Inhibitory effect of all-trans retinoic acid on proliferation of microvascular endothelial cells

AIM: To evaluate the effects of all-trans retinoic acid (atRA) on the proliferation in cultured mouse cerebral microvascular endothelial cells (bEnd.3). METHODS: Cultured cells were divided into five groups randomly, one as control group, the other four groups were 10-9, 10-8, 10-7 and 10-6 mol/L group. Effects of atRA on proliferation in bEnd.3 cells were detected by flow cytometry and immunocytochemitry of PCNA and MTT at 24 h, 48 h and 72 h. The effects of atRA (10-6 mol/L group) on the expressions of angiogenic genes in bEnd.3 cells were studied using microarray. RESULTS: The results of MTT and flow cytometry showed that all-trans retinoic acid at concentration of 10-6 mol/L significantly inhibited the proliferation of bEnd.3 cells. Immunocytochemical staining showed the expression of PCNA was markedly decreased in bEnd.3 cells at 24 h after treatment with atRA. Microarray results demonstrated that there were 11 down-regulated angiogenic genes and 2 up-regulated angiogenic genes in 10-6mol/L atRA group. CONCLUSION: All-trans retinoic acid at concentration of 10-6mol/L may significantly inhibit the proliferation of bEnd.3 cells treated for 24 h in vitro via down-regulation of angiogenic genes and PCNA expression.     

Study on the culture of rat myocardium microvascular endothelial cells and microarray analysis

AIM: To study the cytological characteristics of rat myocardium microvascular endothelial cells (RMMVEC) by microarray. METHODS: The RMMVEC were cultured by the method of planting myocardium tissue. The morphology of RMMVEC was studied by light and electronic microscopy. Its molecular markers were observed by immunocytochemistry. Cell proliferation kinetic was analyzed by counting the number of cells. The gene expression of the RMMVEC was studied by endothelial cell biology gene microarray and compared the change of gene expression among the cultured cells of primary, 2nd and 5th passage. RESULTS: The RMMVEC showed morphological characteristics of microvascular endothelial cells (MVEC): growing in a cobblestone pattern, forming tube-like structure or capillary network and having microvilli on cell surface. At the same time, the RMMVEC showed positive staining for vWF, CD34, CD31, CD105 and Tie-2. Gene microarray analysis indicated expression of VEGFR, ICAM-1, VCAM-1, angiopoietin1, PECAM1 (CD31) and other genes closely related to microvascular endothelial functions at relatively high level. But in cultured cells of 5th passage the characteristic gene expression of microvascular endothelial cells disappeared. CONCLUSION: The RMMVEC cultured by this method possess typical characteristics of MVEC. The cytological characteristics are steady in the cultured cells of primary and 2nd passage. It can be utilized to study the mechanisms of some cardiovascular diseases.     

Effects of adrenomedullin on proliferation of vascular smooth muscle cells induced by urotensin Ⅱ

AIM:To observe the effect of adrenomedullin(ADM)on proliferation of vascular smooth muscle cells(VSMC) induced by urotensin Ⅱ(UⅡ). METHODS:DNA synthesis of cultured rat aortic VSMC was measured by [³H]-TdR incorporation. The activities of mitogen activated protein kinase(MAPK) were determined by isotope tagged with [γ-³²P]-ATP. RESULTS:UⅡ(10^-8mol/L) significantly increased [³H]-TdR incorporation of VSMC and MAPK activities by 38%(P＜0.05) and 260%(P＜0.01) respectively compared with control group. Compared with UⅡ group, 10^-10,10^-9,10^-8mol/L ADM decreased [³H]-TdR incorporation of VSMC by 7%(P＞0.05), 32%(P＜0.05)and 41%(P＜0.01),respectively, and diminished MAPK activities by 24%(P＞0.05), 32%(P＜0.05)and 36%(P＜0.05),respectively. CONCLUSION:ADM inhibits proliferation of VSMC induced by urotensin Ⅱ through inhibiting MAPK activation.     

