Abstract:Aim To investigate the effect of overexpression of Bax inhibitor-1(BI-1) gene in cardiomyocytes on ischemia-reperfusion injury and its mechanism. Methods Thirty SD rats were randomly divided into sham operation group (sham group), ischemia-reperfusion group (I/R group), adenovirus control group (Ad-EGFP group), overexpression of BI-1 genome group (Ad-BI-1 group), cyclosporine A group (CsA group). After the establishment of myocardial ischemia-reperfusion injury(MIRI)model in rats, TTC staining was used to observe myocardial infarction area, TUNEL staining was used to observe myocardial apoptosis, ultrastructural changes of myocardial cells were observed under electron microscope; qRT-PCR was used to detect the expression of BI-1 mRNA in the myocardium of rats in each group. The myocardial cells of neonatal rats were isolated and cultured and divided into control group, hypoxia/reoxygenation group (H/R group), adenovirus control group (Ad-EGFP group), overexpression of BI-1 genome group (Ad-BI-1 group) and cyclosporine A group (CsA group) according to different cell treatment. The subcellular localization of BI-1 was detected by immunocytochemistry, the expression of BI-1 protein was detected by Western blot, and the MPTP opening level of myocardial mitochondria was detected by Calcein-AM; and the expression of apoptosis related proteins Bcl-2, Bax, CytC, Caspase-3 and Caspase-9 were detected by Western blot. Results Compared with sham group, myocardial infarct area, apoptosis number, mitochondrial structure damage in I/R group, Ad-EGFP group, Ad-BI-1 group and CSA group increased significantly (P＜0.05), while those in Ad-BI-1 group and CsA group decreased significantly (P＜0.05) compared with I/R group. qRT-PCR results showed that compared with sham group, BI-1 mRNA expression decreased significantly in I/R group, Ad-EGFP group and CsA group, while it increased significantly in Ad-BI-1 group; Subcellular location showed that BI-1 was mainly located in the endoplasmic reticulum of cardiomyocytes, and the fluorescence intensity of Calcein-AM was significantly lower in H/R group, Ad-EGFP group, Ad-BI-1 and CsA group than that in control group, while that of Ad-BI-1 and CsA group was significantly higher than that of H/R group; Compared with control group, the expression level of BI-1 protein was significantly decreased in H/R group, Ad-EGFP group and CsA group, but it was significantly increased in Ad-BI-1 group. Western blot results showed that compared with sham group, the ratio of Bcl-2/Bax decreased significantly in I/R group, Ad-EGFP group, Ad-BI-1 group and CsA group (P＜0.05), the expression of Caspase-3 and Caspase-9 increased significantly (P＜0.05); while the ratio of Bcl-2/Bax increased significantly in Ad-BI-1 group and CsA group compared with I/R group (P＜0.05), and the expression of Caspase-3 and Caspase-9 decreased significantly (P＜0.05). The expression of CytC was significantly higher in I/R group, Ad-EGFP group, Ad-BI-1 group and CsA group than that of sham group (P＜0.05). However, the expression of CytC in the mitochondria of myocardial cells was significantly lower in I/R group, Ad-EGFP group, Ad-BI-1 group and CsA group than that of sham group (P＜0.05), and the expression of CytC was significantly higher in the Ad-BI-1 group and the CsA group than that of I/R group (P＜0.05). Conclusion Overexpression of BI-1 gene can reduce the myocardial infarct area of MIRI rats, reduce the apoptosis of myocardial cells and improve the mitochondrial structure and function damage. The mechanism may be that overexpression of BI-1 can play the above role by changing the Bcl-2/Bax ratio, inhibiting the opening of MPTP, reducing the release of CytC, and reducing the activation of Caspase-3 and Caspase-9.

