Transforming growth factor beta 1 down-regulates vascular endothelial growth factor receptor 2/flk-1 expression in vascular endothelial cells

Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel-derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor beta 1 (TGF-beta 1) reduced the high affinity 125I-VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0-2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in 125I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-beta 1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-beta 1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells.     

Dual pathways of tubular morphogenesis of vascular endothelial cells by human glioma cells: vascular endothelial growth factor/basic fibroblast growth factor and interleukin-8

In this study, we examined whether human glioma cells are angiogenic in a model using human microvascular endothelial cells, and also which factor is responsible for the glioma-dependent angiogenesis. Tubular morphogenesis in type I collagen gel by human microvascular endothelial cells was stimulated in the presence of 10 and 100 ng/ml of vascular endothelial growth factor (VEGF), 10 ng/ml basic fibroblast growth factor (bFGF) and 10 ng/ml of interleukin-8 (IL-8). Tube formation of the microvascular endothelial cells was assayed in the glioma cell lines IN157 and IN301, co-cultured using the double chamber method. IN301 cells had much higher levels of VEGF, bFGF and transforming growth factor-beta mRNA than IN157 cells, whereas the two had similar levels of transforming growth factor-alpha mRNA. By contrast, IN157 cells had much higher levels of IL-8 mRNA than IN301 cells. IN301-dependent tubular morphogenesis was inhibited by anti-VEGF or anti-bFGF antibody, and the inhibition was almost complete when anti-VEGF and anti-bFGF antibodies were present. On the other hand, IN157-dependent tubular morphogenesis was inhibited by anti-IL-8 antibody, but not by anti-VEGF or anti-bFGF antibodies. These findings demonstrated dual paracrine controls of tumor angiogenesis by human glioma cells. One is mediated through VEGF and/or bFGF, and the other, through IL-8.     

Hypoxia induces vascular endothelial growth factor gene and protein expression in cultured rat islet cells

The formation of new microvasculature by capillary sprouting at the site of islet transplantation is crucial for the long-term survival and function of the graft. Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, may be a key factor in modulating the revascularization of islets after transplantation. In this study, we examined the gene expression of VEGF mRNA in three tumor cell lines and in isolated whole and dispersed rat islets in vitro by Northern blot hybridization in normoxic (5% CO2, 95% humidified air) and hypoxic (1% O2, 5% CO2, 94% N2) culture conditions. Increased expression of VEGF mRNA was observed in beta-TC3, RAW 264.7, and IC-21 tumor cell lines when subjected to hypoxia. With isolated whole islets in normoxic culture, a threefold increase in VEGF mRNA (P < 0.001) was seen at 48 h as compared with freshly isolated islets. This response was similar to the 3.8-fold increase observed with islets subjected to hypoxia. Dispersed rat islet cell clusters cultured on Matrigel for 24 h under hypoxic conditions showed a 3.4-fold increase (P < 0.01) in VEGF mRNA compared with those cultured in normoxia. This correlated with increased VEGF secretion as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies revealed the presence of increased expression of VEGF protein near the center of islets after 24 h of normoxic culture. Islet cell clusters on Matrigel showed intense cellular localization of VEGF in both beta-cells and non-beta-cells. These findings suggest that rat islet cells, when subjected to hypoxia during the first few days after transplantation, may act as a major source of VEGF, thereby initiating revascularization and maintaining the vascular permeability of the grafted islets.     

Expressions of vascular endothelial growth factor in nonparenchymal as well as parenchymal cells in rat liver after necrosis

