Human Foreskin Microvascular Endothelial Cells Research Articles

Differential gene expression analysis of tubule forming and non-tubule forming endothelial cells: CDC42GAP as a counter-regulator in tubule formation

The formation of new tubular structures from a quiescent endothelial lining is one of the hallmarks of sprouting angiogenesis. This process can be mimicked in vitro by inducing capillary-like tubular structures in a three-dimensional (3D) fibrin matrix. We aimed to analyze the differential mRNA expression in two phenotypically distinct cell populations from the same culture, namely in tubule-forming endothelial cells and monolayer endothelial cells not participating in tubule formation. A fibrin-rich 3D matrix derived from human plasma was used to facilitate tubule formation by human foreskin microvascular endothelial cells (hMVEC). After 7 days of stimulation with VEGF, bFGF, and TNF-alpha, the culture consisted of a monolayer and capillary-like sprouts that had grown into the fibrinous matrix. A method was developed to separate the monolayer and tubule-forming populations of hMVEC, keeping their cellular integrity intact to ensure mRNA extraction and cDNA production. Subsequent array analysis resulted in an inventory of differentially expressed genes that were associated with either tube-forming (angiogenic) or non-angiogenic capacity. Differential gene expression was verified by real-time PCR on the original RNA samples as well as on RNA obtained from laser-capture microdissected cross sections of monolayers and capillary structures in the 3D fibrinous matrix. The expression of CDC42GAP, an inhibitor of active-state small Rho GTPases, was reduced in tubular hMVEC. Overexpression of CDC42GAP in hMVEC attenuated endothelial tubule formation, while its suppression by siRNA slightly enhanced this process. Thus, CDC42GAP was identified as a counter-regulatory mediator for tubule formation.

Activation of human endothelial cells by tumor necrosis factor‐α results in profound changes in the expression of glycosylation‐related genes

The endothelium plays a central role in the logistics of the immune system by allowing the selective transmigration of leukocytes, as well as the maintenance of the circulation and coagulation homeostasis. Evidence is increasing that the carbohydrate composition of the endothelial cell surface is critical for the cells to exert their physiological function. The major aim of this study is to unravel the mechanisms underlying the expression of carbohydrate structures by endothelial cells, which are involved in leukocyte adhesion and migration. Using quantitative real-time PCR, the expression profile of a selected group of 74 glycosylation-related genes has been determined in human umbilical vein endothelial cells (HUVEC) and human foreskin microvascular endothelial cells (FMVEC) under control and TNFalpha-induced conditions. The set of genes comprised 59 glycosyltransferases, 6 mannosidases and 9 sulfotransferases. In parallel, the overall cell surface glycan profile has been assessed by the use of glycan-specific lectins and monoclonal antibodies. The results demonstrate that HUVEC and FMVEC differ substantially in the expression of glycosylation-related genes and, accordingly, also in the presence of different glycan epitopes on the cell membrane. Induction of an inflamed phenotype of the cells by treatment with TNFalpha differentially modulates a set of these genes in HUVEC and FMVEC resulting in a change in the cell membrane associated glycans that are of importance in inflammation-related endothelial cell-surface processes.

Enhanced angiogenic capacity and urokinase-type plasminogen activator expression by endothelial cells isolated from human endometrium.

The endometrium is a tissue unique for its cyclic destruction and rapid regeneration of blood vessels. Angiogenesis, indispensable for the regeneration process, provides a richly vascularized, receptive endometrium fundamental for implantation, placentation, and embryogenesis. Human endometrial microvascular endothelial cells (hEMVEC) were isolated to better understand the properties and angiogenic behavior of these cells. Unlike human foreskin microvascular endothelial cells (hFMVEC), which proliferated better upon stimulation by basic fibroblast growth factor, hEMVEC were much more sensitive to vascular endothelial growth factor A (VEGF-A) stimulation, probably due to enhanced VEGF receptor 2 expression. In addition, hEMVEC displayed an enhanced expression of the urokinase-type plasminogen activator (u-PA) compared with hFMVEC. No differences were found in tissue-type PA, PA inhibitor-1, and u-PA receptor expression. The high expression of u-PA by hEMVEC was also found in tissue sections. hEMVEC formed capillary-like structures when cultured in 20% human serum on top of three-dimensional fibrin matrices, and VEGF-A or basic fibroblast growth factor increased this tube formation. This is in contrast with hFMVEC, which formed tubes only after simultaneous stimulation by a growth factor and tumor necrosis factor-alpha. The high basal level of u-PA contributes to and may explain the higher angiogenic properties of hEMVEC (in vitro).