Abstract:Aim To investigate the epigenetic regulation mechanism of oxidized lipoprotein(a)[ox-Lp(a)] injury on vascular endothelial cells. Methods Bioinformatics and luciferase reporter gene were used to screen candidate microRNA binding to 0,1- translocation enzyme 2 (TET2) mRNA 3’-UTR and verify their targeted binding tendency. 0 mg/L, 25 mg/L, 50 mg/L and 100 mg/L of ox-Lp(a) were incubated with HUVEC-12 vascular endothelial cell line for 24 h, or incubated with HUVEC-12 vascular endothelial cell line with 100 mg/L ox-Lp(a) for 0 h, 6 h, 12 h, 24 h, 48 h respectively. qRT-PCR and Western blot were used to detect TET2 mRNA and protein expression levels. The expression of hsa-miR-125a-5p was detected by qRT-PCR. The change of TET2 activity was analyzed by detecting 5hmc level. Transwell was used to detect the permeability of monoclonal vascular endothelial cells. Results Bioinformatic analysis and luciferase reporter assay showed that TET2 was the target gene of hsa-miR-125a-5p, and the binding energy of hsa-miR-125a-5p to the 3’-UTR of TET2 mRNA was low (－30.1 kcal/mol). The activity of TET2 protein and mRNA was inhibited by ox-Lp(a) in the dose and time-dependent manner. The best reaction dose and time of ox-Lp(a) was 100 mg/L and 24 h. The activity of TET2 was down-regulated by 100 mg/L ox-Lp(a), while the expression of hsa-miR-125a-5p was significantly up-regulated, and anti-hsa-miR-125a-5p could reverse it. Ox-Lp(a) significantly increased the permeability of monolayer endothelial cells, but could be partially reversed by anti-hsa-miR-125a-5p. Conclusion Ox-Lp(a) inhibited the expression and activity of TET2 and increased the permeability of monolayer vascular endothelial cells by up-regulating hsa-miR-125a-5p expression which targeted binding to 3’-UTR of TET2 mRNA.

Abstract:Vascular endothelial dysfunction plays an important role in the pathogenesis of atherosclerosis. As a protective barrier of vascular endothelium, glycocalyx plays a series of protection role, including regulation of vascular permeability, mediating the release of nitric oxide (NO) induced by shear stress, inhibition of the adhesion between leukocytes, platelets and endothelial cells, anticoagulation. This article reviews the important meaning of glycocalyx’s integrity and its endothelial protection, and its relationship with atherosclerosis.

Abstract:Aim To determine whether advanced glycation end products(AGE)altered the morphology and distribution of the adherens junction protein vascular endothelial(VE)-cadherin in human umbilical vein endothelial cells(hUVEC)and to understand the mechanism of this alteration.Methods Human umbilical vein endothelial cells(hUVEC)were respectively incubated with AGE-HSA in different concentrations and timing.In some other cases,hUVECs were pretreated with mitogen activated protein kinase(MAPK)pathway inhibitors(PD98059,SP600125,SB203580),or transfected with adenovirus.To visualize the morphological changes of VE-cadherin,cells were incubated with rabbit anti-VE-cadherin primary antibody and then FITC anti-rabbit IgG secondary antibody.The morphological changes of VE-cadherin were observed with confocal microscope.Results The morphological of VE-cadherin was significantly changed in coincident with an increase of the dose and time of AGE-HSA.These changes could be inhibited with MAPK and Rho inhibitors.Conclusion These observations suggested that AGE modified proteins can induce morphological changes of VE-cadherin in endothelial cell.Activations of MAP kinase and Rho kinase pathways play an important role in AGEs-induced hUVECs VE-cadherin distribution.

