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IntroductionVascular calcification (VC) is an independent risk factor for cardiovascular diseases. VC increases mortality of all-causes. VC is one of most common cardiovascular complications in type II diabetes. So far, no therapy has been proven to be effective in treatment of clinical VC. The present study investigated the therapeutic effects of MR409, an agonistic analog of growth hormone-releasing hormone (GHRH-A), on VC in diabetic db/db mice.Method and resultDiabetic mice were injected with MR409 subcutaneously every day for 8 weeks. Long-term treatment with MR409 improved serum lipid profile and endothelium-dependent relaxation to acetylcholine, and reduced vascular structural injury in diabetic mice without affecting serum growth hormone level. Echocardiography showed that calcium plaques present in heart valve of diabetic mice disappeared in diabetic mice after treatment with MR409. MR409 inhibited vascular calcium deposition associated with a marked reduction in the expressions of osteogenic-regulated alkaline phosphatase (ALP) and transcription osteogenic marker gene Runx2 in diabetic mice. MR409 also inhibited vascular reactive oxygen species (ROS) generation and upregulated the expressions of anti-calcifying protein Klotho in diabetic mice.DiscussionOur results demonstrate that GHRH-A MR409 can effectively attenuate VC and heart valve calcification, and protect against endothelial dysfunction and vascular injury in diabetic mice without significantly affecting pituitary-growth hormone axis. The mechanisms may involve upregulation of anti-calcifying protein Klotho and reduction in vascular ROS and the expression of redox sensitive osteogenic genes Runx2 and ALP. GHRH-A may represent a new pharmacological strategy for treatment of VC and diabetics associated cardiovascular complications.

Objective: Hypertension is associated with a low-grade systemic inflammation in cardiovascular system. Macrophage infiltration may initiate an inflammatory process that contributes to vascular and ventricular remodeling in hypertensive human and mice. The present study investigated the effect of chemical depletion of macrophage using liposome encapsulated clodronate (LEC) on cardiac hypertrophy and remodeling in angiotensin (Ang) II hypertensive mice. Methods: C57BL/6 mice received an Ang II (1.1 mg/kg/day with a minipump) infusion for 2 weeks to induce hypertension. Endothelium-dependent relaxation (ED) was examined by organ bath, hematoxylin and staining and Masson-Trichrome staining were used to evaluate aorta and cardiac hypertrophy and fibrosis. Results: Ang II infusion significantly increased systolic blood pressure (SBP), cardiac hypertrophy and fibrosis, and impaired EDR accompanied by increased macrophage infiltration in the heart. Treatment with LEC significantly lowered Ang II-induced cardiac hypertrophy and fibrosis and cardiac macrophage infiltration, and improved EDR with a mild reduction in SBP. Ang II increased the expression of inflammatory cytokines tumor necross factor alpha and interleukin 1 beta and profibrotic factors transforming growth factor beta 1 and fibronectin in the heart, with was reduced by LEC treatment. Treatment with LEC prevented Ang II-induced the phosphorphorylation of ERK1/2 and c-Jun-N-terminal kinase. Conclusions: Our study suggests that cardiac macrophage may be critical for hypertensive cardiac hypertrophy and remodeling, the underlying mechanisms may involve initial heart inflammation and the activation of hypertrophic MAPKs pathway.

Activation of the renin–angiotensin system has been implicated in hypertension. Angiotensin (Ang) II is a potent proinflammatory mediator. The present study investigated the role of myeloid angiotensin type 1 receptor (AT1R) in control of macrophage phenotype in vitro and vascular injury in deoxycorticosterone acetate (DOCA)/salt hypertension. In human THP-1/macrophages, Ang II increased mRNA expressions of M1 cytokines and decreased M2 cytokine expressions. Overexpression of AT1R further increased Ang II-induced expressions of M1 cytokines and decreased M2 cytokines. Silenced AT1R reversed Ang II-induced changes in M1 and M2 cytokines. Ang II upregulated hypoxia-inducible factor (HIF)1α, toll-like receptor (TLR)4, and the ratio of pIκB/IκB, which were prevented by silenced AT1R. Silenced HIF1α prevented Ang II activation of the TLR4/NFκB pathway. Furthermore, Ang II increased HIF1α via reactive oxygen species-dependent reduction in prolyl hydroxylase domain protein 2 (PHD2) expression. The expressions of AT1R and HIF1α and the ratio of pIκB/IκB were upregulated in the peritoneal macrophages of DOCA hypertensive mice, and the specific deletion of myeloid AT1R attenuated cardiac and vascular injury and vascular oxidative stress, reduced the recruitment of macrophages and M1 cytokine expressions, and improved endothelial function without significant reduction in blood pressure. Our results demonstrate that Ang II/AT1R controls the macrophage phenotype via stimulating the HIF1α/NFκB pathway, and specific myeloid AT1R KO improves endothelial function, vascular inflammation, and injury in salt-sensitive hypertension. The results support the notion that myeloid AT1R plays an important role in the regulation of the macrophage phenotype, and dysfunction of this receptor may promote vascular dysfunction and injury in salt-sensitive hypertension.

