Meng-xiong Tang

Differential regulation of collagen types I and III expression in cardiac fibroblasts by AGEs through TRB3/MAPK signaling pathway

Abstract.Advanced glycation end products (AGEs) play an important role in collagen deposition in diabetic cardiomyopathy. TRB3, a mammalian homolog of Drosophila tribbles, functions to increase glucose intolerance and regulates cell proliferation. We demonstrated that AGEs induce collagen type I expression but inhibit collagen type III expression, accompanied by increased TRB3 expression. Furthermore, the collagen type I induced byAGEs was down-regulated after inhibition of ERK and p38-MAPK, the collagen type III reduced by AGEs was up-regulated after inhibition of ERK. The expression of collagen types I and III regulated by AGEs through MAPK was partly reversed after treatment with TRB3 siRNA. It suggests that the TRB3/MAPK signaling pathway participates in the regulation of collagen types I and III by AGEs and may provide new therapeutic strategies for diabetic cardiomyopathy.

The role of thrombospondin-1-mediated TGF-β1 on collagen type III synthesis induced by high glucose

Transforming growth factor-β1 (TGF-β1) has been thought to play a major role during cardiac fibrosis in the development of diabetic cardiomyopathy, and cardiac fibrosis mainly as a result of an increase of collagen type III occurs in the human hearts with diabetes. Thrombospondin-1 (TSP-1) has been reported to activate the latent complex of TGF-β1. We examined the effects of TSP-1 on the expression of TGF-β1 and collagen type III by rat cardiac fibroblasts in high ambient glucose. We demonstrated that high glucose induces the mRNA and protein expression of collagen type III, TGF-β1, and TSP-1. Furthermore, the mRNA and protein expression of collagen type III induced by high glucose was downregulated after treatment with TGF-β1 antibody, or TSP-1 siRNA. The expression of TGF-β1 increased by high glucose was also reversed after treatment with TSP-1 siRNA. Our findings suggest that the TSP-1 participates in the upregulation of TGF-β1, collagen type III by high glucose and may provide new therapeutic strategies for diabetic cardiomyopathy.

Activin receptor-like kinase 7 mediates high glucose-induced H9c2 cardiomyoblast apoptosis through activation of Smad2/3

Cardiomyocyte apoptosis is an important pathological change of diabetic cardiomyopathy. How the elevated glucose levels cause cell apoptosis remains unknown. The aim of our study was to investigate whether activin receptor-like kinase 7 (ALK7)-Smad2/3 signaling pathway plays an important role in high glucose-induced cardiomyocyte apoptosis. H9c2 cardiomyoblasts and neonatal rat cardiomyocytes were treated with 33mmol/l glucose. The expression of ALK7, Smad2 and Smad3 were inhibited by small interfering RNA respectively. The level of ALK7, total Smad2/3, phosphorylated Smad2/3, B-cell lymphoma-2 (Bcl-2) and cleaved Caspase3 were evaluated using western blot. The apoptosis rate was detected by flow cytometer. High glucose treatment caused the apoptosis of H9c2 cardiomyocyte and the inhibition of Smad2 or Smad3 attenuated this apoptosis. ALK7 existed in both H9c2 cardiomyoblasts and neonatal rat cardiomyocytes and high ambient glucose upregulated its expression. The increased expression level of cleaved Caspase3 and apoptosis rate and decreased expression of Bcl-2 were reversed after ALK7 was inhibited. The expression of phosphorylated Smad2/3 also decreased after the knockdown of ALK7. Our findings suggest that ALK7 mediates high ambient glucose-induced H9c2 cardiomyoblasts apoptosis through the activation of Smad2/3.

Overexpressing STAMP2 Improves Insulin Resistance in Diabetic ApoE−/−/LDLR−/− Mice via Macrophage Polarization Shift in Adipose Tissues

STAMP2 is a counterregulator of inflammation and insulin resistance. The aim of this study is to investigate whether activation of STAMP2 improves insulin resistance by regulating macrophage polarization in adipose tissues. The diabetic ApoE−/−/LDLR−/− mouse model was induced by high-fat diet and low-dose streptozotocin. Samples were obtained from epididymal, subcutaneous and brown adipose tissues. Infiltration of M1/M2 macrophages and inflammatory cytokines were investigated by immunohistochemistry. We then used gene overexpression to investigate the effect of STAMP2 on macrophages infiltration and polarization and inflammatory cytokines expression. Our results showed that infiltration of macrophages, the ratio of M1/M2 macrophages and the expression of pro-inflammatory cytokines were enhanced and STAMP2 was downregulated in adipose tissues of diabetic ApoE−/−/LDLR−/− mice compared with control mice. STAMP2 gene overexpression could significantly reduce macrophages infiltration, the ratio of M1/M2 macrophages and the expression of pro-inflammatory cytokines in epididymal and brown adipose tissues, improving insulin resistance. Our results suggested that STAMP2 gene overexpression may improve insulin resistance via regulating macrophage polarization in visceral and brown adipose tissues.

