Effects of indoxyl sulfate on adherens junctions of endothelial cells and the underlying signaling mechanism

Uremic patients have a much higher risk of cardiovascular diseases and death. Uremic toxins are probably involved in the development of vascular endothelial dysfunction. Indoxyl sulfate (IS) is a uremic toxin that accumulates with deterioration of renal function. This study explored the effects of IS on the adherens junctions of vascular endothelial cells and revealed the underlying mechanism. Bovine pulmonary artery endothelial cells (BPAECs) were treated with IS, and the distribution of vascular endothelial cadherin (VE-cadherin), p120-catenin, β-catenin, and stress fibers was examined by immunofluorescence. IS treatment resulted in disruption of intercellular contacts between BPAECs with prominent parallel-oriented intracellular stress fiber formation. Intracellular free radical levels which measured by flow cytometry increased after IS treatment. The antioxidant, MnTMPyP, and an ERK pathway inhibitor, U0126, both significantly prevented IS-induced disruption of intercellular contacts. Western blotting analyses demonstrated that IS-induced phosphorylation of myosin light chain kinase (MLCK) and myosin light chains (MLC) as well as activation of extracellular-signal-regulated protein kinase (ERK1/ERK2). Pretreatment with MnTMPyP prevented ERK1/2 phosphorylation. U0126 prevented the IS-induced MLCK and MLC phosphorylation. MEK-ERK acted as the upstream regulator of the MLCK-MLC pathway. These findings suggest that the superoxide anion-MEK-ERK-MLCK-MLC signaling mediates IS-induced junctional dispersal of BPAECs.     

3,4‐dimethoxystilbene,a resveratrol derivative with anti‐angiogenic effect,induces both macroautophagy and apoptosis in endothelial cells

Angiogenesis plays an important role in many pathological processes. Identification of novel anti‐angiogenic agents will provide new insights into the mechanisms for angiogenesis as well as potential lead compounds for developing new drugs. In the present study, a series of resveratrol methylated derivatives have been synthesized and screened. We found trans‐3,4‐dimethoxystilbene (3,4‐DMS) with the fullest potential to develop as an anti‐angiogenic agent. In vitro and in vivo analyses suggested that 3,4‐DMS could effectively inhibit endothelial cell proliferation, migration, tube formation, and endogenous neovascularization. Our results showed that 3,4‐DMS exerted its anti‐angiogenic effect likely through induction of endothelial cell apoptosis via a pathway involving p53, Bax, cytochrome c, and caspase proteases. Moreover, 3,4‐DMS also induced macroautophagy in endothelial cells through activation of AMPK and the downstream inhibition of mTOR signaling pathway. Further studies indicated that intracellular calcium ([Ca²⁺]_i) might bridge the 3,4‐DMS‐induced apoptosis and macroautophagy through modulating reactive oxygen species (ROS) levels in endothelial cells. Combination of 3,4‐DMS with inhibitor of autophagy, such as 3‐methyladenine (3‐MA) and autophagy‐related gene (ATG) 5 small interfering RNA (siRNA), potentiated the pro‐apoptotic and anti‐angiogenic effects of 3,4‐DMS. Our study provides a novel angiogenic inhibitor and a useful tool in exploring the molecular mechanisms for the crosstalk between apoptosis and macroautophagy in endothelial cells. 3,4‐DMS could be served as a potential lead compound for developing a class of new drugs targeting angiogenesis‐related diseases. J. Cell. Biochem. 114: 697–707, 2013. © 2012 Wiley Periodicals, Inc.     

The inhibitory effect of alacepril,an angiotensin-converting enzyme inhibitor,on endothelial inflammatory response induced by oxysterol and TNF-α

Abstract

The objectives were to determine the effects of alacepril, an angiotensin-converting enzyme inhibitor, on the expression of adhesion molecules and monocyte adherence to endothelial cells induced by 7-ketocholesterol (7-KC) and tumor necrosis factor (TNF)-α. We used human aortic endothelial cells (HAECs) and U937 monocytic cells. Surface expression and mRNA levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were determined by EIA and RT-PCR. Adherence of U937 to HAECs was assessed by adhesion assay. Incubation of HAEC with 7-KC increased the surface expression of protein and mRNA levels of ICAM-1 and VCAM-1 on HAECs and the production of reactive oxygen species (ROS) in HAECs. Pretreatment with alacepril reduced the enhanced expression of these molecules in a dose-dependent manner. The inhibitory effect of alacepril against 7-KC or TNF-α-induced CAMs expression was stronger than that of captopril or enalapril. Alacepril inhibited the production of ROS in HAECs stimulated by 7-KC or TNF-α. These results suggest that alacepril works as anti-atherogenic agent through inhibiting endothelial-dependent adhesive interactions with monocytes induced by 7-KC and TNF-α.     