Dual-direction effect of crenulatin on apoptosis of cerebral microvascular endothelial cells and it's mechanism

) ［ABSTRACT］AIM: To study the effect and the mechanism of crenulatin, an effective constituent of Chinese traditional medicine, on apoptosis of cerebral microvascular endothelial cells. METHODS: The following terminal concentrations of crenulatin were used in the study: 25 mg/L and 100 mg/L. Apoptosis of mouse cerebral microvascular endothelial cells (bEnd.3 cell line) was evaluated by flow cytometer, immunocytochemical assay (Fas, Bcl-2) and Western blotting (caspase-3) after culture for 24 h. RESULTS: Compared with control group, apoptosis of bEnd.3 cells in 25 mg/L group was significantly inhibited (P<0.05), but apoptosis in the 100 mg/L group was significantly increased (P<0.05). In apoptosis inhibited group, the Fas immunocytochemical staining was weaker, the positive cells were significantly decreased (P<0.05) and caspase-3 expression was decreased compared with control group; however, the Bcl-2 staining was stronger and the positive cells were significantly increased (P<0.05). On the other hand, in apoptosis increased group (100 mg/L group), the changes were just opposite. CONCLUSIONS: The effect of crenulatin on apoptosis of mouse cerebral microvascular endothelial cells possesses a dual-direction change, inhibitive effect in 25 mg/L and stimulative effect in 100 mg/L group, respectively. The mechanism is related to the alterations of Fas/Bcl-2 expression and caspase-3 activity.     

Effects of hypoxia on endothelial nitric oxide synthase expression in cerebral artery endothelial cells

AIM: To investigate the molecular mechanism by which hypoxia affect the endothelial nitric oxide synthase (eNOS) expression in cerebral artery endothelial cells (CAECs). METHODS: Primary cultured porcine CAECs were exposed to hypoxia for 2 h, 6 h, 12 h, 24 h and 48 h. The eNOS mRNA level was determined by RT-PCR. The level of eNOS protein was detected by Western blotting. After specific PKC inhibitors BIM Ⅰ(1 μmol/L) and G6983 (1 μmol/L) were added, CAECs were exposed to hypoxia for 24 h. The effect of hypoxia on eNOS mRNA stability was analyzed after actinomycin D was added. RESULTS: After exposure to hypoxia for 2 h, the levels of eNOS mRNA and protein in CAECs were increased. The levels of eNOS mRNA and protein reached peak after 12 h of hypoxia (about 2.5 fold and 2.0 fold, respectively, compared to control), and remained at higher level even after 48 h of hypoxia. Moreover, hypoxia did not change the stability of eNOS mRNA. The specific PKC inhibitors BIM Ⅰ and G6983 attenuated significantly the effects of hypoxia on eNOS gene expression. CONCLUSION: These results suggest that hypoxia may enhance the expression of eNOS gene in CAECs through PKC signaling pathway, which might be one of the mechanisms of cerebral artery dilation and neuroprotection during cerebral hypoxia.     

Effects of high-glucose on proliferation and apoptosis in endothelial progenitor cells of type 2 diabetes mellitus

AIM: This study aimed to observe the effects of high-glucose on proliferation and apoptosis of endothelial progenitor cells (EPCs) in type 2 diabetes mellitus patients,and tried to elucidate their possible role.METHODS: Various concentrations of glucose were added to the culture system of EPCs from 25 cases of type 2 diabetes mellitus patients (DM group) and 25 cases of healthy volunteers (control group).MTT assays were used to detect the proliferative rates.Annexin-V/PI stains were used to detect the apoptotic rates,and RT-PCR to detect the expression level of bcl-2 and bax.RESULTS: Proliferative activity of EPCs in both control group and DM group were attenuated when concentration of glucose was 33 mmol/L,while apoptotic rates increased.No significant change of proliferative rate and apoptotic rate of EPCs in DM group and control group in the presence of 5 mmol/L glucose was observed.The expression level of bax of EPCs in both DM group and control group increased while expression level of bcl-2 did not change much in the presence of 33 mmol/L glucose.CONCLUSION: High-glucose attenuates proliferative activity of EPCs and increases the apoptotic rate.Upregulation of bax may be its possible role.     

