Effect of sulodexide on apoptosis of human dermal microvascular endo-thelial cells exposed to hypoxia / 中国病理生理杂志

AIM:To investigate the effect of sulodexide(SDX)on the apoptosis of human dermal microvascu-lar endothelial cells(HDMECs)exposed to hypoxia and its underlying mechanism.METHODS:The HDMECs were cul-tured and divided into normoxia control group cultured under normoxic condition;hypoxia control group cultured in a humid incubator maintained at 37 ℃with 5% CO2and 1% O2for 24 h;treatment groups treated with SDX at 0.25,0.5 and 1 LSU/mL for 24 h under hypoxic condition.The cell viability was measured by CCK-8 assay.The apoptotic rate of the HD-MECs was analyzed by flow cytometry.The activity of caspase-3 in HDMECs was examined by caspase-3 activity assay kit. The expression of pro-apoptotic factor P53,caspase-3,Bax and anti-apoptotic factor Bcl-2 at mRNA and protein levels was determined by real-time PCR and Western blot.RESULTS:SDX increased the viability of HDMECs exposed to hypoxia, but also decreased the apoptosis.Furthermore, SDX down-regulated the expression of pro-apoptotic factor P53, Bax and caspase-3 at mRNA and protein levels as well as the activity of caspase-3,while the expression of anti-apoptotic factor Bcl-2 was up-regulated.CONCLUSION:SDX significantly increases the viability and decreases the apoptosis of HDMECs ex-posed to hypoxia.Inhibition of the mitochondrial apoptosis pathway may be involved in the underlying mechanism.

Hypoxia promotes the migration and apoptosis of human dermal micro-vascular endothelial cells / 基础医学与临床

Objective To study the effects of hypoxia on the cell migration,apoptosis and expression of related genes and proteins of human dermal microvascular endothelial cells(HDMEC). Methods To culture HDMEC in hypoxia condition by putting CoCl2in the cell incubator and all the subjects were divided to two groups which included normal group and hypoxia group.To find the appropriate CoCl2dose by CCK-8 experiment and the 200 μmol/L CoCl2was the best to establish hypoxia model.The migration of cells was measured by wound healing test.Apoptosis rate was detec-ted by flow cytometry. The gene expressions were detected by RT-PCR and the protein levels were detected by West-ern blot. Results The migration ability of cells was enhanced in hypoxia condition (P<0.05). The apoptosis rate was increased in hypoxia condition(P<0.05).The gene expressions of HIF-1α,HIF-1β,VEGF,iNOS were increased comparing with normal groups depending on time(P<0.05).The protein expressions of HIF-1α,VEGF,iNOS were in-creased comparing with normal groups depending on time(P<0.05). Conclusions Cell migration, apoptosis of HDMEC were influenced by hypoxia. It may be explained by increasing expression of related gene and proteins like HIF-1α,VEGF,iNOS in hypoxia condition.

Hypoxia promotes the migration and apoptosis of human dermal micro-vascular endothelial cells / 基础医学与临床

Objective To study the effects of hypoxia on the cell migration,apoptosis and expression of related genes and proteins of human dermal microvascular endothelial cells(HDMEC). Methods To culture HDMEC in hypoxia condition by putting CoCl2in the cell incubator and all the subjects were divided to two groups which included normal group and hypoxia group.To find the appropriate CoCl2dose by CCK-8 experiment and the 200 μmol/L CoCl2was the best to establish hypoxia model.The migration of cells was measured by wound healing test.Apoptosis rate was detec-ted by flow cytometry. The gene expressions were detected by RT-PCR and the protein levels were detected by West-ern blot. Results The migration ability of cells was enhanced in hypoxia condition (P<0.05). The apoptosis rate was increased in hypoxia condition(P<0.05).The gene expressions of HIF-1α,HIF-1β,VEGF,iNOS were increased comparing with normal groups depending on time(P<0.05).The protein expressions of HIF-1α,VEGF,iNOS were in-creased comparing with normal groups depending on time(P<0.05). Conclusions Cell migration, apoptosis of HDMEC were influenced by hypoxia. It may be explained by increasing expression of related gene and proteins like HIF-1α,VEGF,iNOS in hypoxia condition.

The Study of Expression of E-selectin of Fetal Skin Vessel and Cultured Fetal Dermal Microvascular Endothelial Cell

Inflammation is the characteristics of scar formation which is abscent in fetal wound healing. The adhesion molecules such as selectin groups are believed to have key roles for migration of inflammatory cells through the microvascular endothelial cells to the wound 77re purpose of this study was to evaluate the expression of E-selectin on the cultured human fetal and neonate dermal microvascular endothelial cells. The back skin of spontaneously delivered dead fetus (IUP 18-22 wks) and circumcised prepuce skin of neonate were used Human fetal dermal microvascular endothelial cells (HFDMEC) were isolated by extracting microvascular segment pom trypsin treated fetal and neonate skin tissue and isolated by sieving with nylon mesh and then by 35% Perocoll gradient centrifugation. Further purification was done with the Ulex europaeus I coated magnetic dynabead To confirm the fetal and neonatal endothelial cells, expression of factor VIII antigen on cell surface and uptake of acetylated low-density lipoprotein were checked Expression of E-selectin on cultured fetal and neonatal endothelial cells in response to IL-1alpha TNF-alpha INF-gamma was examined by ELISA. And the expression of E-selectin on fetal and neonatal dermal microvascular endothelial cells was examined by immunohistochemical study using monoclonal 3B7 anti E-selectin antibody in cultured fetal and neonatal skin. The expression of E-selectin on endothelial cells was not significantly digerent between fetal and neonatal endothelial cells. This expression was augmented 10 times more by IL-1alpha, TNF-alpha, INF-gamma. Augmented endothelial E-selectin expression by IL-1alpha, TNF-alpha, INF-gamma showed peak level 4 hours after stimulation and return to baseline level after 48 hrs. This time course was similar in both fetal and neonatal endothelial cells. Immunohistochemically, the expression of E-selectin molecule of unstimulated fetal and neonatal tissue was not observed However, on both fetal and neonatal tissue cultured for 4 hours after stimulation by 100 u/ml of IL-1 and 100 u/ml of TNF, expression of E-selectin molecule in microvasculature of upper dermis was observed and this expression persisted for up to 16 hours of culture. Also after culturing for 48hrs with 500 qlml of IFN, expression of E-selectin was observed in the microvessels of upper dermis. In conclusion, we could not find any digerences between the fetal and neonate skin in the expression of E-selectin on the endothelial cells spontaneously or stimulated by IL-1alpha, TNF-alpha or INF-gamma in vivo and vitro which means the expression of E-selectin vny not be an important mechanism of scarless wound healing in fetus.