Actin realignment and cofilin regulation are essential for barrier integrity during shear stress

Vascular endothelial cells and their actin microfilaments align in the direction of fluid shear stress (FSS) in vitro and in vivo. To determine whether cofilin, an actin severing protein, is required in this process, the levels of phospho‐cofilin (serine‐3) were evaluated in cells exposed to FSS. Phospho‐cofilin levels decreased in the cytoplasm and increased in the nucleus during FSS exposure. This was accompanied by increased nuclear staining for activated LIMK, a cofilin kinase. Blocking stress kinases JNK and p38, known to play roles in actin realignment during FSS, decreased cofilin phosphorylation under static conditions, and JNK inhibition also resulted in decreased phospho‐cofilin during FSS exposure. Inhibition of dynamic changes in cofilin phosphorylation through cofilin mutants decreased correct actin realignment. The mutants also decreased barrier integrity as did inhibition of the stress kinases. These results identify the importance of cofilin in the process of actin alignment and the requirement for actin realignment in endothelial barrier integrity during FSS. J. Cell. Biochem. 114: 782–795, 2013. © 2012 Wiley Periodicals, Inc.     

Phytoestrogen calycosin-7-O-β-D-glucopyranoside ameliorates advanced glycation end products-induced HUVEC damage

Vasculopathy including endothelial cell (EC) apoptosis and inflammation contributes to the high incidence of stroke and myocardial infarction in diabetic patients. The aim of the present study was to investigate the effect of calycosin-7-O-β-D-glucopyranoside (CG), a phytoestrogen, on advanced glycation end products (AGEs)-induced HUVEC damage. We observed that CG can significantly ameliorate AGEs-induced HUVEC oxidative stress and apoptosis. The ratio of SOD/MDA was significantly increased to the normal level by CG pretreatment. CG preincubation dramatically increased anti-apoptotic Bcl-2 while decreased pro-apoptotic Bax and Bad expressions as detected by immunocytochemistry. Moreover, CG ameliorated macrophage migration and adhesion to HUVEC; the monocyte chemotactic protein-1 and interleukin-6 levels in the culture supernatant were dramatically reduced by CG as determined by ELISA; the expressions of inflammatory proteins including ICAM-1, TGF-β1, and RAGE in both protein and mRNA levels were significantly reduced to the normal level by CG pretreatment as determined by immunocytochemistry and real-time RT-PCR. The intracellular investigation suggests that CG can reverse AGEs-activated ERK1/2 and NF-κB phosphorylation, in which estrogen receptors were involved in. Our results strongly indicate that CG can modulate EC dysfunction by ameliorating AGEs-induced cell apoptosis and inflammation.     

Type 1 IFN inhibits the growth factor deprived apoptosis of cultured human aortic endothelial cells and protects the cells from chemically induced oxidative cytotoxicity

It has been shown that the genesis of atherosclerotic lesions is resulted from the injury of vascular endothelial cells and the cell damage is triggered by oxygen radicals generated from various tissues. Human vascular endothelial cells can survive and proliferate depending on growth factors such as VEGF or basic FGF and are induced apoptosis by the deprivation of growth factor or serum. It was found that type 1 IFN inhibits the growth factor deprived cell death of human aortic endothelial cells (HAEC) and protects the cells from chemically induced oxidative cytotoxicity. The anti‐apoptotic effects of type 1 IFN were certified by flow cytometry using annexin‐V‐FITC/PI double staining and cell cycle analysis, fluorescence microscopy using Hoechst33342 and PI, colorimetric assay for caspase‐3 activity, p53 and bax mRNA expressions, and cell counts. It was considered that IFN‐β inhibits the executive late stage apoptosis from the results of annexin‐V‐FITC/PI double staining and the inhibition of caspase‐3 activity, and that the anti‐apoptotic effect might be owing to the direct inhibition of the apoptotic pathway mediated by p53 from the transient down‐regulation of bax mRNA expression. Whereas, type 1 IFN protected the cells from the oxidative cytotoxicity induced by tertiary butylhydroperoxide (TBH) under the presence of Ca²⁺. The effects of IFN‐β is more potent inhibitor of cell death than IFN‐α. These results indicate that type 1 IFN, especially IFN‐β may be useful for the diseases with vascular endothelium damage such as atherosclerosis or restenosis after angioplasty as a medical treatment or a prophylactic. J. Cell. Biochem. 113: 3823–3834, 2012. © 2012 Wiley Periodicals, Inc.