal formation induced by carotid artery ligation was suppressed by 54%, and in vitro PDGF‐induced proliferation was greatly reduced (79%) in sm‐STIM1‐KO. Notably, the Ca2+ store‐refilling rate in STIM1‐KO SMCs was substantially reduced, and sustained PDGF‐induced Ca2+ entry was abolished. This defective Ca2+ homeostasis prevents PDGF‐induced NFAT activation in both contractile and proliferating SMCs. We conclude that STIM1‐regulated Ca2+ homeostasis is crucial for NFAT‐mediated transcriptional control required for induction of SMC proliferation, development, and growth responses to injury.—Mancarella, S., Potireddy, S., Wang, Y., Gao, H., Gandhirajan, K., Autieri, M., Scalia, R., Cheng, Z., Wang, H., Madesh, M., Houser, S. R., Gill, D. L. Targeted STIM deletion impairs calcium homeostasis, NFAT activation, and growth of smooth muscle. FASEB J. 27, 893–906 (2013). www.fasebj.org

Immunosuppressive/anti-inflammatory cytokines directly and indirectly inhibit endothelial dysfunction--a novel mechanism for maintaining vascular function.

Endothelial dysfunction is a pathological status of the vascular system, which can be broadly defined as an imbalance between endothelium-dependent vasoconstriction and vasodilation. Endothelial dysfunction is a key event in the progression of many pathological processes including atherosclerosis, type II diabetes and hypertension. Previous reports have demonstrated that pro-inflammatory/immunoeffector cytokines significantly promote endothelial dysfunction while numerous novel anti-inflammatory/immunosuppressive cytokines have recently been identified such as interleukin (IL)-35. However, the effects of anti-inflammatory cytokines on endothelial dysfunction have received much less attention. In this analytical review, we focus on the recent progress attained in characterizing the direct and indirect effects of anti-inflammatory/immunosuppressive cytokines in the inhibition of endothelial dysfunction. Our analyses are not only limited to the importance of endothelial dysfunction in cardiovascular disease progression, but also expand into the molecular mechanisms and pathways underlying the inhibition of endothelial dysfunction by anti-inflammatory/immunosuppressive cytokines. Our review suggests that anti-inflammatory/immunosuppressive cytokines serve as novel therapeutic targets for inhibiting endothelial dysfunction, vascular inflammation and cardio- and cerebro-vascular diseases.

Resistance to salt-induced hypertension in catechol-O-methyltransferase-gene-disrupted mice

Background Previous studies have indicated that catechol-O-methyltransferase (COMT) can modulate renal dopaminergic tone. Objective To test the hypothesis that COMT blockade protects from salt-induced hypertension. Methods COMT gene-disrupted (−/−) mice and wild-type controls received a high-sodium diet (NaCl 6%) for 3 weeks. Blood pressure and heart rate were recorded by radiotelemetry. Tissue and urine samples were assessed by light microscopy and high-performance liquid chromatography. The effects of nitecapone treatment were also examined. Systolic blood pressure and heart rate during normal sodium diet were similar in COMT (−/−) and wild-type mice. The high-sodium diet increased night-time systolic and diastolic blood pressures in wild-type mice, whereas blood pressure in COMT (−/−) mice remained unaltered. In wild-type mice, the sodium-induced increase in blood pressure was completely normalized by treatment with the COMT inhibitor, nitecapone. At baseline, 24-h urinary excretion of levodopa (l-DOPA), dopamine and noradrenaline was increased by 145, 85 and 74%, respectively, in COMT (−/−) mice compared with wild-type controls. In COMT (−/−) and wild-type mice, a high-sodium diet increased urinary l-DOPA excretion by 405 and 660% (reflected as 102 and 212% increases in dopamine excretion), respectively. The absolute amounts of urinary l-DOPA and dopamine remained 60 and 20% greater in COMT (−/−) mice. The high-sodium diet did not influence renal cortical COMT activity. Conclusion Our findings suggest that COMT deficiency in mice increases the availability of l-DOPA, leading to enhanced dopaminergic tone, which may be associated with resistance to salt-induced hypertension. The findings of the present study also underline the importance of COMT in the regulation of blood pressure, sodium excretion and renal dopaminergic tone.

Lipoic acid supplementation prevents cyclosporine-induced hypertension and nephrotoxicity in spontaneously hypertensive rats.