Proteolysis of the urokinase-type plasminogen activator receptor by metalloproteinase-12: implication for angiogenesis in fibrin matrices

AbstractPericellular proteolysis plays an important role in cell migration and the formation of new capillary structures. The plasminogen activator/plasmin and matrix degrading metalloproteinase (MMP) cascades act together in the remodeling of matrix and cell-matrix contacts. Previously we have shown that the formation of capillary structures by human foreskin microvascular endothelial cells (hMVECs) in a 3-dimensional fibrin matrix requires a functional urokinase-type plasminogen activator receptor (u-PAR). Here we report on the unexpected finding that inhibition of hMVEC-derived MMP activity by BB94 (batimastat) increased the outgrowth of capillary structures in a fibrin matrix. BB94 prevented the release of the u-PA binding domain D1 of u-PAR and thereby increased the number of functional u-PARs on hMVECs without affecting the u-PAR messenger RNA levels. Comparison of various types of protease inhibitors pointed to the prime involvement of MMP activity. Using recombinant MMPs it was shown that MMP-12 activity was able to release the D1 domain of cellularly expressed u-PAR. In addition, the expression of MMP-12 in control and basic fibroblast growth factor/tumor necrosis factor-α–stimulated hMVECs was shown by reverse transcriptase–polymerase chain reaction, suggesting that endothelial cell–derived MMP-12 may be involved in angiogenesis-related u-PAR shedding. This new mechanism of u-PAR cleavage provides new insights into the mutual interactions between the MMP and u-PA/plasmin systems. Moreover, it may be helpful in the interpretation of recent data on the use of specific MMP inhibitors in the treatment of several types of cancer.

Transcriptional profiling of human microvascular endothelial cells in the proliferative and quiescent state using cDNA arrays.

Endothelial cells are known to be a rich source of transcriptional gene expression. Recent technological advances now permit the detailed profiling of mRNA expression using arrays of known cDNAs on blots. We have used such arrays to examine expression of mRNA by primary isolates of human foreskin microvascular endothelial cells in the proliferative and quiescent state. Cells were stimulated by a combination of known growth factors for these cells including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and 'endothelial cell growth supplement (ECGS)' either alone or in combination. Analysis showed the expression of many mRNAs but of the 588 examined, only one, namely monocyte chemotactic protein-1 (MCP-1), showed a decrease on treatment with EGF. A combination of image grabbing followed by subtractive densitometry enabled identification of the mRNAs upregulated in proliferating endothelium. In consideration of the possibility of selective vascular targeting, of particular interest was the increase in expression of the mRNA for the cell surface proteins vascular endothelial (VE-) and neural (N-) cadherin and alpha5, alphav, beta1 and beta3 integrins. The alpha5 integrin offers a previously unrecognized opportunity for vascular targeting.

Interleukin-4 Stimulates Expression of Urokinase-Type-Plasminogen Activator in Cultured Human Foreskin Microvascular Endothelial Cells

AbstractThe effect of interleukin-4 (IL-4) on the fibrinolytic system of human microvascular and macrovascular endothelial cells in culture was studied. Only foreskin microvascular endothelial cells (EC) responded to IL-4 treatment with a dose- and time-dependent increase in urokinase-type plasminogen activator (u-PA) (control: 3.0 ± 0.8 ng/105 cells/24 h; 200 U/mL IL-4: 6.7 ± 0.8 ng/105 cells/24 h), whereas human macrovascular EC remained unaffected. A maximum effect was achieved with 200 U/mL IL-4. Little u-PA activity was detected in the conditioned media of human foreskin microvascular EC (HFMEC) treated without and with IL-4 before plasmin treatment (control: 0.03 ± 0.003 IU/105 cells/20h; 200 U/mL IL-4: 0.09 ± 0.007 IU/10s cells/20 h). These values increased to 0.18 ± 0.02 IU/105 cells/20 h and 0.53 ± 0.04 IU/105 cells/20 h, respectively, after plasmin treatment, indicating that u-PA is released by HFMEC predominantly in its inactive precursor form single-chain u-PA (scu-PA). u-PA activity increased also in the cell lysates of HFMEC up to 2.5-fold after IL-4 treatment. Plasminogen activator inhibitor type-1 (PAI-1) levels produced by HFMEC remained unaffected by IL-4, whereas tissue-type plasminogen activator (t-PA) levels were slightly decreased when HFMEC were treated with IL-4. These findings were also reflected in the specific mRNA levels as determined by Northern blotting, u-PA-specific mRNA increased significantly in HFMEC in the presence of IL-4, whereas t-PA mRNA and PAI-1-specific mRNA in HFMEC and u-PA specific mRNA in human saphenous vein EC (HSVEC) remained unaffected by IL-4 treatment. Our findings suggest a role for IL-4 in the process of angiogenesis, in addition to its known proliferative effect on human microvascular EC, by increasing the fibrinolytic potential of such EC, thereby facilitating extracellular proteolysis and cell migration.

