Human Omental Microvascular Endothelial Research Articles

Human Omental Microvascular Endothelial and Mesothelial Cells: Characterization of Two Distinct Mesodermally Derived Epithelial Cells

Human omental microvascular endothelial (HOME) and mesothelial (MESO) cells share many phenotypic properties, but can be characterized from one another based upon a comprehensive panel of endothelial and mesothelial markers. Traditional cell markers such as vonWillebrand factor, DiI-Ac-LDL, andUlex europaeusI lectin are not sufficient to distinguish between HOME and MESO cells. Furthermore, immunoreactivity to a panel of endothelial cell-specific monoclonal antibodies, including representatives from the known clusters of differentiation (CD), indicate that some of these antigens are coexpressed in HOME and MESO cells. In distinguishing between the two cell types, HOME and not MESO cells express E-selectin, E/P-selectin, P-selectin (CD62), Le-y, and VLA-6 (CDw49f*). Moreover, HOME cells and not MESO cells form tube-like structures when cultured on Matrigel. MESO cells differ from HOME cells based upon (1) the expression of cytokeratins; (2) their rapid proliferation in response to platelet-derived growth factor; and (3) a change from an epitheliod to fibroblast-like morphology in response to tumor necrosis factor and epidermal growth factor. Both HOME and MESO cells express tissue plasminogen activator and plasminogen activator inhibitor, but urokinase activity is only expressed by MESO cells. As there is no one universal endothelial or mesothelial cell marker that can specifically confirm the identity of these cells, it appears necessary to employ a comprehensive panel of cell markers to rule out the possibility of misidentifying a cell culture.

Tubulogenesis by microvascular endothelial cells is mediated by vascular endothelial growth factor (VEGF) in renal cell carcinoma.

To investigate the role of vascular endothelial growth factor (VEGF) in tumour angiogenesis in renal cell carcinoma (RCC). The expression of VEGF was examined in tissue samples from 25 patients with RCC using the reverse-transcriptase polymerase chain reaction (RT-PCR). Cellular localization of VEGF was studied in normal kidney and RCC tissues. Tube formation by human omental microvascular endothelial (HOME) cells co-cultured with A498 RCC cells was quantified in a three-dimensional collagen gel using computer-image analysis. RT-PCR detected VEGF m-RNA in tissue from 20 of 25 patients with RCC. An immunohistochemical study revealed that VEGF was primarily localized in the cytosol of normal renal tubule cells and RCC cells. Tube formation by HOME cells was increased in the presence of A498 cells overexpressing VEGF mRNA, induced by exogenous VEGF in a dose-dependent manner and completely inhibited by an anti-VEGF antibody. VEGF, which is produced and released from RCC cells, may elicit tumour angiogenesis by inducing microvessel tubulogenesis in patients with RCC. The co-culture system may be useful for screening inhibitors of tumour angiogenesis in RCC.

Induction of Tubulogenesis of Microvascular Endothelial Cells by Basic Fibroblast Growth Factor from Human SN12C Renal Cancer Cells

To investigate the role of basic fibroblast growth factor (b-FGF) in angiogenesis in renal cell carcinoma. We examined b-FGF expression in 25 patients with renal cell carcinoma (RCC) by reverse transcriptase polymerase chain reaction (RT-PCR). We measured the tube formation by human omental microvascular endothelial (HOME) cells induced by b-FGF in a three-dimensional collagen gel in the absence or presence of SN12C human renal cancer cells by means of computer-image analysis. RT-PCR analysis detected b-FGF m-RNA in 20 (80%) of 25 patients with RCC. The tube formation of HOME cells was increased in the presence of SN12C cells overexpressing b-FGF mRNA, induced by exogenous b-FGF in a dose dependent manner and completely inhibited by anti-b-FGF antibody. Basic-FGF may develop tumor angiogenesis by inducing microvessel tubulogenesis in patients with RCC.

Expression of tissue‐type plasminogen activator and its inhibitor couples with development of capillary network by human microvascular endothelial cells on matrigel