FP-receptor gene silencing ameliorates myocardial fibrosis and protects from diabetic cardiomyopathy

Prostaglandin F2α-F-prostanoid (PGF2α-FP) receptor is closely related to insulin resistance, which plays a causal role in the pathogenesis of diabetic cardiomyopathy (DCM). We sought to reveal whether PGF2α-FP receptor plays an important part in modulating DCM and the mechanisms involved. We established the type 2 diabetes rat model by high-fat diet and low-dose streptozotocin (STZ) and then evaluated its characteristics by metabolite tests, Western blot analysis for FP-receptor expression, histopathologic analyses of cardiomyocyte density and fibrosis area. Next, we used gene silencing to investigate the role of FP receptor in the pathophysiologic features of DCM. Our study showed elevated cholesterol, triglyceride, glucose, and insulin levels, severe insulin resistance, and FP-receptor overexpression in diabetic rats. The collagen volume fraction (CVF) and perivascular collagen area/luminal area (PVCA/LA) were higher in the diabetic group than the control group (CVF% 10.99 ± 0.99 vs 1.59 ± 0.18, P < 0.05; PVCA/LA% 17.07 ± 2.61 vs 2.86 ± 0.69, P < 0.05). We found that the silencing of FP receptor decreased cholesterol, triglyceride, glucose, and insulin levels and ameliorated insulin resistance. The CVF and PVCF/LA were significantly downregulated in FP-receptor short hairpin RNA (shRNA) treatment group (FP-receptor shRNA group vs vehicle group: CVF% 5.59 ± 0.92 vs 10.97 ± 1.33, P < 0.05, PVCA/LA% 4.74 ± 1.57 vs 14.79 ± 2.22, P < 0.05; FP-receptor shRNA + PGF2α group vs vehicle group : CVF% 5.19 ± 0.79 vs 10.97 ± 1.33, P < 0.05, PVCA/LA% 5.96 ± 1.15 vs 14.79 ± 2.22, P < 0.05, respectively). Furthermore, with FP-receptor gene silencing, the activated protein kinase C (PKC) and Rho kinase were significantly decreased, and the blunted phosphorylation of Akt was restored. FP-receptor gene silencing may exert a protective effect on DCM by improving myocardial fibrosis, suggesting a new therapeutic approach for human DCM.Key messagesFP-receptor gene silencing improves glucose tolerance and insulin resistance in type 2 diabetes (T2D).FP-receptor gene silencing modulates the activities of PKC/Rho and Akt signaling pathways in T2D.FP-receptor gene silencing decreases collagen expression and ameliorates myocardial fibrosis in T2D.FP-receptor gene silencing protects from diabetic cardiomyopathy in T2D.

Gas6 Delays Senescence in Vascular Smooth Muscle Cells through the PI3K/ Akt/FoxO Signaling Pathway

Background/Aims: Growth arrest-specific protein 6 (Gas6) is a cytokine that can be synthesized by a variety of cell types and secreted into the extracellular matrix. Previous studies have confirmed that Gas6 is involved in certain pathophysiological processes of the cardiovascular system through binding to its receptor, Axl. In the present study, we investigated the role of Gas6 in cellular senescence and explored the mechanisms underlying its activity. Methods: We used vascular smooth muscle cells (VSMCs) to create two cellular senescence models, one for replicative senescence (RS) and one for induced senescence (IS), to test the hypothesis that Gas6 delays senescence. Results: Gas6-treated cells appear relatively younger compared with non-Gas6-treated cells. In particular, Gas6-treated cells displayed decreased staining for SA-β-Gal, fewer G1 phase cells, and decreased levels of p16INK4a and p21Cip1 expression; conversely, Gas6-treated cells displayed more S phase cells and significantly increased proliferation indexes. Furthermore, in both the IS and RS models with Gas6 treatment, the levels of PI3K, p-Akt, and p-FoxO3a decreased following Axl inhibition by R428; similarly, the levels of p-Akt and p-FoxO3a also decreased following PI3K inhibition by LY294002. Conclusion: Gas6/Axl signaling is essential for delaying the cellular senescence process regulated by the PI3K/Akt/FoxO signaling pathway.