Effects of high glucose on proliferation,migration, adhesion and secretion of rat late endothelial progenitor cells

AIM: To investigate the effects of high glucose on the proliferation, adhesion, migration and secretion potentials of late endothelial progenitor cells (EPCs) from bone marrow. METHODS: Mononuclear cells were collected from rat bone marrow by density gradient centrifugation and cultured with M199 medium. The early EPCs were identified by DiI-ac-LDL and FITC-UEA-1 double staining. The late EPCs were identified by RT-PCR to detect the expression of von Willebrand factor (vWF) and VE-cadherin. Moreover, the cells were identified by FACS to detect the expression of CD133 and vascular endothelial growth factor receptor-2(VEGFR-2). The 3rd generation of EPCs was harvested and incubated with glucose in a series of concentrations (5, 10, 20 or 40 mmol/L). The cell proliferation, adhesion, migration and the secretion of chemokines such as monocyte chemoattractant protein-1(MCP-1) and interleukin-8 (IL-8) were assayed with MTT, adhesion test, modified Boyden chamber assay and ELISA, respectively. RESULTS: Compared with normal glucose (5 mmol/L)treatment, high glucose (10, 20, 40 mmol/L) dose-dependently degraded the proliferation and migration of late EPCs (P<0.05 or P<0.01). At the same time, treatment with glucose at the concentration of 40 mmol/L decreased the adhesion of EPCs (P<0.05) and increased the release of MCP-1 and IL-8 by late EPCs. CONCLUSION: High glucose inhibits proliferation, adhesion and migration of late EPCs, and enhances the secretion of inflammatory factors, indicating that the high glucose correlates with the vascular complications of patients with diabetes.     

Hypoxic preconditioning protects rat myocardial microvascular endothelial cells against endoplasmic reticulum stress-induced injury

AIM：To investigate the effect of hypoxic preconditioning (HPC) on endoplasmic reticulum stress (ERS)-induced injury in cultured microvascular endothelial cells (MVECs) from rat hearts. METHODS：MVECs injury was induced by an ERS inductor thapsigargin (TG). Lactate dehydrogenase (LDH) leakage and apoptotic rate were detected to evaluate the injury of MVECs. Cytoskeleton and endoplasmic reticulum (ER) in MVECs were observed by phalloidin-FITC fluorescence staining and ER staining, respectively. Two-dimensional electrophoresis and mass spectrometry (MS) were used to identify proteomic profile in MVECs treated with TG. Western blotting was used to detect the expression of ERS markers, calreticulin (CRT) and glucose-regulated protein 78 (GRP78). RESULTS：TG induced the increase in LDH activity in medium and the apoptosis of MVECs in a dose-dependent manner. TG treatment up-regulated the expression of CRT and GRP78, while HPC attenuated the ERS-induced injury and the up-regulation of ERS markers in MVECs. CONCLUSION：HPC protects MVECs from ERS-induced injury.     

Study on COX activity and its mRNA expression,and PGE2 release from cerebral microvascular endothelial cells stimulated by IL-1β