Human omental microvascular endothelial (HOME) cells seeded on Matrigel begin to migrate within 1 h, forming honeycomb-like structures and capillary-like networks within 18 h. Cross-sections of the capillary networks show them to be tube-like structures. Northern blot analysis showed that tissue-type plasminogen activator (t-PA) mRNA synthesis increased from the initial state at 0 h after seeding on Matrigel, reaching a steady state after 4 h. This elevated cellular t-PA mRNA level decreased markedly at 24 h. In contrast, the cellular plasminogen activator inhibitor-1 (PAI-1) mRNA level demonstrated biphasic curves during the 24 h after seeding on Matrigel: the PAI-1 mRNA level was increased eightfold initially at 4 h over that at 0 h, then declined, and again secondarily increased to greater than tenfold at 18 h. Cellular levels of both 72 kD type IV collagenase and tissue inhibitor of metalloproteinase (TIMP-2) mRNA were increased only a slightly within 2-4 h. These elevated mRNA levels were maintained for 18 h, while the TIMP-1 mRNA level increased up to 18 h, reaching around three times the level at 0 h. However, on collagen-coated dishes, cellular levels of t-PA, PAI-1, 72 kD type IV collagenase, TIMP-1, and TIMP-2 mRNA were not greatly changed during incubation for 24 h. On Matrigel, the cellular t-PA mRNA level at 18 h after seeding was greatly increased when treated with specific anti-transforming growth factor-beta (TGF-beta) antibody. In contrast, both PAI-1 and TIMP-1 mRNA levels at 18 h were reduced in the presence of anti-TGF-beta antibody. Development of the capillary network on Matrigel was inhibited in the presence of anti-t-PA antibody. Epidermal growth factor (EGF) enhanced t-PA gene expression and TGF-beta inhibited its expression in HOME cells cultured on collagen-coated dishes. On the other hand, TGF-beta enhanced cellular expression of the PAI-1 gene. The formation of a capillary network by HOME cells on Matrigel appears to be balanced by angiogenic EGF and anti-angiogenic TGF-beta through modulation of PA activity.

Inhibition of tubular morphogenesis in human microvascular endothelial cells by co-culture with chondrocytes and involvement of transforming growth factor β: a model for avascularity in human cartilage

Tube formation in collagen gel was induced in human omental microvascular endothelial (HOME) cells in the presence of epidermal growth factor (EGF) or transforming growth factor-α (TGF-α). TGF-α enhanced the expression of the tissue plasminogen activator (t-PA) gene, whereas TGF-β increased the expression of the PA inhibitor-1 (PAI-1) gene and inhibited that of the t-PA gene. TGF-β inhibited the tube formation of HOME cells in type I collagen gel that was enhanced in response to TGF-α. We have recently established an angiogenesis model in vitro in which vascular endothelial cells on type I collagen gel in an inner chamber are co-cultured with other types of cells in an outer chamber. Here we examined whether the EGF/TGF-α-induced tube formation in HOME cells was modulated by human chondrocytes co-culture in the outer chamber. TGF-α-dependent tube formation of HOME cells was inhibited when human chondrocytes were co-cultured in the outer chamber. This chondrocyte-induced inhibition of tube formation was partly abrogated by co-administration of anti-TGF-β antibody. These findings suggest that TGF-β is partly involved in the human chondrocyte-dependent inhibition of tube formation by human microvascular endothelial cells. This is the first model system demonstrating that avascularity of human chondrocytes is partly due to the TGF-β family produced from them.

Cross Talk of Tumor Necrosis Factor-α and Epidermal Growth Factor in Human Microvascular Endothelial Cells

Our previous studies imply that tumor necrosis factor-α (TNF-α) and epidermal growth factor (EGF) might share a common signal transduction pathway in human omental microvascular endothelial (HOME) cells. Exposure of cultured HOME cells to TNF-α for 10 min enhanced EGF receptor phosphorylation at a rate comparable to EGF. Apparent phosphorylation of tyrosine residues was observed in addition to serine/threonine of the EGF receptor by EGF, but only a slightly if any tyrosine phosphorylation by TNF-α. In vitro kinase activity of EGF receptor was also enhanced by TNF-α as well as by EGF. Furthermore, expression of the c- fos gene was enhanced in response to either EGF or TNF-α. Pretreatment of HOME cells with EGF for 12 h almost completely blocked the induction of the c- fos gene by EGF anti partially blocked the c- fos induction by TNF-α. TNF-α-induced c- fos gene expression appeared to be partly due to its transactivatlon of EGF receptor. EGF and TNF-α could enhance c- fos gene expression when protein kinase C was down-regulated by phorbol ester myristate (PMA). Gel retardation assay with the NF-κB consensus sequence showed that NF-κB binding activity was dramatically activated by TNF-α, but not by EGF or PMA. The binding of another transcription factor, AP-1 (Jun/Fos), was enhanced by EGF, TNF-α, and PMA, whereas TNF-α could still activate AP-1 after longer exposure to EGF. TNF-α-induced activation of c- fos gene appears to be mediated through pleiotropic mechanisms and partly through a common signal with EGF, possibly through EGF receptor in microvascular endothelial cells.