Vascular endothelial growth factor (VEGF) can induce proliferation of sinusoidal endothelial cells. Its mRNA expression was increased in proliferating rat hepatocytes in primary culture. To clarify a role of VEGF in liver after necrosis, expressions of VEGF and its receptors were measured in the liver or liver cells isolated from rats after carbon tetrachloride intoxication. Hepatic VEGF mRNA expression increased later than 24 h after the intoxication and became prominent at 168 h when liver necrosis disappeared, while hepatic mRNA expressions of its receptors increased between 24 and 72 h. VEGF mRNA expression was increased in Kupffer cells, hepatic macrophages and stellate cells isolated from rats between 24 and 72 h after the intoxication and in hepatocytes at 168 h compared to those cells from normal rats. Immunohistochemical VEGF stains were comparable to such results. Vascular endothelial cells existed abundantly in the necrotic areas, and sinusoidal endothelial cells appeared following disappearance of the necrotic areas. VEGF mRNA expression in hepatocytes isolated from 70% resected liver was increased at 12 h after the operation and became marked between 72 and 168 h. Similar increase of hepatic VEGF expression was immunohistochemically seen. In conclusion, VEGF derives from nonparenchymal as well as parenchymal cells in rat liver after necrosis. The former might contribute to vascular endothelial cell proliferation and the latter to sinusoidal endothelial cell regeneration.     

Human neutrophils secrete vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a pivotal role in mediating neovascularization as well as other endothelial cell alterations during inflammation. In this study, we demonstrate that human neutrophils are a source of VEGF. We observed that isolated blood neutrophils released VEGF in response to different stimuli and we demonstrated by immunohistochemistry that neutrophils infiltrating inflamed tissues contain VEGF. These results indicate that neutrophil-derived VEGF may be instrumental in regulating vascular responses during acute and chronic inflammation.     

Retinoids downregulate vascular endothelial growth factor/vascular permeability factor production by normal human keratinocytes

PURPOSE: We describe the clinical presentation, angiographic findings, and clinical outcome in a group of patients with pseudoaneurysms treated by a new endovascular technique using Guglielmi electrolytically detachable platinum coils (GDCs). METHODS: We retrospectively reviewed the angiographic and clinical findings in a series of 11 patients with pseudoaneurysms occurring in a variety of locations: seven in the cavernous carotid artery, one in the petrous carotid artery, two in the anterior cerebral artery, and one in the cervical vertebral artery. RESULTS: All aneurysms were cured with GDC embolization. The only complication was a branch occlusion, which resolved with heparinization and produced no clinical sequelae. CONCLUSION: Pseudoaneurysms can be safely and effectively treated by embolization with GDCs. Consideration needs to be given to the anatomic location of the pseudoaneurysm and the acuity of onset. Treatment efficacy may by improved if there are bony confines around the aneurysm or if therapy takes place in the subacute period, when the wall of the pseudoaneurysm has matured and stabilized.     

Ligand-dependent development of the endothelial and hemopoietic lineages from embryonic mesodermal cells expressing vascular endothelial growth factor receptor 2

The existence of a common precursor for endothelial and hemopoietic cells, termed the hemangioblast, has been postulated since the beginning of the century. Recently, deletion of the endothelial-specific vascular endothelial growth factor receptor 2 (VEGFR2) by gene targeting has shown that both endothelial and hemopoietic cells are absent in homozygous null mice. This observation suggested that VEGFR2 could be expressed by the hemangioblast and essential for its further differentiation along both lineages. However, it was not possible to exclude the hypothesis that hemopoietic failure was a secondary effect resulting from the absence of an endothelial cell microenvironment. To distinguish between these two hypotheses, we have produced a mAb directed against the extracellular domain of avian VEGFR2 and isolated VEGFR2+ cells from the mesoderm of chicken embryos at the gastrulation stage. We have found that in clonal cultures, a VEGFR2+ cell gives rise to either a hemopoietic or an endothelial cell colony. The developmental decision appears to be regulated by the binding of two different VEGFR2 ligands. Thus, endothelial differentiation requires VEGF, whereas hemopoietic differentiation occurs in the absence of VEGF and is significantly reduced by soluble VEGFR2, showing that this process could be mediated by a second, yet unidentified, VEGFR2 ligand. These observations thus suggest strongly that in the absence of the VEGFR2 gene product, the precursors of both hemopoietic and vascular endothelial lineages cannot survive. These cells therefore might be the initial targets of the VEGFR2 null mutation.     