Yes-associated protein (YAP) and its associated coactivator of PDZ-binding motif (TAZ) are co-transcriptional regulators and down effectors of the Hippo signaling pathway. Recent studies have shown that the Hippo/YAP signaling pathway may play a role in mediating vascular homeostasis. This study investigated the role of YAP/TAZ in endothelial dysfunction and vascular inflammation in angiotensin (Ang)II hypertensive mice. The infusion of AngII (1.1 mg/kg/day by mini-pump) for 3 weeks induced the activation of YAP/TAZ, manifested by decreased cytosolic phosphor-YAP and phosphor-TAZ, and increased YAP/TAZ nuclear translocation, which were prevented by YAP/TAZ inhibitor verteporfin. AngII significantly increased systolic blood pressure (SBP), macrophage infiltration, and expressions of proinflammatory cytokines, and impaired endothelial function in the aorta of the mice. Treatment with verteporfin improved endothelial function and reduced vascular inflammation with a mild reduction in SBP. AngII also induced YAP/TAZ activation in human umbilical vein endothelial cells in vitro, which were prevented by LB-100, an inhibitor of protein phosphatase 2A (PP2A, a major dephosphorylase). Treatment with LB-100 reversed AngII-induced proinflammatory cytokine expression and impairment of phosphor-eNOS expression in vitro. Our results suggest that AngII induces YAP/TAZ activation via PP2A-dependent dephosphorylation, which may contribute to the impairment of endothelial function and the induction of vascular inflammation in hypertension. YAP/TAZ may be a new target for hypertensive vascular injury.

Puerarin, a major isoflavonoid compound from Chinese herb Kudzu roots, has been widely used for the treatment of hypertensive and cardiovascular diseases in China. Here, we investigated puerarin’s beneficial effects on the cardiovascular system in angiotensin (Ang) II-induced hypertensive rats. Sprague–Dawley rats were treated with Ang II for 5 days or with puerarin for 10 days followed by Ang II and puerarin for 5 days. Endothelium-dependent relaxation (EDR) to acetylcholine was determined using an organ chamber bath. Ang II increased the systolic blood pressure (SBP: 178 ± 5 mmHg vs. 112 ± 3 mmHg in control, p < 0.05), aortic (30%, p < 0.05), and left ventricular (LV) weight (23%); puerarin reduced SBP (160 ± 2 mmHg, p < 0.05), aortic, and left ventricular weight in Ang II-infused rats. Puerarin also reduced aortic medial thickness and myocardial cell surface area in Ang II-infused rats. Compared with control rats, Ang II infused rats exhibited an impaired EDR with reduction in the protein expression of phosphor-eNOS at Ser 1177 and an increase in the expression of gp91phox (85%), p22phox (113%), transforming growth factor β1 (145%) and vascular cell adhesion molecule 1 (82%). Puerarin improved EDR and reversed the changes in Ang II-induced protein expression of above molecules. Our results demonstrate that in Ang II-induced hypertensive rats, puerarin protects against endothelial dysfunction and end organ damage with a mild reduction in SBP, and that the cardiovascular beneficial effects of puerarin may be in part attributed to its anti-oxidant and upregulation of phosphor-eNOS.

Background: Endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of obesity, insulin resistance and cardiovascular diseases (CVDs). Impairment of insulin vascular action may represent a mechanism linking insulin resistance and CVDs. The present study tested the hypothesis that adipocyte-derived resistin inhibits insulin-stimulated endothelial NO production through the induction of ER stress.Methods and Results: Human umbilical vein endothelial cells (HUVC) were incubated with tunicamycin (an inducer of ER stress, 1–20 μg/mL) or resistin (10–100 ng/mL) for 1 h. Either tunicamycin or resistin increased GRP78 (an ER stress marker) expression associated with the impairment of insulin-stimulated Akt/eNOS phosphorylation, which were prevented by TUDCA (an ER stress suppressor). Resistin increased reactive oxygen species (ROS) production, antioxidant treatment inhibited resistin-induced GRP78 expression and impairment of insulin Akt/eNOS signaling, suggesting that ROS may involve resistin-induced ER stress. Resistin also increased JNK phosphorylation, which was prevented by TUDCA. JNK inhibitor SP600125 relieved the resistin inhibitory effects on endothelial insulin Akt/eNOS signaling. In ex vivo experiments, the incubation of aortic rings with resistin impaired insulin- but not acetylcholine-induced vasodilation, which was restored by TUDCA. LNAME (a NOS inhibitor) abolished insulin-induced vasorelaxation in the control or the resistin-treated aortic rings. In addition, resistin increased the mRNA expressions of proinflammatory cytokines tumor nuclear factor (TNF)α and interleukin (IL)-1β, which were also prevented by TUDCA.Conclusion: Our results support the ideal that ER stress may play an important role for resistin impairment of vascular insulin signaling and insulin action. The mitigation of ER stress may represent a new strategy for prevention and treatment of CVDs in obesity and insulin resistant-related diseases.