Cooperative roles of hepatocyte growth factor and plasminogen activator in tubular morphogenesis by human microvascular endothelial cells.

Epidermal growth factor (EGF) or transforming growth factor‐α (TGF‐α) stimulated cell migration, chemotaxis, and the expression of tissue‐type plasminogen activator (t‐PA) in human omental microvascular endothelial (HOME) cells. Hepatocyte growth factor (HGF) stimulated cell proliferation, but had a negligible stimulatory effect on cell migration, the expression of t‐PA and tube‐like formation into collagen gel in HOME cells. Basic fibroblast growth factor stimulated cell proliferation, cell migration, tubulogenesis and the expression of urokinase‐type plasminogen activator (u‐PA) in bovine aortic endothelial (BAE) cells. HOME and BAE cells had both high‐ and low‐affinity receptors for HGF. In BAE cells, u‐PA activity and tube‐like structures in collagen gel were induced in the presence of HGF alone. In contrast, in HOME cells, t‐PA activity and tube‐like structures were induced in the presence of TGF‐a alone, but not in the presence of HGF alone. However, we observed a marked induction of tube formation by HOME cells when both t‐PA and HGF were added simultaneously. In the model system for tumor angiogenesis, when HOME cells were co‐cultured with a renal cancer cell line, KPK13, tube‐like structures were induced in the presence of HGF: KPK13 cells expressed large amounts of t‐PA mRNA. Our present study suggested that HGF in concert with active t‐PA could be angiogenic in HOME cells.

Anti-angiogenic activity of arachidonic acid metabolism inhibitors in angiogenesis model systems involving human microvascular endothelial cells and neovascularization in mice.

We have established an in vitro angiogenesis model using human omental microvascular endothelial (HOME) cells, in which epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) stimulated cell migration and tube formation. In this study, we examined whether alpha-guaiaconic acid (GR-12) and its synthetic 20 derivatives showed inhibition of cell migration and tubular formation of HOME cells. We found that GR-12 inhibits arachidonic acid metabolism, while GR-12 and one derivative, GS-01, inhibit tubular formation of endothelial cells in our model system. Confluent monolayers of HOME cells were damaged with a razor blade and incubated with or without TGF-alpha; HOME cell migration was stimulated about 1.5-fold over control values in the presence of TGF-alpha. Treatment of HOME cells with GR-12 or GS-01 inhibited both spontaneous and TGF-alpha-stimulated migration. GR-12 or GS-01 inhibited TGF-alpha-induced HOME-cell tube formation in type-1 collagen gels. We examined whether these compounds could modulate tubular formation of HOME cells induced by human cancer cells. Enhanced tube formation of HOME cells by co-cultured esophageal cancer cells was almost completely inhibited by co-administration of GR-12 or GS-01. Both compounds also inhibited formation of tubular networks of HOME cells on Matrigels. We also examined anti-angiogenic activity of these compounds in an in vivo model system of tumor angiogenesis in mice. In this system, GS-01 inhibited development of capillary networks at a rate comparable to that of a well-known anti-angiogenic compound, fumagillin, but GR-12 did not. The inhibitor of arachidonic acid metabolism is thus expected to modulate tumor angiogenesis.

Morphological Change and Destabilization of β-Actin mRNA by Tumor Necrosis Factor in Human Microvascular Endothelial Cells

Tumor necrosis factor-α (TNF-α) produced morphological changes from a cobblestone-like shape into a spindle shape in human omental microvascular endothelial (HOME) cells and also a drastic rearrangement of actin filaments. Expression of β-actin gene was diminished in HOME cells treated with TNF-α for 24 h. Northern blot analysis of the β-actin gene demonstrated that the cellular level of β-actin mRNA was decreased at 6-12 h after exposure to TNF-α. However, there appeared to be no changes in cellular mRNA levels of β-tubulin, fibronectin, laminin B1, laminin B2, and laminin binding protein genes after treatment with TNF-α. Nuclear run-on assays showed increased transcription of the low-density lipoprotein receptor gene, but not of the β-actin gene. These data suggested that the TNF-α-induced inhibition of β-actin gene expression was not due to altered transcription activity. The degradation rates of β-actin, plasminogen activator inhibitor-1, and epidermal growth factor receptor mRNAs were examined in the presence of actinomycin D. β-Actin mRNA was found to be specifically destabilized in TNF-αtreated HOME cells, while other mRNA species were not. Coadministration of cycloheximide blocked the TNF-α-induced degradation of β-actin mRNA. The TNF-α-induced destabilization of β-actin mRNA and rearrangement of actin filaments are discussed in relation to the morphological changes in human microvascular endothelial cells.