AIM: To study the cyclooxygenase(COX) activity and its mRNA expression, and PGE₂ release from rats cerebral microvascular endothelial cells (rCEMC) stimulated by IL-1β(30 μg/L) at different times. METHODS: rCMEC were cultured, and identified by immunohistochemistry for von Willebrand factor (Ⅷ factor, a marker for all endothelial cells) in cytoplasm of the cells. After rCEMC grew to confluency, they were stimulated with IL-1β for 0.5, 1, 2, 4, 8, 12 and 24 h, respectively. Activity of COX-1 and COX-2 in rCEMC and production of PGE₂ in the conditioned media were detected by ELISA. COX-1 and COX-2 mRNA expressions were measured by real-time quantity PCR. The amplification product was tested by melting curve and identified by electrophoretic gel. RESULTS: ① Positive immunostaining for Ⅷ factor was present diffusely in the cytoplasm in more than 90% rCMEC. ② Compared to the cells without IL-1β stimulation, the production of PGE₂ increased significantly (P<0.05) at 4 h after rCEMC were incubated with IL-1β and reached the top level at 12 h (P<0.01), then declined thereafter at 24 h (P<0.05). ③ There was no significant difference on COX-1 activity between IL-1β group and non-IL-1β group. COX-2 activity increased significantly compared with those in non-IL-1β (P<0.05) at 8 h after rCEMC were incubated with IL-1β and reached the top level at 12 h (P<0.01), then declined thereafter at 24 h (P<0.05). ④ There was no significant difference on COX-1 mRNA expression between IL-1β group and non-IL-1β group. COX-2 mRNA was induced and became detectable at 1 h, and reached the top level at 4 h, then declined thereafter at 8 h and became undetectable by 12 h and 24 h after incubation with IL-1β. The melting curve showed there was no nonspecific amplification and electrophoretic gel showed the lengths of amplification products accorded with the predicted lengths. CONCLUSION: While rCEMC are stimulated by IL-1β, the excretion of PGE₂ increases and reaches the top level at 12 h, which is related with its induction on COX-2 mRNA expression and COX-2 activity.     

Effects of rat tail collagen and C-erbB-2 antibody on the adhesion and proliferation in hepatocellular carcinoma cells

AIM: To explore the effect of collagen and C-erbB-2 protein on the adhesion and the proliferation in hepatocellular carcinoma cells. METHODS: Hepatocellular carcinoma cell line (HepG-2) identified to positive for C-erbB-2 gene was used to study the adhesion and the growth feature by the action of rat tail collagen and C-erbB-2 antibody.RESULTS: The action of rat tail collagen to potentiated the adhesion in HepG-2 cells was significantly but no proliferation effect was observed. C-erbB-2 antibody inhibited the adhesion and proliferation of HepG-2 cells and also abolished the potentiated effect of rat tail collagen on the adhesion in HepG- 2 cells. CONCLUSION: The signaling transduction mediated by C-erbB-2 protein was correlated to the adhesion and the proliferation of HepG-2. The blockage of C-erbB-2 gene signal transduction may be a strategic target to the treatment of liver cancer in the future.     

Effects and possible mechanism of recombinant human erythropoietin on proliferation of endothelial progenitor cells

AIM: To observe the effects of recombinant human erythropoietin on proliferation of endothelial progenitor cells (EPCs) from healthy volunteers and patients with renal failure,and tried to elucidate the possible mechanism.METHODS: Various concentrations of rhEPO were added to the culture system of EPCs from 15 cases of patients with renal failure (RF group) and 15 cases of healthy volunteers (control group).MTT assays were used to detect proliferative rates.Annexin-V/PI stains were used to measure the apoptotic rates.Western blotting was used to determine the expression of Akt protein kinase.RESULTS: Numbers and proliferative ability of EPCs from control group and RF group were improved in dose-dependent manner when concentrations of rhEPO were 100 U/L,600 U/L and 1 200 U/L.However,compared to the control group,numbers and function of EPCs from RF group were remarkably decreased.The apoptosis rate of EPCs was decreased and the activity of Akt protein kinase was improved in the presence of 1 200 U/L rhEPO.Wortmannin was able to block the effects.CONCLUSION: rhEPO improves the number and function of EPCs from both healthy volunteer and patients with renal failure.PI3K/Akt might play an important role in it.     