Visceral adiposity index score indicated the severity of coronary heart disease in Chinese adults

BackgroundVisceral adiposity contributes to cardiometabolic risk, and visceral adiposity index (VAI) had significant correlation with visceral adiposity. We aimed to explore whether VAI was associated with cardiac structure and function and assess the impact of the cut-off points of VAI defining visceral adipose dysfunction (VAD) on the severity of coronary heart disease (CHD).MethodsA total of 95 patients with CHD were divided into Control (nondiabetic CHD patients) and DM group (diabetic CHD patients). Then the two groups were respectively divided into VAD absent and VAD groups. Clinical, echocardiographic and coronary artery angiographic indexes were acquired to examine in relation to VAI.ResultsA significant increasing trend among the four groups of patients (Control + VAD absent, Control +VAD, DM + VAD absent and DM +VAD groups) were observed for waist circumference (WC), body mass index (BMI), systolic blood pressure (SBP), glucose, VAI and Gensini score (P<0.05 for all). The following variables were associated with VAI: total cholesterol, nonesterified fatty acid, Waist-Hip ratio and SBP. VAI was independently associated with Gensini score.ConclusionsThe extent of CHD was more severe in diabetes, and VAI as a simple indicator of visceral adipose mass was strongly associated with the severity of CHD. The cut-off points of VAI used for defining VAD were more useful in diabetic CHD patients in identifying the severity of CHD.

Overexpression of STAMP2 suppresses atherosclerosis and stabilizes plaques in diabetic mice

Our research aims to evaluate the function of the STAMP2 gene, an important trigger in insulin resistance (IR), and explore its role in macrophage apoptosis in diabetic atherosclerotic vulnerable plaques. The characteristics of diabetic mice were measured by serial metabolite and pathology tests. The level of STAMP2 was measured by RT‐PCR and Western blot. The plaque area, lipid and collagen content of brachiocephalic artery plaques were measured by histopathological analyses, and the macrophage apoptosis was measured by TUNEL. Correlation of STAMP2/Akt signaling pathway and macrophage apoptosis was validated by Ad‐STAMP2 transfection and STAMP2 siRNA inhibition. The diabetic mice showed typical features of IR, hyperglycaemia. Overexpression of STAMP2 ameliorated IR and decreased serum glucose level. In brachiocephalic lesions, lipid content, macrophage quantity and the vulnerability index were significantly decreased by overexpression of STAMP2. Moreover, the numbers of apoptotic cells and macrophages in lesions were both significantly decreased. In vitro, both mRNA and protein expressions of STAMP2 were increased under high glucose treatment. P‐Akt was highly expressed and caspase‐3 was decreased after overexpression of STAMP2. However, expression of p‐Akt protein was decreased and caspase‐3 was increased when STAMP2 was inhibited by siRNA. STAMP2 overexpression could exert a protective effect on diabetic atherosclerosis by reducing IR and diminishing macrophage apoptosis.

Pitavastatin calcium improves endothelial function and delays the progress of atherosclerosis in patients with hypercholesterolemia