Abstract:Aim To explore whether paraoxon,an active component of organophosphate,can directly injure vascular endothelial cells in vitro and to explore the potential mechanisms. Method The thorac aortic rings of healthy sprague-dawley rats and cultured human umbilical vein endothelial cells(hUVEC) were exposed to medium contained different concentrations(36.3 nmol/L～36.3 μmol/L) of paraoxon and co-incubation different time.Both endothelial-dependent and non-dependent relaxation of aortic rings in rats and endothelial monolayer permeability in acetylcholine(Ach)-induced endothelium dependent relaxation(EDR) and increased hUVEC was assayed. Results Paraoxon concentration and time-dependently inhibited permeability of the endothelial monolayer in hUVEC.Paraoxon also simultaneously resulted in a reduction of both superoxide dismutase(SOD) activity and nitric oxide(NO) content and an increase of malondialdehyde(MDA) content in both aortic tissues and cultured cellular medium.But sodium nitroprusside-induced endothelium-independent relaxation of aortic rings were not affected by paraoxon.The injurious effects of paraoxon to EDR was partly lessened by added L-arginine,but not done by pretreatment of atropine. Conclusion Paraoxon could directly injure vascular endothelium cells and EDR.The mechanisms of endothelial dysfunction induced by paraoxon may relate to trigger oxidative stress and formation of lipid peroxidation by oxidation-stress,and the increase of endothelial cell monolayer permeability.

Abstract:Aim To investigate the relationship between aquaporin-4(AQP4)mRNA expression and blood-brain barrier(BBB) permeability after experimental cerebral hemorrhage in rats. Methods The cerebral hemorrhage models were established by stereotaxic injection of auto-non-anticoagulant artery blood into the caudate nucleus.AQP4 mRNA expression was detected by RT-PCR,BBB permeability by Evens Blue method and brain water contents by dry-wet weight method. Results In contrast to the control group,AQP4 mRNA expression increased in cerebral hemorrhage group at 6 hours(1.06±0.12),reached peak at 3 days(1.34±0.14),then was still higher than normal at 7 days(p<0.05);BBB permeability increased at 6 hours after cerebral hemorrhage(0.5955±0.0956),reached peak at 1～3 days(0.8889±0.0968,0.7914±0.0520),and decreased at 5～7 days gradually(p<0.05).There was a significantly positive correlation between AQP4 mRNA expression and BBB permeability(r=0.686,p<0.01),which was consistent with the change of cerebral edema. Conclusion Cerebral edema may be formed through the upregulation of aquaporin-4 mRNA expression and the increase of BBB permeability after cerebral hemorrhage.

Abstract:Aim To investigate the effect of advanced glycation end products modified human serum albumin(AGE-HSA) on morphological changes of tight junction associated protein ZO-1 in endothelial cell and the mechanism in this pathological procedure. Methods Human umbilical vein endothelial cell(hUVEC)-derived cell line(ECV304) were incubated with AGE-HSA in different concentrations and timing. To visualize the morphological changes of tight junction protein ZO-1,the treated cell were incubated with mouse anti-ZO-1 primary antibody and then FITCanti-mouse IgG secondary antibody.The morphological changes of ZO-1 were observed with confocal microscope.The cell were pre-administrated with PD98059,a specific inhibitor of MEK1(ERK upstream kinase)or SB203580,a specific inhibitor of p38 MAP kinase,respectively,before exposed to AGE-HSA,then the cell were rinsed with DMEM for three times and exposed to AGE-HSA. The cell were transfected with dominant negative MEK1 or MEK6b(p38 upstream kinase) mutant adenoviruse,then exposed to AGE-HSA.And the cell were transfected with constitutively active MEK1 or MEK6b mutant adenoviruse. Results In normal control group,ZO-1 staining appeared as a continuous and smooth line along the regions of cell cell contact.Under the stimulation of AGE HSA,morphology of ZO-1 in endothelial cell were changed greatly in a concentration and time-dependent manner.The changes were partially blocked by PD98059 and SB203580.The transfection of dominant negative MEK1 and MEK6b mutant adenoviruse had the similar effects.The transfection of constitutively active MEK1 and MEK6b disrupted the structure of ZO-1. Conclusion AGE modified proteins can induce morphological changes of ZO-1 in endothelial cell.Activations of ERK and p38 MAP kinase pathways play an important role in this procedure.