Monocyte/macrophage recruitment is closely associated with the degree of hypertensive renal injury. We investigated the direct role of macrophages using liposome-encapsulated clodronate (LEC) to deplete monocytes/macrophages in hypertensive renal injury. C57BL/6 mice were treated with a pressor dose of angiotensin (Ang, 1.4 mg/kg/day) II plus LEC or the PBS-liposome for 2 weeks. Ang II mice developed hypertension, albuminuria, glomerulosclerosis, and renal fibrosis. LEC treatment reduced systolic blood pressure (SBP), albuminuria, and protected against renal structural injury in Ang II mice. Ang II significantly increased renal macrophage infiltration (MOMA2+ cells) and the expression of renal tumor necrosis factor α and interleukin β1, which were significantly reduced in Ang II/LEC mice. Ang II increased renal oxidative stress and the expression of profibrotic factors transforming growth factor (TGF) β1 and fibronectin. Ang II also inhibited the phosphorylation of endothelial nitric oxide synthase [phospho-endothelial nitric oxide synthesis (eNOS), ser1177]. LEC treatment reduced renal oxidative stress and TGFβ1 and fibronectin expressions, and increased phospho-eNOS expression in the Ang II mice. In Dahl rats of salt-sensitive hypertension, LEC treatment for 4 weeks significantly attenuated the elevation of SBP induced by high salt intake and protected against renal injury and fibrosis. Our results demonstrate that renal macrophages play a critical role in the development of hypertension and hypertensive renal injury and fibrosis; the underlying mechanisms may be involved in the reduction in macrophage-driven renal inflammation and restoration of the balance between renal oxidative stress and eNOS. Therefore, macrophages should be considered as a potential therapeutic target to reduce the adverse consequences of hypertensive renal diseases.

Obesity and cigarette smoke are major cardiovascular (CV) risk factors and, when coexisting in the same individuals, have additive/synergistic effects upon CVD. We studied the mechanisms involved in nicotine enhancement of CVD in Sprague Dawley rats with diet-induced obesity. The rats were fed either a high fat (HFD) or normal rat chow diet with or without nicotine (100 mg/L in drinking water) for 20 weeks. HFD rats developed central obesity, increased systolic blood pressure (SBP), aortic superoxide (O2-) production, and impaired endothelial nitric oxide synthase (eNOS) and endothelium-dependent relaxation to acetylcholine (EDR). Nicotine further increased SBP, O2- and impaired eNOS and EDR in obese rats. In the peritoneal macrophages from obese rats, tumor necrosis factor (TNF) α, interleukin 1β and CD36 were increased, and were further increased in nicotine-treated obese rats. Using PCR array we found that 3 of 84 target proinflammatory genes were increased by 2-4 fold in the aorta of obese rats, 11 of the target genes were further increased in nicotine-treated obese rats. HUVECs, incubated with conditioned medium from the peritoneal macrophages of nicotine treated-obese rats, exhibited reduced eNOS and increased NADPH oxidase subunits gp91phox and p22phox expression. Those effects were partially prevented by adding anti-TNFα antibody to the conditioned medium. Our results suggest that nicotine aggravates the CV effects of diet-induced obesity including the oxidative stress, vascular inflammation and endothelial dysfunction. The underlying mechanisms may involve in targeting endothelium by enhancement of macrophage-derived TNFα.

Ming-Sheng Zhou, Ivonne Hernandez SchulmanNephrology-Hypertension Section, Veterans Affairs Medical Center and Division of Nephrology and Hypertension and Vascular Biology institute, University of Miami Miller School of Medicine, Miami, FL, USAAbstract: A growing number of experimental and clinical studies have provided evidence indicating that pharmacological blockade of the renin–angiotensin system (RAS) by either angiotensin-converting enzyme inhibitors or angiotensin type 1 receptor blockers reduces the incidence of new onset type 2 diabetes in subjects with hypertension and/or cardiovascular disease, independently of antihypertensive and cardiovascular protective effects. The beneficial effects of RAS inhibition on the development of diabetes have been largely attributed to improvements in peripheral insulin sensitivity and glucose metabolism. This review focuses on recent experimental and clinical evidence supporting the role of RAS inhibition in the reduction of new onset type 2 diabetes and the mechanisms that may be involved.Keywords: renin–angiotensin system, antihypertensive, diabetes