Role of post-hemorrhagic shock mesenteric lymph in enhancement of vascular permeability

AIM：To investigate the role of post-hemorrhagic shock mesenteric lymph (PHSML) in the enhancement of vascular permeability. METHODS：Eighteen Wistar rats were randomized into sham group, shock group, and shock plus mesenteric lymph drainage (shock + drainage) group. The rats in shock group and shock + drainage group were routinely subjected to hemorrhagic shock and hypotension ［(40±2） mmHg］ was maintained for 90 min, and then the fluid resuscitation was performed. Mesenteric lymph was drained in the rats in shock+drainage group from resuscitation finished to 6 h, for the observation of PHSML drainage on the vascular permeability in multiple tissues of hemorrhagic shock rats. Afterwards, human umbilical vein endothelial cells (HUVECs) were incubated with the PHSML in vitro to observe the effects of PHSML on the morphology and permeability of HUVECs. RESULTS：The degree of blue color and concentrations of Evens blue in the lung, myocardium, kidney, liver, spleen and small intestine were significantly increased in the shocked rats than that in sham group, while the ratios of the dry weight to the wet weight were decreased. The mesenteric lymph drainage reversed these changes. Meanwhile, 4% and 10% of PHSML at 0~3 h and 3~6 h after resuscitation, and lipopolysaccharide (10 mg/L) all caused the damage of HUVECs, decreased the viability and trans-endothelial electrical resistance of HUVECs, and increased the permeability of HUVECs to fluorescein isothiocyanate-labeled albumin. CONCLUSION：PHSML is a vital factor in the enhancement of vascular permeability.     

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.     

Role of VE-cadherin/catenins complex in microvascular permeability during inflammation

VE-cadherin forms VE-cadherin/catenins complex by interacting with catenins, and VE-cadherin/catenins complex is an important component of adherens junctions (AJ). During inflammation, different kinds of factors can increase the microvascular permeability eventually by causing the disassembly of VE-cadherin/catenins complex.     

Mechanism of exogenous nitric oxide in attenuating endotoxin-induced increase of rat pulmonary microvascular permeability

AIM and METHODS:The animal model of acute lung injury (ALI) caused by intratracheal instillation of lipopolysaccharides(LPS) in vivo and human peripheral blood polymorphonuclear neutrophil (PMN) in vitro were used to study the effects of sodium nitroprusside (SNP), nitric oxide (NO) donor, on LPS-induced PMN accumulation, microvascular permeability and PMN apoptosis. RESULTS:①In vivo, PMN accumulation in lung, the protein content in bronchoalveolar lavage fluid (BALF) and the Evans blue dye and monastral blue dye extravasation in lung tissue of LPS group were markedly higher than those of both sham operation group and LPS+SNP group. ②In vitro, the apoptotic percentage of SNP group was much higher than that of control group, while compared with LPS group, SNP+LPS group has significantly higher apoptotic percentage. CONCLUSIONS:SNP intratracheal instillation attenuated LPS-induced microvascular permeability and alleviated ALI. PMN apoptosis induced by SNP may be one of the potential mechanisms underlying the decrease of PMN accumulation in lung tissue.     

Effects of insulin and glucose on secretion of tissue-type plasminogen activator and its inhibitor-1 in hypoxic vascular endothelial cells

AIM: To study the effects of insulin and glucose on tissue-type plamingen activator (tPA) and its inhibitor-1 (PAI-1) secretion in cultured human endothelial cells. METHODS: Human endothelial cell line ECV-304 was cultured with glucose and/or insulin at different concentrations with or without hypoxic exposure. RESULTS: The tPA, PAI-1 secretion and ratio of tPA/PAI-1 increased in endothelial cells during hypoxia. Insulin and glucose increased the tPA and PAI-1 secretion in endothelial cells exposed to hypoxia, and increase in tPA/PAI-1 ratio was also observed at 4 h and 8 h. CONCLUSION: Hypoxia stimulates the release of tPA and PAI-1. Insulin and glucose also stimulate the tPA and PAI-1 secretion during hypoxia.     

