Autophagy attenuates injury of human pulmonary microvascular endothelial cells under mimic ischemia/reperfusion microenvironment

AIM: To detect the autophagic changes of human pulmonary microvascular endothelial cells (HPMVECs) under ischemia/reperfusion (I/R) microenvironment, and to clarify the effects of autophagy on the HPMVECs survival and endothelial barrier integrity under I/R condition. METHODS: Rapamycin (RAP) was applied to promote autophagy of HPMVECs. These cells were then incubated under the condition of oxygen-glucose deprivation/oxygen-glucose restoration (OGD). After exposure to OGD, the changes of autophagy, cellular death and permeability of the cells were determined by transmission electron microscopy, flow cytometry and transwell assay, respectively. RESULTS: Compared with the control cells, OGD-challenged cells had a much higher level of autophagy. The apoptotic rate was much higher and endothelial permeability was more serious in OGD group than those in control group. Preconditioning with RAP effectively improved OGD induced autophagy, it did not affect the cell survival and endothelial permeability under normal living condition, but obviously decreased the cells apoptotic rate, and remarkably lowered OGD-induced high permeability of the cells. CONCLUSION: Autophagy protects HPMVECs against I/R-induced injury. Promotion of autophagy is helpful for attenuating I/R-induced cell death and sustaining the endothelial barrier integrity.     

Comparative analysis of cultured endothelial progenitor cells in vitro from PBMCs and enriched CD133+ cells

AIM: To compare the methods of two currently employed isolation methods for endothelial progenitor cells (EPCs): from total peripheral blood mononuclear cells (PBMCs) and from enriched CD133+ cells, by defining the cell morphology, phenotype, reproductive activities and function in vitro, providing a reference for clinic application. METHODS: PBMCs from the healthy subjects were used for CD133+ sorting or not. The two groups of isolated cells were suspended in complete medium M199 for 7 d to 14 d. EPCs phenotype were characterized by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and VEGF concentration was measured using an ELISA kit. Matrigel experiment and migration assay were imitated vascularization in vivo. RESULTS: PBMCs produced more colony-forming units (CFU) than CD133+ cells from the same volume of blood (P<0.01). From 7 d to 14 d, the two groups show decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144+ cells in CD133+ group were lower than those in PBMCs groups (P<0.01). Cells in PBMCs group secreted more VEGF than that in CD133+ group on 7 d (P<0.01). Compared to CD133+ group, PBMCs group showed more potential of proliferation and vascularization in vitro. CONCLUSION: CD133+ sorted cells show a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, which is unable to differentiate to mature endothelial cells, indicating that its not a preferential way to obtain EPCs for clinic therapy.     

Mechanism of endothelial microparticle generation in hypertension and its pathophysiological roles

Hypertension is closely related to many target organ damage. Endothelial microparticles (EMPs), derived from endothelial cells in response to endothelial cell activation or apoptosis, are complex vesicular structures with a membrane skeleton and express various antigens specific to parental endothelial cells. EMPs circulate in human plasma and show elevated levels in many vascular damage diseases, such as hypertension, atherosclerotic vascular diseases, sepsis and diabetes. Recent studies have shown that EMPs could be a comprehensive index for endothelial homeostasis monitoring, such as vasomotor activity, anti-inflammatory status and so on. Especially, more and more evidence suggests that EMPs play an important role in hypertension. Patients with hypertension show higher circulating levels of EMPs compared with healthy controls. Furthermore, increasing evidence demonstrates that EMPs can induce endothelial dysfunction in vitro and in vivo, and then further promote the development of hypertension and its complications. This review will summarize the progress in the definition and formation mechanisms of EMPs, levels of EMPs and their phenotypes in patients with hypertension, and the pathophysiological roles of EMPs in hypertension.     

Advances on studies of miR-21 in vascular endothelial function and angiogenesis

MicroRNAs (miRNAs)are a class of non-coding, endogenous, single-stranded small RNA molecules composed of 19~25 nucleotides. miRNAs are widely involved in the process of human life activities. Recent studies have shown that part of miRNAs regulate the vascular endothelial function and angiogenesis. High expression of miRNA-21 is found to play important roles in the cell proliferation, cell apoptosis, cell growth and death of vascular endothelial cells. This review will focus on the recent progress related to miRNAs in vascular endothelial function and angiogenesis, providing a new insight in cardiovascular disease prevention, clinical diagnosis, prognosis and target therapeutics.     