Effect of CoQ10 on apoptosis and proliferation of cultured mouse microvascular endothelial cells and it's mechanism

AIM: To study the inhibitory effect of CoQ₁₀ on the apoptosis of microvascular endothelial cells and it's probable mechanism. METHODS: Using serum pharmacology method and cytoflowmetery, the effects of CoQ₁₀ at different concentrations on apoptosis and proliferation in cultured mouse brain microvascular endothelial cells (bEnd.3) were investigated. The expression of Fas protein and Bcl-2 protein were observed with immunocytochemical method (ABC). RESULTS: The cell apoptosis was inhibited significantly in CoQ₁₀ groups (50 μL and 25 μL) in cultured bEnd.3 cells. The results of immunocytochemical staining showed that the expressions of Fas protein was inhibited and Bcl-2 protein was stimulated significantly in CoQ10 group with above concentration. But there was no significant change in cell proliferation. CONCLUSIONS: CoQ₁₀ may inhibit apoptosis of microvascular endothelial cells (bEnd.3) via up-regulation of Bcl-2 and down-regulation of Fas. Authors suggest that this is one of the protection mechanisms of CoQ₁₀ from dysfunction of microvascular endothelial cells.     

Effects of Norrin gene on the proliferation and cell cycle of human retinal capillary endothelial cells

AIM: To isolate, cultivate and identify human retinal capillary endothelial cells (HRCECs), and to assess the effects of high expression of Norrin gene on the proliferation and cell cycle of HRCECs. METHODS: The cultured cells were identified with anti-factor VIII related antigen. AP-3myc-hNorrin/pRK5 were transfected into cultured HRCECs in vitro by lipofectamine 2000. Their transfection efficiency were measured by RT-PCR, immunohistochemistry and Western blotting，respectively. Its effects on cell proliferation and cell cycle were detected. RESULTS: The cultured cells were identified with immunochemically positive brown staining. In comparison with those of the controls, the Norrin expression in experimental group was significantly increased on mRNA and protein levels (showed by the myc tag) after 48 h. The cell number of experimental group was larger than that in the control group with statistically significant differences. Flow cytometry showed the cells in G2 phase were mainly increased (P<0.01). CONCLUSION: The plasmid AP-3myc-hNorrin/pRK5 is successfully transfected into HRCECs by lipofectamine 2000. Norrin gene improves the proliferation ability of HRCECs by promoting the synthesis of DNA. Norrin may have an important role in the retinal angiogenesis, which may provide a new gene target in the treatment of retinal vascular disorders.     

Effects of peroxynitrite on the endothelial cells

Endothelial cells produce both superoxide and nitric oxide. Nitric oxide and superoxide are known to react rapi dly to formthe stable peroxynitrite anion. Peroxynitrite mediates the oxidation of protein, lipid, deoxyribose and inhibits mitochondrial electron transport. Peroxynitrite may break DNA strands and activate poly(ADP-ribose) synthetase. If the reactionis excessive, it results in a depletion of intracellular NAD⁺ and ATP. There is ultimately cell death.     

The expression of NF-κB and ICAM-1 in rat brain of experimental intracerebral hemorrhage and cerebral microvascular endothelial cells injured by hydrogen peroxide

AIM: To discuss the possible mechanism of the inflammation after intracerebral hemorrhage (ICH) and the relationship of nuclear factor-kappa B (NF-κB) and intercellular adhesion molecule-1 (ICAM-1). METHODS: The expression of NF-κB and ICAM-1 were detected by immunohistochemistry, in situ hybridization, immunocytochemistry and Western blotting techniques in rat brain of experimental ICH and cerebral microvascular endothelial cells (RCMECs) injured by hydrogen peroxide. RESULTS: The expression of NF-κB p65 and ICAM-1 were up-regulated in rat brain after ICH. The ICAM-1 reached the peak at 1 day while the NF-κB at 4th day. NF-κB p65 expressed remarkably in cultured RCMECs immediately after injured by hydrogen peroxide, while ICAM-1 expressed remarkably　2 hours later. PDTC, an inhibitor of NF-κB, down-regulated the expression of NF-κB and ICAM-1. CONCLUSION: NF-κB induces the expression of ICAM-1 in RCMECs injured by reactive oxygen species (ROS).     