Shear stress regulates NO production in vascular endothelial cells through Pim1/eNOS signaling pathway

AIM: To determine whether laminar shear stress regulates nitric oxide (NO) production in vascular endothelial cells through Pim1/endothelial nitric oxide synthase (eNOS) signaling pathway. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to laminar shear stress using a parallel-plate flow system. NO production is evaluated by NO assay kit. Pim1 protein expression and eNOS phosphorylation were determined by Western blot. A specific small interfering RNA was used to knock down Pim1 gene expression, and then the changes of above indicators were detected. RESULTS: After 15-min exposure of HUVECs to laminar shear stress (15 dyn/cm²), rapid increases in Pim1 protein expression and NO production were observed (P < 0.05). Shear stress also caused time-dependent stimulation of eNOS phosphorylation (P < 0.05). The shear-induced Pim1 expression and NO production were abrogated in the HUVECs transfected with siPim1 (P < 0.05). Pim1 silencing also prevented shear-induced rise of eNOS-Ser1177 phosphorylation (P < 0.05). CONCLUSION: Pim1 may account for shear-induced NO production in endothelial cells due to phosphorylation activation of eNOS.     

Effects of myosin light chain kinase on regulation of endothelial barrier function

Myosin light chain kinase (MLCK) activates the regulatory light chain of myosin II, and the phosphorylated myosin light chain leads to actomyosin contractile activity, as well as the cell contraction and increasing intercellular gap, which finally results in endothelial barrier dysfunction. MLCK-dependent hyperpermeability occurs in response to multiple cell signaling molecules and signaling pathways, including Ca²⁺, Src, PKC, NO, cGMP and mitogen activated protein kinases (MAPK). In this review, different mechanisms of endothelial hyperpermeability mediated by MLCK are discussed.     

Effect of Sini decoction on expression of caveolin-1 and eNOS in EAhy926 cells injured by homocysteine

AIM：To investigate the mechanism of Sini decoction in treating human vascular endothelial cell injury and the roles of caveolin-1 and nitric oxide (NO) system in this procedure. METHODS：Model of human umbilical vein endothelial EAhy926 cells injured by homocysteine (Hcy) was established. The protective effect of Sini decoction on the injured EAhy926 cells was observed, and the expression of caveolin-1 and endothelial nitric oxide synthase (eNOS) was detected by real-time fluorescence quantitative PCR and Western blotting. RESULTS：Compared with control group, the Hcy-treated EAhy926 cells showed reduced adherent cell number and NO concentration in culture supernatant, decreased expression of eNOS mRNA and protein, and increased expression of caveolin-1 mRNA and protein (all P<0.05). Compared with Hcy group, better growth of adherent cells, elevated NO concentration in culture supernatant, attenuated expression of caveolin-1 mRNA and protein, and enhanced expression of eNOS mRNA and protein in Sini decoction groups were observed (all P<0.05). CONCLUSION：Homocysteine may injure EAhy926 cells by enhancing the expression of caveolin-1 and suppressing the expression of eNOS, while Sini decoction may protect EAhy926 cells by suppressing the expression of caveolin-1 and enhancing the expression of eNOS.     

Effects of taurine-magnesium complex on vascular endothelium and hemorheology in rats in vivo

AIM: To study the protective effects of taurine-magnesium complex (TMC) on endothelium and hemorheology in rats. METHODS: A model of the endothelial damage was made by means of giving rats an injection of adrenaline and making them swim in ice-cold water, then number of circulating endothelial cells (CEC) in whole blood, plasma ET-1 and NO₂^-/NO₃^- content, NOS activity and rheology were determined, respectively. The protective effects of TMC were also observed. RESULTS: An increase in the number of CEC accompanied by abnormal whole blood viscosity, and endothelium-derived ET-1 were observed in model rats. Both the NO₂^-/NO₃^- content and NOS activity were declined significantly in model rats. TMC reduced the number of CEC, resumed NO₂^-/NO₃^- content and NOS activity, and improved the whole blood viscosity in a dose-dependent manner in model rats. CONCLUSION: Ice-cold water bath with adrenaline causes an acute damage of vascular endothelium and abnoramal rheology. TMC protects against the injury of vascular endothelium and improves the hemorheology.     

Role of endothelial cell glucose metabolism in vascular development

As an important organ in the body, blood vessels transport oxygen and nutrients to the tissues of the body. Endothelial cells are layers of cells lined with blood vessels whose metabolism is involved in regulating multiple links of vascular development and vascular diseases. Glucose metabolism provides the main energy for endothelial cells, and changes in glucose metabolism regulation affect vascular development and even lead to various diseases. Recently, strategies have been proposed to target endothelial cell metabolism for the treatment of vascular diseases. This article reviews the role of endothelial cell glucose metabolism in vascular development and vascular diseases in recent years.     