Association of endothelial lipase gene Thr111Ile and Gly26Ser polymorphism with lipoprotein in patients with coronary heart disease

AIM: To investigate the association of endothelial lipase gene (LIPG) Thr111Ile and Gly26Ser polymorphism with lipoprotein in patients with coronary heart disease (CHD) in Chinese.METHODS: 438 patients were classified as 242 CHD group and 196 controls group by selective coronary angiography.Plasma level of lipoprotein was determined and the Thr111Ile and Gly26Ser polymorphism was screened by PCR-RELP.RESULTS: The frequencies of Thr111Ile genotype in Chinese were CC 76.7%,CT 23.3％,TT 0.0％.The frequencies of allele were C 88.3％,T 11.7%.The plasma level of HDL-c in CT group was significantly higher than that in CC group (P<0.05) on logistic regression analysis.However,logistic regression analysis revealed that there was no significant difference between CHD group and control group for Thr111Ile polymorphism (P>0.05).No Gly26Ser mutation was observed in this study.CONCLUSION: The polymorphism of Thr111Ile is present in patients with CHD in Chinese,and T allele is related to high HDL-c level.There is no significant association between the polymorphism of Thr111Ile and CHD.The Gly26Ser mutation has not found in this study.     

Role of sphingosine 1-phosphate on high glucose-induced vascular endothelial cell dysfunction

AIM: To explore the role of sphingosine 1-phosphate (S1P) in the dysfunction of vascular endothelial cells exposed to high glucose. METHODS: In human aortic endothelial cells cultured under high-glucose (22 mmol/L glucose) medium, nitric oxide (NO) level, polymorphonuclear neutrophil-endothelial cell adhesion rate, protein level of intercellular adhesion molecule-1 (ICAM-1), migration of endothelial cells and Akt/endothelial nitric oxide synthase (eNOS) pathway activation were observed after S1P, sphingosine kinase-1 inhibitor and/or Akt inhibitor treatments. RESULTS: S1P decreased NO level, increased polymorphonuclear neutrophil adhesive rate, enhanced ICAM-1 protein level, and inhibited migration of endothelial cells and activation of Akt/eNOS pathway in endothelial cells cultured under high-glucose condition. Sphingosine kinase-1 inhibitor, which reduced S1P content, significantly improved the above endothelial cell function indexes and restored the activation of Akt/eNOS pathway. CONCLUSION: S1P promoted high glucose-induced dysfunction of endothelial cells probably by inhibiting the activation of Akt/eNOS signal pathway. Targeting S1P is expected to become one of potential treatment strategies to reduce endothelial cell dysfunction.     

Regulatory effect of miRNA enfolded in microparticles on endothelial cells

Endothelial cells are the cells lining the inner surfaces of blood vessels. They build up a single cell layer and play an important role in the regulation of vascular functions and the maintenance of homeostasis. The activation or injury of endothelial cells is associated with multiple diseases, including hypertension, atherosclerosis, cancer, diabetes, etc. Recent studies have found that microparticles are involved in the process of endothelial cell injury, thus playing a part in the development of many diseases. miRNAs enfolded in the microparticles have become importance in recent years. Moreover, recent studies also found that the concentration of the miRNAs enfolded in the microvesicles is higher than that of the miRNAs in the plasma, which means that the microparticles are the main form of miRNAs present in peripheral circulatory system. This article is to review the recent research progress in microparticle-encapsulated miRNAs and their regulatory effect on endothelial cell injury.     

Preliminary study of endothelial progenitor cells formed vessel-like structure in vitro

AIM: Endothelial progenitor cells (EPCs) are a group of stem cells/progenitor cells, which exist in postnatal body and can be of specially homing to the foci of angiogenesis and then differentiate into endothelial cells.This investigation was to study the method for culturing endothelia progenitor cells (EPCs) in vitro, and to observe its feasibility and condition formed vessel-like structure.METHODS: The cells were isolated from born marrow, peripheral blood, umbilical cord blood or spleen in different laboratories.The EPCs derived from human umbilical cord blood and rabbit peripheral blood were cultured in vitro through adhesion selection and were differentiated into endothelial cells under the induction of special cytokines.The expression of CD34, VEGFR-2, AC133 and VE-cadherin were detected by fluorescence-activated cell sorting.The endothelial cell lineage was confirmed by DiI-ac-LDL up-taking and Ⅷ factor immunocy tochemistry.RESULTS: The EPCs derived from human umbilical cord blood and rabbit peripheral blood were cultured in vitro successfully, forming cord-like and tube-like structure.The EPCs derived from rabbit peripheral blood differentiated more mature and formed vessel-like structure.CONCLUSION: The EPCs derived from human umbilical cord blood and rabbit peripheral blood formed vessel-like structure in vitro.EPCs may be a potential resource of vessel tissue engineering.     