BackgroundStatins have proven efficacy in inhibiting the onset and progress of atherosclerosis. The effectiveness of pitavastatin in reversing carotid atherosclerosis associated with hypercholesterolemia (HC) is unknown.ObjectivesTo explore the simultaneous effects of pitavastatin calcium on brachial arterial flow-mediated vasodilatation (FMD), carotid intima-media thickness (IMT), and arterial stiffness (β), three surrogate markers of atherosclerosis were studied in HC patients.MethodsA randomized, double-blind trial was performed with 40 HC subjects who fulfilled the inclusion/exclusion criteria. Patients were given pitavastatin calcium 1 mg/d (Group 1) or 2 mg/d (Group 2) for 8 weeks. There were 20 patients in each group, and 30 gender- and age-matched healthy subjects as controls were recruited. FMD of the brachial artery, carotid IMT, and arterial stiffness indicated by β were measured at baseline and at 8 weeks after starting pitavastatin calcium therapy using ultrasound techniques. Biochemical tests were also made on all subjects.ResultsAt baseline, higher total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), reduced FMD, and increased β and IMT were observed in HC patients (P<0.001 for all) compared with controls. After 8 weeks, TC was decreased by 20.59%/27.56% and LDL-C 30.92%/35.64%, respectively, in comparison to baseline groups; the HC groups had reduced β and improved endothelial function over the 8-week follow-up (P<0.05–0.001); nonetheless, no significant alterations of IMT were found (P>0.05). Significant negative interactions between TC/LDL and FMD (P<0.05–0.001), positive interactions between TC and IMT (P=0.003) and between TC/LDL and β (P<0.001–0.000) were found.ConclusionsTreatment with pitavastatin calcium exerted favorable effects on endothelial function and arterial stiffness. It also improved carotid atherosclerosis in patients with HC.概要目的观察匹伐他汀钙对高胆固醇血症患者外周血管的影响。创新点首次在国内发现匹伐他汀钙能够改善高胆固醇血症患者肱动脉和颈动脉血管内皮功能而且延缓其动脉粥样硬化发展, 并首次证实改善内皮功能是匹伐他汀钙延缓其动脉粥样硬化发展的重要原因。方法按照入选排除标准, 选取本院高胆固醇血症患者 (HC), 完成超声心动图检查的40 例。根据剂量不同, 分为两个剂量组: 1 mg 剂量组20 例 (男性5 例, 女性15 例, 平均年龄(55.20±8.35)岁), 2 mg 剂量组20 例 (男性9 例, 女性11 例, 平均年龄(57.56±6.09)岁)。访视结束后完成超声心动图检查的HC 组36 例, 两个剂量组分别有2 人失访。治疗后1 mg 剂量组18 例 (男性3 例, 女性15 例, 平均年龄(56.00±7.85)岁), 2 mg 剂量组18 例 (男性7 例, 女性11 例, 平均年龄(57.79±6.46)岁)。选择本院同期体检中心30 例正常人作为对照 (年龄和性别均与病例组匹配, 男性14 例, 女性16 例, 平均年龄(54.94±6.90)岁)。所有研究对象, 均经隔夜禁食12∼14 小时, 次日清晨抽取空腹肘静脉血, 测定临床生化指标。采用Sequia512 彩色多普勒超声诊断仪, 应用高分辩率外周血管超声技术, 检测HC 治疗前后肱动脉血流介导性舒张功能 (FMD)、颈动脉结构和功能。结论经匹伐他汀钙治疗8 周后, 高胆固醇血症患者血管功能明显改善, 表现为FMD 升高, 僵硬度减小; 颈动脉僵硬度和内中膜厚度 (IMT) 延缓进展与其内皮功能改善密切相关。

Testosterone delays vascular smooth muscle cell senescence and inhibits collagen synthesis via the Gas6/Axl signaling pathway

Testosterone deficiency is associated with a higher incidence of cardiovascular diseases in men. However, its effect on cell senescence, which plays a causal role in vascular aging, remains unclear. Here, we tested the hypothesis that testosterone alleviated vascular smooth muscle cell (VSMC) senescence and collagen synthesis via growth arrest-specific protein 6 (Gas6)/Axl- and Akt/FoxO1a-dependent pathways. Testosterone significantly ameliorated angiotensin II-induced VSMC senescence and collagen overexpression. In addition, testosterone inhibited angiotensin II-induced matrix metalloproteinase-2 (MMP-2) activity, which played a pivotal role in facilitating age-related collagen deposition. Testosterone increased the expression of tissue inhibitor of metalloproteinase-2 but decreased the expression of MMP-2 and membrane type-1 metalloproteinase which contributed to increase MMP-2 activity. The effects on VSMCs senescence and collagen synthesis were mediated by restoration of angiotensin II-induced downregulation of Gas6 and Axl expression and a subsequent reduction of Akt and FoxO1a phosphorylation. The effects of testosterone were reversed by a Gas6 blocker, Axl-Fc, and a specific inhibitor of Axl, R428. Treatment of VSMCs with PI3K inhibitor LY294002 abrogated the downregulating effect of testosterone on MMP-2 activity. Furthermore, when FoxO1a expression was silenced by using a specific siRNA, the inhibitory effect of testosterone on MMP-2 activity was revered as well, that indicated this process was Akt/FoxO1a dependence. Taken together, Gas6/Axl and Akt/FoxO1a were involved in protective effects of testosterone on VSMCs senescence and collagen synthesis. Our results provide a novel mechanism underlying the protective effect of testosterone on vascular aging and may serve as a theoretical basis for testosterone replacement therapy.