Effects of cellular repressor of E1A-stimulated genes on VEGF release and monolayer permeability of human vascular endothelial cells

AIM: To study the effects and mechanism of cellular repressor of E1A-stimulated genes (CREG) on VEGF release and monolayer permeability of human vascular endothelial cells (ECs). METHODS: The monolayer permeability of ECs was measured by transwell chamber model. The expression and localization of F-actin and VE-cadherin were examined by immunofluroscence using Olympus IX-70 fluorescent microscope. Enzyme-linked immunosorbent assay (ELISA) were performed to determine the concentration of vascular endothelial growth factors(VEGF) in the culture medium. VEGF neutrilization antibody was used to block the expression of VEGF in the cells. RESULTS: The monolayer permeability of CREG over-expressing ECs (EO group) was significantly higher than that of the normal control ECs (EN group, P<0.05). The monolayer permeability of CREG suppressing ECs (ES group) was lower than that in EN group (P<0.05). F-actin cytoskeleton in EO group showed disorganized, polymerized and bundled obviously to form large quantity of stress fibers in the central portion of the cells, whereas F-actin in EO group was mainly observed in the peripheral portion of the cells and only small amounts in the central portion of the cells. A widespread gap formation and a loss of VE-cadherin staining at the periphery were found in the cells of EO group. Inversely, the cells in ES group showed the localization of VE-cadherin at the cell-cell contacts tightly and the formation of zipper-like structures. Compared with EN group, the secretion of VEGF in the cell culture supernatants increased in EO group, but decreased in ES group (P<0.05). Furthermore, the changes of ECs permeability, cytoskeleton reorganization and loss of VE-cadherin induced by CREG were abolished by the addition of anti-VEGF neutralizing antibody. CONCLUSION: CREG over-expression increases the monolayer permeability of ECs, induces the cytoskeleton reorganization and reduces VE-cadherin expression by enhancing the secretion of VEGF in vitro.     

BYHWD promotes endothelial progenitor cells to repair damaged vascular endothelium

AIM: To investigate the role of Buyanghuanwu decoction (BYHWD) in promoting endothelial progenitor cells (EPCs)-induced recovery of damaged vascular endothelium. METHODS: The endothelial damaged rats were lavaged with BYHWD and injected with EPCs through vena caudalis. The repaired situation of damaged endothelium was observed. RESULTS: Compared with EPCs group and BYHWD group, the endothelial thickness was reduced, the levels of calcium, triglycerides and total cholesterol were decreased, but the high density lipoprotein levels were increased. In addition, the protein expression of vascular endothelial nitric oxide synthase and vascular stromal cell-drived factor-1 was sig-nificantly increased, but the expression of CXC chemokine receptor-4 was significantly reduced in BYHWD+EPC group. CONCLUSION: BYHWD promotes EPCs repairing damaged endothelium, the mechanism may be related to improve the internal environment and promotes the EPCs homing.     

ZNF580 is up-regulated in S1P-induced migration and proliferation of endothelial cells

AIM: To investigate whether a novel human C2H2-type zinc finger protein ZNF580 is involved in the proliferation and migration of endothelial cells induced by sphingosine 1-phosphate (S1P). METHODS: The cDNA of EA.hy926 cells was analyzed by RT-PCR to determine the S1P receptor expression profile. The cells were incubated with S1P at different concentrations and for different time intervals. Total RNA and protein in treated EA.hy926 cells were analyzed by RT-PCR and Western blotting. SB203580, a chemical inhibitor of p38 MAPK, was used to determine whether p38 MAPK pathway had any effect on the up-regulation of ZNF580 expression by S1P. The plasmid pEGFP-ZNF580 or the synthetic ZNF580-siRNA was transfected into EA.hy926 cells with Lipofectamine 2000 for 48 h. Cell migration assay and MTT colorimetric assay were used to investigate the effects of ZNF580 on the motility and growth of endothelial cells. RESULTS: EA.hy926 endothelial cells expressed S1P1, S1P3 and S1P5 receptors. Furthermore, S1P up-regulated ZNF580 at mRNA and protein levels in a concentration- and time-dependent manner. The p38 MAPK pathway specific inhibitor SB203580 blocked the S1P-induced up-regulation of ZNF580 expression. Moreover, overexpression/silencing of ZNF580 in EA.hy926 cells led to enhancement/decrease of the migration and proliferation of the cells. CONCLUSION: S1P-induced migration and proliferation of endothelial cells are critical for angiogenesis. ZNF proteins usually play an essential role in altering gene expression and regulating the angiogenesis.     