Injuring effect of DMSO-soluble particles from cigarette smoke on human umbilical vein endothelial cell line EA. hy 926 in vitro

AIM: To investigate the injuring effect of DMSO-soluble particles from cigarette smoke(DSP) on human umbilical vein endothelial cells. METHODS: Human umbilical vein endothelial cell line EA. hy 926 was used as target cells in the study. The growth and viability of the cells treated with various dosages (1, 2, 4 or 4 mL/L) of DSP and low dose (2 mL/L) of DSP at different time points were evaluated by MTT colorimetric assay and celllular protein assay in 96-well plates. Transmission electron microscopy study was carried out to observe the ultrastructure of human umbilical vein endothelial cells under DSP treatment.RESULTS: DSP inhibited the proliferation of human umbilical vein endothelial cell line EA. hy 926. Under DSP treatment, the reducing cellular protein and increasing cell death(mainly necrosis) were observed in time-dependent and dosage-dependent manners.CONCLUSIONS: These results indicated that the toxic effect of DSP caused functional disturbance and structural damage of human endothelial cells.     

Effects of the Bushen Ningxin Chinese herb-containing serum on the adherence of human monocytes to endothelial cells

AIM: To study effect of the Bushen Ningxin decoction, a Chinese medicine, on the adherence of monocytes to endothelial cells and its mechanism. METHODS: Using cultured human umbilical vein endothelial cells (HUVECs) as target cells, oxidized low density lipoprotein (ox-LDL) was added to the endothelial cell culture to prepare the model of human endothelial cell injury. The serum of rabbits having been treated with Bushen Ningxin decoction was added to trial architecture, the adherence of monocyte-like cell line U937 to HUVECs was analyzed using Rose Bengal staining. In addition, the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1(VCAM-1) and E-selectin in HUVECs was measured by flow cytometry. RESULTS: Treatment of HUVEC with ox-LDL (100 mg/L) for 24 hours significantly increased adhesion of U937 to HUVECs. If serum of the animal treated with Bushen Ningxin decoction was added to trial architecture, the adhesion decreased significantly. The flow cytometry analysis showed that ox-LDL could induce the expression of ICAM-1, VCAM-1 and E-selectin in HUVECs. Serum of the animal treated with Bushen Ningxin decoction significantly decreased the expression of ICAM-1, VCAM-1 and E-selectin in HUVECs. CONCLUSION: The Bushen Ningxin Chinese herb-containing serum has an inhibitory effect on the adherence of monocytes to endothelial cells, probably by way of down-regulating the expression of ICAM-1, VCAM-1 and E -selectin in endothelial cells.     

Effect of endothelial Rho and Rho kinase in extravasation migration of sarcoma cell

AIM: To evaluate the effect of endothelial Rho and Rho kinase in extravasation migration of sarcoma cell. METHODS: We used an in vitro model of sarcoma cell transmigration across a monolayer of HUVEC cultured on collagen gel to observe extravasation migration of sarcoma cells and calculated the electrical resistance of HUVEC monolayer in extravasation migration of sarcoma cells. RESULTS: Sarcoma cells migrated through endothelial cells into collagen gel, the electrical resistance of a HUVEC monolayer reduced in extravasation migration of sarcoma cells.Endothelial Rho inhibitor(C3 transferase) and Rho kinase inhibitor(Y-27632) inhibited extravasation migration of sarcoma cells and inhibited reduction of electrical resistance of a HUVEC monolayer in extravasation migration of sarcoma cells. CONCLUSION: Endothelial Rho and Rho kinase regulates sarcoma cell transendothelial migration through modification of endothelial cytoskeleton.     

Effect of basic fibroblast growth factor on synthesis of C-type natriuretic peptide in cultured human umbilical vein endothelial cells

AIM: To investigate the effect of basic fibroblast growth factor (bFGF) on C-type natriuretic peptide (CNP) production, release and mRNA expression. METHODS: Human endothelial cell cultured;CNP was mea sured by radioimmunoassay method;CNP mRNA expression was determined by RT-PCR technique.RESULTS: bFGF could augment CNP synthesis in human endothelial cells. Compared with control group,25 ng, 50 ng, 100 ng bFGF increased CNP contents in endothelial cells by 88% (P＜0.05), 95% (P＜0.05), 187% (P＜0.01), respectively.100 ng bFGF also stimulated CNP release from cultured human endothelial cell. In addition, 25 ng, 50 ng and 100 ng bFGF stimulated CNP mRNA expression of cultured human endothelial cells in a dose-dependent manner. CONCLUSION: bFGF might regulate CNP synthesis,release and mRNA expression in cultured umbilical human endothelial cells.     