Thrombomodulin-dependent anticoagulant activity is regulated by vascular endothelial growth factor

Thrombomodulin (TM) is a cell-surface receptor that plays a critical role in endothelial cell anticoagulant activity through its cofactor role in the thrombin-catalyzed activation of human protein C. In this study, we examined the effect of vascular endothelial growth factor (VEGF), a potent angiogenic factor, on surface anticoagulant activity and thrombomodulin expression. We show that thrombin-dependent activation of human protein C, measured on the endothelial cell surface, increased from 50 to 80% following exposure of cells to VEGF for 24 h. The effect was concentration dependent with the half-maximal stimulatory effect at approximately 100 pM. This increase in thrombin-dependent aPC generation correlated with a proportional and concentration-dependent increase in the level of cell-surface TM antigen. Both the total cellular TM antigen and the total cellular TM mRNA levels increased approximately 2.5-fold in VEGF-treated cells suggesting that most if not all of the regulation was at the message level. We further show that VEGF blocked IL-1 beta-induced suppression of both TM surface antigen and mRNA and was similarly capable of antagonizing the down-regulation of TM by TGF-beta and from cell activation by LPS. Our data suggest that VEGF regulation of TM may contribute to mechanisms that would maintain local hemostasis during angiogenesis and revascularization and could play a role in minimizing loss of vessel anticoagulant function during inflammatory processes.     

Thalidomide inhibits corneal angiogenesis induced by vascular endothelial growth factor

BACKGROUND: Ocular diseases caused by neovascularization are among the leading causes of blindness. No specific pharmacological treatment is available. Among potential drugs, thalidomide deserves special interest since a wide body of clinical experience exists. However, its antiangiogenic effect is controversial. We therefore investigated the effect of thalidomide on corneal angiogenesis induced by vascular endothelial growth factor (VEGF), which has a special role among angiogenic growth factors. METHODS: Corneal neovascularization was induced in NZW rabbits by an intrastromal pellet loaded with 500 or 750 ng VEGF. Animals received two daily feedings of 200 mg/kg thalidomide. RESULTS: Significant inhibition of corneal angiogenesis (P < 0.0001) was caused by the teratogenic dose of thalidomide after the 5th day of treatment and persisted for more than 16 days. No obvious side effects were recorded. CONCLUSIONS: Thalidomide has a significant antiangiogenic effect against VEGF-induced neovasclar growth. Together with earlier findings this observation indicates that the drug inhibits two angiogenic pathways which are mediated through integrin adhesion molecules.     

Expression of vascular endothelial growth factor and placenta growth factor in human placenta

Normal development and function of the placenta requires invasion of the maternal decidua by trophoblasts, followed by abundant and organized vascular growth. Little is known of the significance and function of the vascular endothelial growth factor (VEGF) family, which includes VEGF, VEGF-B, and VEGF-C, and of placenta growth factor (PIGF) in these processes. In this study we have analyzed the expression of VEGF and PIGF mRNAs and their protein products in placental tissue obtained from noncomplicated pregnancies. Expression of VEGF and PIGF mRNA was observed by in situ hybridization in the chorionic mesenchyme and villous trophoblasts, respectively. Immunostaining localized the VEGF and PIGF proteins in the vascular endothelium, which was defined by staining for von Willebrand factor and for the Tie receptor tyrosine kinase, an early endothelial cell marker. VEGF-B and VEGF-C mRNAs were strongly expressed in human placenta as evidenced by Northern blot analysis. These data imply that VEGF and PIGF are produced by different cells but that both target the endothelial cells of normal human term placenta.     