Background Cyclosporine (CsA) has significantly improved long-term survival after organ transplantations. Hypertension and nephrotoxicity are common side effects during CsA treatment and are aggravated by high salt intake. Objective To examine whether lipoic acid (LA), a natural antioxidant that scavenges reactive oxygen species and regenerates/recycles endogenous antioxidants, could prevent CsA-induced hypertension and nephrotoxicity. Methods Six-week-old spontaneously hypertensive rats (SHR) on a high-sodium diet (NaCl 6%) received CsA [5 mg/kg subcutaneously (s.c.)] alone or in combination with LA (0.5% w/w) for 6 weeks. Blood pressure, arterial functions, and tissue morphology were determined. Immunohistochemistry, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and high-pressure liquid chromatography were used for kidney and heart samples. Results CsA induced severe hypertension, cardiac hypertrophy, endothelial dysfunction, and pronounced albuminuria. Histologically, the kidneys showed severe thickening of the media of the afferent arteries with fibrinoid necrosis, perivascular monocyte/macrophage infiltration and nitrotyrosine overexpression. CsA induced the expression of fibrogenic connective tissue growth factor both in the heart and kidneys. The detrimental effects of CsA were associated with upregulation of myocardial atrial natriuretic peptide (ANP) mRNA expression, paradoxical activation of the renin–angiotensin system (RAS), induction of renal reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and overexpression of oxidative stress-induced transcription factor NRF2. LA lowered blood pressure, ameliorated cardiac hypertrophy and endothelial dysfunction, and totally normalized albuminuria. In LA-treated rats, renal and cardiac morphologies were indistinguishable from those of SHR controls. CsA-induced myocardial ANP and connective tissue growth factor (CTGF) mRNA overexpression, RAS activation, NADPH oxidase induction, and NRF2 overexpression were prevented by LA. LA induced the mRNA expression of γ-glutamylcysteine ligase, the rate-limiting enzyme in glutathione synthesis, and markedly increased hepatic cysteine and glutathione concentrations. Conclusions Our findings suggest a salutary role for lipoic acid supplementation in the prevention of CsA-induced hypertension and nephrotoxicity, and underscore the importance of increased oxidative stress in the pathogenesis of CsA toxicity.

Effects of a Western diet versus high glucose on endothelium-dependent relaxation in murine micro- and macro-vasculature

Vascular contractility and endothelium-dependent vasodilatation were studied in mesenteric, aorta and coronary vasculature from male and female LDL receptor deficient (LDLR(-/-)) and wild type C57BL/6 mice fed either a high-fat Western Diet (WD) or regular animal chow (RD). Endothelium-dependent vasodilatation was also studied in small mesenteric arteries and aorta from C57BL/6 mice following a 20 h exposure in vitro to 30 mM glucose. Compared with RD-fed animals, WD-fed LDLR-/- animals had increased body weights, elevated triglycerides and total cholesterol, but not glucose. Control C57BL6 animals had elevated body weight without increased cholesterol, triglyceride or glucose levels. The contractile sensitivity to cirazoline (pD(2)) of small mesenteric arteries was the same for RD-fed LDLR-/- and RD-fed C57BL6 mice, but was reduced in WD-fed male LDLR-/- and WD-fed female C57BL/6 mice. Maximum mesenteric contractile values for cirazoline (Emax) were unchanged; however, the Emax for phenylephrine in the aorta from WD-fed male C57BL/6 (but not LDLR-/- or female C57BL/6) mice was reduced. The Emax for acetylcholine-mediated endothelium-dependent vasodilatation in micro- and macro vessels (small mesenteric artery, coronary artery and aorta) from WD-fed LDLR-/- and C57BL/6 mice was unaltered, in contrast to the reduction in Emax for glucose-exposed tissues. Furthermore, the component of acetylcholine-mediated vasodilatation resistant to the combination of inhibitors of nitric oxide synthase, cyclooxygenase and guanylyl cyclase (nitro L-arginine methyl ester - 100 microM; indomethacin 10 microM and 1H-[1,2,4]-oxadiazolo[4,3,-a]quinoxalin-1-one, ODQ - 10 microM, respectively) was generally greater in WD-fed mice. Thus, vasculature from WD-fed mice with short-term dyslipidaemia do not exhibit reduced endothelium-dependent vasodilatation, but the WD is associated with changes in the overall endothelial-dependent relaxation and contractile responses thus suggesting an impact of diet rather than dyslipidaemia on cellular signalling pathways in vascular tissue. In contrast, acute hyperglycaemia resulted in endothelial dysfunction in both small mesenteric arteries and thoracic aorta.

Hyperhomocysteinemia Potentiates Hyperglycemia-Induced Inflammatory Monocyte Differentiation and Atherosclerosis

Hyperhomocysteinemia (HHcy) is associated with increased diabetic cardiovascular diseases. However, the role of HHcy in atherogenesis associated with hyperglycemia (HG) remains unknown. To examine the role and mechanisms by which HHcy accelerates HG-induced atherosclerosis, we established an atherosclerosis-susceptible HHcy and HG mouse model. HHcy was established in mice deficient in cystathionine β-synthase (Cbs) in which the homocysteine (Hcy) level could be lowered by inducing transgenic human CBS (Tg-hCBS) using Zn supplementation. HG was induced by streptozotocin injection. Atherosclerosis was induced by crossing Tg-hCBS Cbs mice with apolipoprotein E-deficient (ApoE−/−) mice and feeding them a high-fat diet for 2 weeks. We demonstrated that HHcy and HG accelerated atherosclerosis and increased lesion monocytes (MCs) and macrophages (MØs) and further increased inflammatory MC and MØ levels in peripheral tissues. Furthermore, Hcy-lowering reversed circulating mononuclear cells, MC, and inflammatory MC and MC-derived MØ levels. In addition, inflammatory MC correlated positively with plasma Hcy levels and negatively with plasma s-adenosylmethionine–to–s-adenosylhomocysteine ratios. Finally, l-Hcy and d-glucose promoted inflammatory MC differentiation in primary mouse splenocytes, which was reversed by adenoviral DNA methyltransferase-1. HHcy and HG, individually and synergistically, accelerated atherosclerosis and inflammatory MC and MØ differentiation, at least in part, via DNA hypomethylation.