Indispensable Role of Tissue-Type Plasminogen Activator in Growth Factor-Dependent Tube Formation of Human Microvascular Endothelial Cells in Vitro

Epidermal growth factor (EGF) stimulates the migration and proliferation of, and tissue-type plasminogen activator (tPA) synthesis in, human omental microvascular endothelial (HOME) cells in culture, as well as inducing tube formatin by these cells. In the present study, we examined the effects of various growth factors, i.e., transforming growth factor-α (TGF-α), insulin-like growth factor 1 (IGF-1), and hepatocyte growth factor (HGF) on HOME cells, and compared their effects with that of EGF. IGF-1 stimulated the proliferation and migration of these cells at a level comparable to EGF. EGF and TGF-α induced expression of tPA in HOME cells, while IGF-1 and HGF did not. EGF and TGF-α induced tube formation by HOME cells in type I collagen gel, while IGF-1 and HGF did not. The stimulatory effect of EGF on tube formation in the gel was blocked by anti-tPA antibody and by a serine protease inhibitor, aprotinin. When exogenous tPA and IGF-1 or HGF were added simultaneously to the culture, a marked induction of tube formation in the gel was observed. Exogenously added tPA alone, however, had no such inducible effect on tube formation. These results indicated an indispensable role of tPA in growth factor-dependent tube formation by HOME cells. Two subsets of growth factors appeared to modulate angiogenesis: One with fully active angiogenic activity which could induce PA (this included EGF and TGF-α), and the other, which could not induce PA and was not angiogenic, but could promote angiogenesis in the presence of PA. This subset included IGF-1 and HGF.

Induction of human microvascular endothelial tubular morphogenesis by human keratinocytes: Involvement of transforming growth factor-α

Transforming growth factor-α(TGF-α), homologous to epidermal growth factor(EGF), is closely involved in hyperproliferation of human keratinocytes. Psoriasis is a common hyperproliferative skin disease characterized by hyperproliferation of keratinocytes and abnormal development of dermal capillary networks. In this study, we have examined whether keratinocytes could enhance angiogenesis. TGF-α or EGF efficiently stimulated formation of tubular-like structures of human omental microvascular endothelial(HOME) cells in type I collagen gels. Human keratinocytes produced TGF-α. To examine whether co-cultured keratinocytes could induce tubulogenesis of HOME cells in collagen gel, we have developed a co-culture system with human keratinocytes. Surprisingly, there appeared new development of many tubular-like structures of HOME cells in collagen gels when co-cultured with keratinocytes. This keratinocytes-dependent tubulogenesis was almost completely blocked when anti-TGF-α-antibody was present. The TGF-α molecules derived from keratinocytes appeared to enhance tubulogenesis of human microvascular endothelial cells. We propose the hypothesis that secretory TGF-α from human keratinocytes may promote an autocrine loop to proliferate the skin keratinocytes and also a paracrine loop to induce the skin angiogenesis.

Induction of low density lipoprotein receptor and a transcription factor SP-1 by tumor necrosis factor in human microvascular endothelial cells.

We have previously reported that tumor necrosis factor-alpha (TNF-alpha) enhances expression of interleukin-6, collagenase, plasminogen activator inhibitor-1, and basic fibroblast growth factor genes in human omental microvascular endothelial (HOME) cells in culture. In this study, we found that treatment of HOME cells with TNF-alpha or interleukin-1 (IL-1) caused enhanced expression of low density lipoprotein (LDL) receptor. A few-fold increase in both LDL binding activity and the receptor mRNA levels was observed when HOME cells were treated with either TNF-alpha or IL-1. Northern blot analysis showed that cellular expression of LDL receptor gene was significantly increased 12-24 h after exposure to TNF-alpha. No significant changes in the life-span of LDL receptor mRNA were observed in untreated and TNF-alpha-treated cells. Scatchard analysis showed an increased receptor number for LDL in TNF-alpha-treated cells. Parallel to increased LDL binding activity, internalization and degradation of LDL were also increased in HOME cells treated with TNF-alpha or IL-1. TNF-alpha-induced enhancement of LDL receptor gene expression was not observed when cycloheximide was present. Cellular mRNA level of SP-1 gene was increased about 3-4-fold at 12 h after treatment with TNF-alpha. Nuclear run-on assays showed increased transcription of LDL receptor gene as well as SP-1 gene by TNF-alpha. Gel retardation assay with the SP-1 consensus fragment showed that SP-1 binding activity was increased about 4-5-fold 12-24 h after treatment with TNF-alpha. NF-kB binding activity was also dramatically increased, but there is no NF-kB motif on the promoter for LDL receptor gene. The induction of LDL receptor by TNF might be mediated through a transcription factor, SP-1.