Neuregulin-1 induces expression of angiogenic factors in coronary artery smooth muscle cells

ATM: To investigate the effects of neuregulin-1 (NRG-1) on the expression of angiogenic factors in human coronary artery smooth muscle cells (HCASMCs). METHODS: HCASMCs were cultured in vitro, and the cells at the 3rd passage were collected to assess the expression and phosphorylation of ErbB by Western blot. After HCASMCs were cultured under normal condition, with hypoxia and serum deprivation, or with NRG-1 treatment, the expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) was determined by Western blot. RESULTS: The expression of ErbB2, ErbB3 and ErbB4 was observed in the HCASMCs, and the phosphorylation of these receptors was increased by NRG-1 treatment. Compared with control group, the expression of VEGF and Ang-1 in the HCASMCs was significantly increased in hypoxia and serum deprivation group (P<0.05﹚,while no difference in the expression of Ang-2 between the 2 groups was found.Compared with hypoxia and serum deprivation group,the expression of VEGF and Ang-1 in the H CASM Cs treated with NRG-1 was furtherincreased﹙P<0.05﹚,and no difference in the expression of Ang-2 between the 2 groups was observed.CONCLUSION: HCASMCs express ErbB2, ErbB3 and ErbB4, and the phosphorylation of the receptors is increased by NRG-1. Hypoxia, serum deprivation and NRG-1 treatment induce the increased expression of VEGF and Ang-1 significantly.     

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.     

Serum from rats at different ages modulates the functional activities of rat bone marrow-derived endothelial progenitor cells

AIM: To investigate the effects of serum from rats at different ages on the functional activities of rat bone marrow-derived endothelial progenitor cells (EPCs). METHODS: Mononuclear cells were obtained from bone marrow of young (1 to 2 month-old) and aged (19 to 26 month-old) Sprague-Dawley rats by Ficoll density gradient centrifugation and cultured with medium DMEM/F12 (containing 10% fetal bovine serum, endothelial cell growth supplement ECGs 100 mg/L, 1×10⁵ U/L of penicillin and streptomycin, respectively), 48 h later, the suspending cells were translocated to be cultured in new flasks coated with fibronectin, the secondary attached cells were used to perform the further experiments. EPCs were characterized as double positive for Dil-ac-LDL uptake and lectin binding. The cells were further identified by CD31 and vWF expression. Serum from young (1 to 2 month-old) and aged (19 to 26 month-old) rats was collected and used to culture EPCs. The experiments were divided into four groups: A= aged rat EPCs + aged rat serum; B= aged rat EPCs + young rat serum; C= young rat EPCs +aged rat serum and D= young rat EPCs + young rat serum. After cultured in DMEM/F12 supplemented with 10% serum from rats at different age (no fetal bovine serum addition in this medium), the average fluorescence intensities of EPCs stained with Dil-ac-LDL were tested by laser scanning co-focal microscopy. Migration and proliferation were assayed by modified Boyden chamber and MTT, respectively. Cell adhesion was performed by replacing EPCs onto cultured DAPI-labeled confluent smooth muscle cell monolayer and the adherent cells were counted. RESULTS: Young rat serum significantly improved the ability of aged rat EPCs for uptake of Dil-ac-LDL (P<0.01), increased the migration (P<0.01), adhesion (P<0.05) and proliferation activity (P<0.01) of aged rat EPCs, whereas aged rat serum obviously decreased the migration (P<0.05) and adhesion (P<0.05) activity of the young rat EPCs. CONCLUSION: Young rat serum significantly improves the activity of aged rat EPCs. On the contrary, aged rat serum partially inhibits the activity of young rat EPCs.     

Effect of hemolysin protein HlyX of Leptospira interrogans serovar Lai on permeability of vascular endothelial cells

AIM: To clone and express the hemolysin gene hlyX of Leptospira interrogans serovar Lai and to investigate the effect of the expression product on the permeability of human umbilical vein endothelial cells (HUVECs).METHODS: The recombinant plasmid pET-hlyX was constructed by inserting the hlyX gene into prokaryotic expression vector pET32a(+), and transformed into E.coli BL21(DH3) to express the fusion protein Trx-HlyX with a His-tag.The fusion protein was purified using HisTrap affinity columns.The permeability of the monolayer HUVECs was measured by enzyme-linked immunosorbent assay for biotin-labeled albumin.Flow cytometry and Hoechst 33258 staining were applied to measure the apoptotic rate of HUVECs after incubation with Trx-HlyX.RESULTS: The recombinant plasmid pET-HlyX was successfully constructed and the fusion protein Trx-HlyX was highly expressed.Compared with the control cells, the purified recombinant protein Trx-HlyX significantly increased the permeability of transfected cells and promoted apoptosis of HUVECs (P<0.05).CONCLUSION: The recombinant plasmid pET-hlyX highly expresses the fusion protein Trx-HlyX.Purified protein Trx-HlyX influences the permeability and has cytotoxicity on HUVECs.