T Lymphocyte Subsets and Binding of T lymphocyte to Human Dermal Microvascular Endothelial Cells in Behcet's Disease / 대한피부과학회지

BACKGROUND: Behcets disease represents a polysymptomatic, recurrent vasculitis' with a chronic course. Its etiology and pathogenesis still remains unclear. Several immunological abnormalities have been described in this disease and altered cell mediated immunity especially has been suggested to play an important role in the pathogenesis. Recently, suppressor-inducer (naive, CD4-CD45RA+ ) and helper-inducer (memory, CD4-CD45RO+) human T cell subsets have been identified by their relevant monoclonal antibodies. It has heen suspected that human dermal microvascular endothelial cells(HDMEC) are an important part in the pathogenesis of Behcets disease. However, there was no report for HDMEC-T lymphocyte adhesion in Behcets disease, OBJECTIVE: We have investigated the subpopulation differences in CD4- T lymphocytes and the adhesiveness of T lymphocytes to cultured HDMEC in the presence of IL-1 alpha, or TNF-alpha using T lymphocytes isolated from normal human subjects and Behcets disease patients respectively. METHODS: T lymphocyte subsets were evaluated by the two-color immuno-fluorescence flow cytometric analysis using anti CD4-, CD8-, CD45RA- and CD45RO monoclonal antibodies. The binding assay of T lymphocytes to HDMEC was performed before and after stimulating HDMEC with IL-l alpha or TNF-alpha. RESULTS: 1. The number of CD4- T cells and the CD4+ to CD8+ ratio decreased in patients with Behcets disease compared to normal controls. 2. In the CD4+ T cell subpopulation, there was a significant decrease in the CD4+CD45RA+cell number with a slight increase in the CD4+ CD45RO+ cell number. 3. After stimulating HDMEC with IL-la and TNF-a, the degree of T lymphocyte-HDMEC adhesion generally increased in an E:T ratio dependent, manner in patients with Behcets disease compared to normal controls. 4. Increased binding of CD4+ CD45RA+ naive T lymphocytes and CD4+CD45RO+ memory T lymphocytes to HDMEC was induced after stimulation with IL-1 alpha and TNF-alpha in both patients and normal controls. The increasing rate was higher in Behcets disease patients than in normal controls. There was no difference in T lymphocyte-HDMEC adhesion between memory and naive T lymphocytes. CONCLUSION: From these findings it can be postulated that the decrease in the CD4+CD45RA+ count may lead to the inactivation of CD8 suppressor cells resulting in abnormal immune suppression shown in Behcets disease. Proinflammatory cytokines may also play an integral role in the pathogenesis of Behcet's disease by activating endothelial cells increasing the interaction between T lymphocytes and endothelial cells. Increasing the interaction between T lymphocytes and HDMEC may be indirect evidence of activation of cell-mediated immunity.

Expression of iC3b Receptor on Candida albicans and Its Role on Adhesion of the Yeast to Human Dermal Microvascular Endothelium / 대한의진균학회지

BACKGROUND: The adherence of microorganisms to host tissue is an important process in the pathogenesis of fungal infection. A protein that shares antigenic and structural homology with the alpha-subunit of leukocyte adhesion glycoprotein CD11b/CD18, also known as iC3b receptor, Mo-1 or Mac-1, has been isolated from the surface of C. albicans. OBJECTIVE: This study was done to observe the changes in the expression of iC3b receptor on C. albicans by glucose or immunosuppressive agents and to elucidate the effect of glucose and anti-iC3b receptor antibodies on adhesion between human dermal microvascular endothelial cells(HDMEC) and C. albicans. METHODS: We utilized immunofluorescence study and immunofluorescent flow cytometry and the binding assay of C. albicans to cultured HDMEC in vitro and blocking assay using monoclonal antibody were performed. RESULTS: Immunofluorescence study and immunofluorescent flow cytometric analysis demonstrated surface expression of iC3b receptor on C. albicans. The expression of surface iC3b receptor on C. albicans in creased in a dose dependent manner with increasing concentrations of glucose, cyclophosphamide and prednisolone. The adherence of C. albicans to HDMEC correlated positively with glucose levels. The adherence of C. albicans to HDMEC decreased significantly by the treatment with anti-iC3b receptor antibodies. CONCLUSION: The results suggest that iC3b receptors should be involved in the adherence of C. albicans to vascular endothelial cells and may be involved in the pathogenesis of hematogenous candidiasis