Decreased response to epidermal growth factor during cellular senescence in cultured human microvascular endothelial cells

We have previously demonstrated that epidermal growth factor (EGF) induces cell migration, tissue-type plasminogen activator synthesis, as well as tubular formation in microvascular endothelial cells from human omental tissue. In this study, we compared the responsiveness to EGF of late passaged (senescent) human omental microvascular endothelial (HOME) cells with that of early passaged (young) HOME cells. We have employed HOME cells derived from surgically resected omental samples from 14 patients. EGF-stimulated cell migration significantly more in the young cells than in the senescent cells during serial cultivation (aging) in vitro. Scatchard analysis demonstrated that the number for both high and low affinity receptors for EGF in HOME cells was decreased dramatically during serial cultivation. The expression of EGF receptor mRNA was also decreased in the senescent HOME cells. Treatment of HOME cells with EGF significantly increased cellular mRNA levels of tissue-type plasminogen activator, and two protooncogenes, c-fos and c-myc, in young HOME cells, but not in senescent HOME cells. Thus HOME cells aged in vitro show a decreased responsiveness to EGF, resulting in decreased migration of human endothelial cells. The serial cultivation of human endothelial cells in vitro may downregulate EGF receptor and decrease responsiveness to exogenous EGF, a potent angiogenic factor.

Endogenous basic fibroblast growth factor-dependent induction of collagenase and interleukin-6 in tumor necrosis factor-treated human microvascular endothelial cells.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to enhance the synthesis of interleukin-6 (IL-6) and collagenase in human omental microvascular endothelial (HOME) cell (Mawatari, M., Kohno, K., Mizoguchi, H., Matsuda, T., Asoh, K., Van Damme, J. V., Welgus, H. G., and Kuwano, M. (1989) J. Immunol. 143, 1619-1627). In the present study, we have examined whether the TNF-alpha-induced synthesis of IL-6 or collagenase in HOME cells is mediated by an inducible growth factor. Among several growth factors examined, we found that the expression of basic fibroblast growth factor (bFGF) mRNA was the one most prominently enhanced when HOME cells were treated with TNF-alpha. The increase of bFGF mRNA by TNF-alpha in HOME cells was observed in both a dose- and time-dependent manner when assayed by Northern blot analysis. The induction of bFGF mRNA was observed by 3 h after incubation with TNF-alpha, and the maximal increase of 5-fold was obtained after 12 h of incubation with 100 units/ml TNF-alpha. Western blot analysis confirmed the enhanced synthesis of bFGF by TNF-alpha. Metabolic labeling and immunoprecipitation assays of bFGF showed that exposure to TNF-alpha enhanced secretion of bFGF into culture medium and also that TNF-alpha stimulated the production of bFGF molecules with molecular masses of 18, 21, and 22.5 kDa in HOME cells. TNF-alpha induced the expression of collagenase mRNA and IL-6 mRNA in HOME cells as well, and the coadministration of neutralizing anti-bFGF antibody almost completely blocked the effects of TNF-alpha. The treatment of HOME cells with exogenous bFGF significantly stimulated the expression of collagenase mRNA and IL-6 mRNA in HOME cells. Therefore, the biological effects of TNF-alpha on HOME cells may be mediated, at least in part, by TNF-alpha-induced bFGF.

Hepatocyte growth factor modulates migration and proliferation of human microvascular endothelial cells in culture

Epidermal growth factor (EGF) induces tubular formation of cultured human omental microvascular endothelial (HOME) cells and EGF also stimulates cell migration as well as expression of tissue type plasminogen activator (t-PA). Here we studied the effects of hepatocyte growth factor (HGF) on cell proliferation, cell migration and expression of t-PA and other related genes. Migration of confluent HOME cells into the denuded space was stimulated by HGF after being wounded with razor blade, but at a reduced rate in comparison with EGF. HOME cells could be proliferated in response to exogenous 100 ng/ml of HGF at rates comparable to that of 20 ng/ml EGF. The chemotactic activity of HOME cells was significantly stimulated by HGF in a dose-dependent manner when assayed by Boyden chamber. HGF did not efficiently enhance expression of both the t-PA gene and a tissue inhibitor of metalloproteinase gene whereas it stimulated expression of plasminogen activator inhibitor-1. Our present study provides a new evidence that some of the biological effects of HGF on HOME cells in culture are similar to those of EGF.