Interleukin-4 stimulates expression of urokinase-type-plasminogen activator in cultured human foreskin microvascular endothelial cells

The effect of interleukin-4 (IL-4) on the fibrinolytic system of human microvascular and macrovascular endothelial cells in culture was studied. Only foreskin microvascular endothelial cells (EC) responded to IL-4 treatment with a dose- and time-dependent increase in urokinase- type plasminogen activator (u-PA) (control: 3.0 +/- 0.8 ng/10(5) cells/24 h; 200 U/mL IL-4: 6.7 +/- 0.8 ng/10(5) cells/24 h), whereas human macrovascular EC remained unaffected. A maximum effect was achieved with 200 U/mL IL-4. Little u-PA activity was detected in the conditioned media of human foreskin microvascular EC (HFMEC) treated without and with IL-4 before plasmin treatment (control: 0.03 +/- 0.003 IU/10(5) cells/20 h; 200 U/mL IL-4: 0.09 +/- 0.007 IU/10(5) cells/20 h). These values increased to 0.18 +/- 0.02 IU/10(5) cells/20 h and 0.53 +/- 0.04 IU/10(5) cells/20 h, respectively, after plasmin treatment, indicating that u-PA is released by HFMEC predominantly in its inactive precursor form single-chain u-PA (scu-PA). u-PA activity increased also in the cell lysates of HFMEC up to 2.5-fold after IL-4 treatment. Plasminogen activator inhibitor type-1 (PAI-1) levels produced by HFMEC remained unaffected by IL-4, whereas tissue-type plasminogen activator (t-PA) levels were slightly decreased when HFMEC were treated with IL-4. These findings were also reflected in the specific mRNA levels as determined by Northern blotting. u-PA-specific mRNA increased significantly in HFMEC in the presence of IL-4, whereas t-PA mRNA and PAI-1-specific mRNA in HFMEC and u-PA specific mRNA in human saphenous vein EC (HSVEC) remained unaffected by IL-4 treatment. Our findings suggest a role for IL-4 in the process of angiogenesis, in addition to its known proliferative effect on human microvascular EC, by increasing the fibrinolytic potential of such EC, thereby facilitating extracellular proteolysis and cell migration.

γ-Interferon counteracts interleukin-1α stimulated expression of urokinase-type plasminogen activator in human endothelial cells in vitro

The effect of γ-interferon (γ-IFN) on the interleukin-1α (IL-1γ) induced stimulation of urokinasetype plasminogen activator (u-PA) expression in human foreskin microvascular endothelial cells (HFMEC) and in human umbilical vein endothelial cells (HFMEC) was investigated. When γ-IFN and IL-1α were added to the cells simultaneously, γ-IFN inhibited the IL-1α induced increase in uPA antigen production in both HFMEC and HUVEC in a dose dependent fashion, with a maximum inhibitory effect achieved between 2.0 and 20.0U/ml of γ-IFN. Pretreatment of HFMEC with γ-IFN for 1 hour before addition of IL-1α resulted in a significant reduction in u-PA synthesis. However, when HFMEC were pretreated for 8 hours with γ-IFN before the addition of IL-1α the reduction in u-PA production was even more significant. When γ-IFN was added to HFMEC 1 hour after IL-1α, a significant inhibition in u-PA synthesis was seen. In contrast only a slight inhibition in IL-1α induced u-PA production was seen when γ-IFN was added to the cells 8 hours after IL-1a. γ-IFN also inhibited significantly the IL-1α induced increase in u-PA specific mRNA in HUVEC and HFMEC.

Effect of Thrombin on the Production of Plasminogen Activators and PA Inhibitor-1 by Human Foreskin Microvascular Endothelial Cells

Human foreskin microvascular endothelial cells synthesize and release tissue-type plasminogen activator (t-PA) in similar amounts as do endothelial cells from umbilical cord artery and vein. Human thrombin increases the production of t-PA by these cells, which could be visualized from 8 h after addition of 0.1-5 units/ml thrombin by fibrin autography after SDS polyacrylamide gel electrophoresis of the endothelial cell conditioned media. Thrombin also increased the secretion of t-PA antigen. Together with t-PA, human microvascular cells release urokinase-type plasminogen activator (u-PA) antigen and endothelial cell-type PA inhibitor, PA inhibitor-1, which were both demonstrated by specific immunoprecipitation from radiolabeled endothelial cell conditioned medium. Thrombin increases the release of u-PA antigen, but no u-PA activity could be demonstrated. Thrombin induced a two-fold stimulation of the synthesis and secretion of PA inhibitor-1 antigen. At 0.1 unit/ml thrombin also an increase in PA inhibitor activity was found. At high concentrations of thrombin a decrease of PA inhibitor activity was found, due to the conversion of the active 46 kD PA inhibitor-1 into a 42 kD product without PA inhibitor activity. Our data indicate that interaction of thrombin with microvascular endothelial cells will shift the balance between t-PA, u-PA and PA inhibitor-1, and thus affects the regulation of fibrinolysis.