Hypoxia and vascular endothelial growth factor stimulate angiogenic integrin expression in bovine retinal microvascular endothelial cells

PURPOSE: Integrins alphavbeta3 and alphavbeta5 are cell-to-matrix adhesion molecules that have been reported to mediate vascular cell proliferation and migration. The authors investigated the regulation of expression of these angiogenic integrins by hypoxia and vascular endothelial growth factor (VEGF) in retinal microvascular endothelial cells in culture. METHODS: Cultured bovine retinal capillary endothelial cells were exposed to human recombinant VEGF under normoxic (95% air, 5% CO2) conditions to assess the effects of VEGF. Hypoxia studies were performed under lower oxygen concentration (0.5%-1.5% O2) induced by nitrogen replacement in constant 5% CO2 conditions. Integrin family mRNA and protein expression were assessed by northern blot analysis and immunoprecipitation. RESULTS: VEGF (25 ng/ml) increased integrin alphav, beta3, and 35 mRNA after 24 hours 6.1+/-0.8-fold (P < 0.001), 5.9+/-1.1-fold (P < 0.001), and 1.9+/-0.2-fold (P < 0.01), respectively. Similarly, hypoxia stimulated gene expression of integrin alphav and beta3 after 24 hours by 5.1+/-1.7-fold (P < 0.01) and 3.0+/-0.5-fold (P < 0.01), respectively, and integrin beta5 after 9 hours 1.4+/-0.2-fold (P < 0.05). This hypoxia-induced, integrin alphav mRNA elevation was inhibited significantly by anti-VEGF neutralizing antibody. Also, a conditioned medium from confluent endothelial cells maintained under hypoxic conditions for 24 hours produced a 7.1+/-1.1-fold increase (P < 0.001) in integrin alphav mRNA expression after 24 hours, which was reversed by anti-VEGF neutralizing antibody. Induction of integrin alphav by VEGF and hypoxia was confirmed in the protein level. CONCLUSIONS: These data suggest that hypoxia stimulates expression of vascular integrins alphavbeta3 and alphavbeta5 in retinal microvascular endothelial cells partially through autocrine-paracrine action of VEGF induced by the hypoxic state.     

Upregulation of Na(+)-dependent alanine transport in vascular endothelial cells by serum: role of intracellular Ca2+

OBJECTIVE: Amino acid transport and its regulation in vascular endothelial cells remains a largely unexplored area. In this study, we evaluated alanine transport in bovine aortic endothelial cells to assess possible mechanisms of regulation. METHODS: Alanine transport into confluent monolayers of endothelial cells was measured using 100 microM [3H]alanine in the presence and absence of external Na+, in cells deprived of serum for 24 hr (SD), and in SD cells exposed to 10% serum (S) for 3 hr (SD + S cells). RESULTS: Our results indicate that although SD did not significantly affect the Na(+)-independent transport of alanine when compared to normal cells, serum addition to serum-deprived cells markedly stimulated the Na(+)-dependent uptake of this amino acid through system A. The stimulation of alanine transport pathway(s) by serum was totally abolished by pretreatment of endothelial cells with 10 microM cycloheximide, suggesting a role of protein synthesis. Serum also induced a marked increase in calcium recycling at the cell membrane, suggesting that calcium is a key element of the serum signaling pathway. Indeed, both BAPTA (20 microM), a cellular calcium chelator, and thapsigargin (1 microM), an agent that depletes intracellular calcium stores, prevented the stimulation of alanine uptake by serum. Finally, pertussis toxin (400 ng/ml), an agent known to inactivate certain G-protein-dependent pathways, significantly reduced the serum-dependent 45Ca uptake and [3H]alanine entry. However, the protein kinase C activator PMA (100 nM), significantly reduced the stimulation of alanine uptake by serum but did not affect the stimulation of calcium uptake. CONCLUSIONS: Altogether these findings suggest that cell calcium is involved in the regulation of system A by serum in vascular endothelial cells.     

Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells

Inadequate presentation of tumor antigens by host professional antigen-presenting cells (APCs), including dendritic cells (DCs), is one potential mechanism for the escape of tumors from the host immune system. Here, we show that human cancer cell lines release a soluble factor or factors that dramatically affect DC maturation from precursors without affecting the function of relatively mature DCs. One factor responsible for these effects was identified as vascular endothelial growth factor (VEGF). Thus, VEGF may play a broader role in the pathogenesis of cancer than was previously thought, and therapeutic blockade of VEGF action may improve prospects for immunotherapy as well as inhibit tumor neovasculature.