VEGF promotes biliary epithelial cell viability and cystic dilation in rats with polycystic kidney

AIM:To explore the promoting effect of vascular endothelial growth factor (VEGF) on the viability of biliary epithelial cells and biliary cystic dilation in rats with polycystic kidney (PCK). METHODS:Immunohistoche-mical staining was used to detect the expression of VEGF (n=6) and CD31 (n=10) in the liver tissue of normal and PCK rats. RT-qPCR and ELISA were used to evaluate the expression levels of VEGF in rat biliary epithelial cells and culture supernatant. WST-1 assay was applied to measure the effect of VEGF on the viability of rat biliary epithelial cells, and the influence of cholangiocyte culture supernatant on the viability of rat vascular endothelial cells. The cell migration assay was employed to observe the effect of cholangiocyte culture supernatant on endothelial cell migration. Tube formation assay was used to assess the impact of cholangiocyte culture supernatant on the angiogenic ability of endothelial cells. RESULTS:The result of immunohistochemical staining manifested that VEGF was highly expressed in the cholangiocytes of the PCK rats (P<0.01). More newly formed blood vessels were observed in the hepatic portal area of PCK rats than that in normal rats (P<0.01). The results of RT-qPCR and ELISA suggested that the mRNA and protein expression levels of VEGF in the cholangiocytes of PCK rats were significantly higher than those in normal rats (P<0.01). VEGF enhanced the viability of cholangiocytes in PCK rats (P<0.01). The culture supernatant of cholangiocytes in PCK rats increased the endothelial cell viability (P<0.01). VEGF siRNA and VEGF receptor inhibitor reduced the viability of cholangiocytes (P<0.01). The results of cell migration assay and tube formation assay indicated that the abilities of endothelial cell migration and tube formation were improved by the culture supernatant of cholangiocytes in PCK rats (P<0.01). CONCLUSION:The bile duct cystic dilation of PCK rats was related to the excessive secretion of VEGF in bile duct epithelial cells.     

Biological characteristics of primarily cultured microvascular endothelial cells in mouse myocardium

AIM：To investigate an effective and stable method to isolate myocardial microvascular endothelial cells (MMVECs) from the mouse heart and to observe their biological characteristics.METHODS：The cells from the mouse heart were isolated by collagenase II digestion followed by different speed adhesion. Then the cells were cultured on the dish coated with polylysine in endothelial cell-specific culture medium. The biological characteristics were observed by trypan blue staining. The cell growth curve and the cell proliferation were also evaluated by MTT assay at different passages. The expression of DiI-ac-LDL, FITC-UEA-1, specific markers of endothelial cells and the expression of CD31, vWF and CD34 were determined by immunofluoresence staining. To evaluate the function of the MMVECs, in vitro tube formation was evaluated under microscope.RESULTS：Two days after the enzyme digestion, the MMVECs formed small and isolated clusters. The MMVECs grew quickly in monolayer with the characteristics of endothelial cell shape at 4~5 d. The cells became confluent and cobblestone-like which were ready for passage at 7~8 d. After passage, the viability of the cells was more than 95%. The first and the third generation of the cells presented an S-shape growth curve. The cell proliferation of the first to the third generation was quick and slowed down after the fifth passage. MMVECs were highly positive for DiI-ac-LDL and FITC-UEA-1 ［(89.2±3.5)%］, indicating the cells were MMVECs. The other relative antigen expression (CD31, vWF and CD34) on the MMVECs was (56.7±3.7)%, (78.5±2.6)% and (67.8±4.2)%, respectively. The MMVECs formed the tubes in vitro after cultured for 6～12 h.CONCLUSION：We can obtain high-purity MMVECs using the combination of collagenase II digestion, the different speed adhesion process and the endothelial cell-specific culture medium for effective and reliable MMVECs isolation and culture.     