A novel mechanism of RhoA activation induced by tumor necrosis factor-α in mouse cerebral microvascular endothelial cells

AIM: To investigate the effects of tumor necrosis factor α (TNF-α) on RhoA activity in mouse cerebral microvascular endothelial cells.METHODS: The bEnd.3 cells, a mouse brain microvascular endothelial cell line, were cultured. RhoA activity was analyzed by pull-down assay 10 min, 30 min and 60 min after TNF-α treatment. Expression of RhoA protein was determined by Western blotting 1 h, 3 h, 6 h, 12 h and 24 h after TNF-α treatment. Small interfering RNA (siRNA) targeting to p115RhoGEF or control nsRNA was transfected into bEnd.3 cells. The expression of p115RhoGEF was determined by Western blotting, and RhoA activity was detected by pull-down assay 30 min after TNF-α treatment.RESULTS: RhoA activity peaked at 30 min after TNF-α treatment(P<0.01) . TNF-α significantly increased the protein expression of RhoA at 12 h and 24 h (P<0.05). Knock-down of p115RhoGEF by siRNA in bEnd.3 cells attenuated TNF-α-induced RhoA activation (P<0.05).CONCLUSION: TNF-α up-regulates RhoA activity and expression. p115RhoGEF may play a role in TNF-α-induced activation of RhoA.     

Effects of HIF-1α silencing on proliferation of rat hepatoma cells

AIM：To study the effect of hypoxia-inducible factor 1α (HIF-1α) silencing on the proliferation of hepatoma cells under hypoxia. METHODS：Rat hepatoma cell line CBRH-7919 was used in this study. Hypoxia model was established by treating the cells with cobalt chloride (CoCl2). The expression of HIF-1α was silenced by small interfe-rence RNA. Real-time RT-PCR and Western blotting were used to detect the mRNA and/or protein expression of HIF-1α, vascular endothelial growth factor (VEGF), p21 and cyclin D1 in CBRH-7919 cells under hypoxia. The proliferation of CBRH-7919 cells was measured by the technique of 5-bromo-2’-deoxyuridine (BrdU) incorporation. RESULTS：The expression of HIF-1α and VEGF at mRNA and protein levels was significantly increased under hypoxia (P<0.05). Silencing of HIF-1α significantly inhibited the expression of HIF-1α, VEGF and cyclin D1 at mRNA and/or protein levels, while increased the protein expression of p21 (P<0.05). The BrdU-positive cells in HIF-1α siRNA transfection group were significantly less than those in control group. CONCLUSION：HIF-1α silencing significantly inhibits the proliferation of hepatoma cells under hypoxia.     

Effect of antioxidant and NFκB on the induction of iNOS gene in rat pulmonary microvascular endothelial cells in vitro

AIM:To investigate the role of nuclear factor κB (NF_κB) in the induction of iNOS gene by TNFα and LPS in endothelial cells and the effect of antioxidant on the induction of iNOS. METHODS:Nitrite was determined based on Griess reaction. iNOS mRNA was analyzed using Northern blot. NF_κB in the cell nucleole was detected with electrophoretic mobility shift assay.RESULTS: (1)NO production and iNOS mRNA expression induced by LPS and TNFα was blocked by pyrrolidine dithiocarbamate(PDTC) or N-acetylcysteine(NAC). (2)LPS and TNFα triggered the activation and translocation of NF_κB, and PDTC or NAC inhibited the activation of NF_κB induced by LPS and TNFα. CONCLUSIONS:(1)The induction of iNOS gene by TNFα and LPS is dependent on the activation of NF_κB.(2)Antioxidants may inhibit the induction of iNOS gene through the inhibition of NF_κB activation.