Role of PARP in endothelial cell apoptosis induced by angiotensin Ⅱ

AIM: To explore the role of poly-(ADP-ribose)polymerase (PARP) in the cultured endothelial cell apoptosis induced by angiotensin Ⅱ.METHODS: The cultured endothelial cells were treated with angiotensin Ⅱ at concentration of 1 μmol/L.The apoptosis of endothelial cells was assessed by TUNEL.Meanwhile,the activity of PARP and the content of nitric oxide (NO) were also measured.RESULTS: Angiotensin Ⅱ induced apoptosis in endothelial cells in a time-dependent manner.The content of NO begun to increase at 6 h (P<0.05),and peaked at 24 h.The activity of PARP also increased at 6 h (P<0.05),peaked at 12 h,and was lower than that in the control at 48 h (P<0.05).CONCLUSION: The cytotoxicity of NO has a relevant role in apoptosis of endothelial cells induced by angiotensin Ⅱ,and can increases the activity of PARP.     

Effect of N,N-dimethylsphingosine on the expression of adhesion molecules in human umbilical vein endothelial cell line EA.hy926

AIM: To investigate whether and how N, N-dimethylsphingosine (DMS) plays a role in modulating the adhesion of monocytes to vascular endothelial cells, and identify whether human umbilical vein endothelial cell line EA.hy926 take place of the vascular endothelial cells.METHODS: Adhesion ratio was measured by flow cytometry, and immunohistochemistry was used to detect the expression of ICAM-1 and P-selectin in HUVEC: EA.hy926 cells after the effect of DMS. RESULTS: DMS inhibited the adhesion of monocytes to HUVEC: EA.hy926 cells in a time-dependent and concentration-dependent manner by reducing the expression of ICAM-1 and P-selectin. CONCLUSIONS: DMS reduced adhesion molecule expression in vascular endothelial cells. DMS may be an important contributor to reduce adhesion ratio, suggesting that DMS plays a negative role in proinflammatory and immune functions of the modified vascular endothelial cells during atherosclerosis and restenosis.     

The feasibility of reendothelialization of the injured arterial wall by autologus endothelial cell transplantation and their effects on neointima proliferation

AIM: To investigate the feasibility of reendothelialization of the injured arterial wall by autologous endothelial cell transplantation and their influences on neointima proliferation. METHODS: New Zealand white rabbits (n=30) were subjected to bilateral iliofemoral artery balloon injury. Cultured, autologous venous endothelial cells were immediately transplanted into one vessel(transplantation group), whereas the contralateral artery received medium only(control group). Reendothelialization of the injured arterial wall was analysed 4 hours or 4 days after cell transplantation by fluorescent tracing、scanning electron microscope(SEM) and Evans blue staining. Pathology analysis was employed 28 days after cell transplantation to evaluate neointima proliferation. RESULTS: The transplanted endothelial cells had adhered into the aterial wall 4 hours after transplantation and began to attach and spread 4 days later. A number of fluorescent labeling endothelial cells were observed in the endothelial injured arterial wall. The vessels in control group were stained nearly completely by Evans blue, whereas about 60% area was not stained in transplantation group. Pathological examination demostrated that neointimal area and maximal intima thickness in transplantation group significant decreased than those in control. CONCLUSION: Autologus endothelial cells were effectively transplanted into the injured arterial wall by balloon catheter, and it can relieve neointima proliferation in the long time.     

Expression of Smoothened and its role in endothelial cells in synovial tissues of rheumatoid arthritis

AIM: To investigate the expression of Sonic Hedgehog signaling pathway-associated factor Smoothened (Smo) and its role in endothelial cells in synovial tissue of active rheumatoid arthritis (RA). METHODS: Smo expression in synovial tissue from 4 RA patients and 4 patients with trauma or meniscal injury (without arthritis, used for control) was detected by the method of immunohistochemistry. Human umbilical vein endothelial cell line EA.hy926 was used as the model of synovial vascular endothelial cells. The expression of Smo was detected by Western blotting after TNF-α treatment. The small interfering RNA (siRNA) specifically targeting Smo gene was synthesized and transfected into EA.hy926 cells. The interference efficiency of the siRNA on the production of Smo protein was determined by Western blotting. The cells were treated with TNF-α and actinomycin D (ActD) 24 h after siRNA transfection. The cell survival rate was determined by CCK-8 assay and the apoptotic rate was examined by flow cytometry. RESULTS: Smo was highly expressed in synovial tissue from active RA patients, especially in endothelial cells as compared with control group. TNF-α significantly increased the protein expression of Smo in EA.hy926 cells. EA.hy926 cells transfected with Smo-siRNA showed a significant decrease in the cell viability with the cell survival rate of (24.30±0.45)% and the apoptotic rate of (48.00±1.96)%, as compared with those in negative control group ［(36.86±0.62）% and （31.70±0.82）%, respectively］. CONCLUSION: Smo may play a role in the regulation of apoptosis in endothelial cells in RA synovium.     

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.