Vascular Endothelial Growth Factor In Endothelial Cells Research Articles

Vascular endothelial growth factor induces protein kinase D-dependent production of proinflammatory cytokines in endothelial cells

Emerging evidence indicates that vascular endothelial growth factor (VEGF) plays a critical role in host inflammatory responses in several disease states, including atherosclerosis, sepsis, and rheumatoid arthritis. In this study, we determined the effect of VEGF on endothelial induction of proinflammatory cytokines and investigated the responsible signal pathways. By using a cytokine antibody array that detects the end point protein products released from endothelial cells (ECs), we found that VEGF, via VEGF receptor 2 (VEGFR2), predominantly induced the production of proinflammatory cytokine interleukin (IL)-6 and CXC chemokines IL-8 and growth-related oncogene-alpha (GRO-alpha) in ECs but not in leukocytes among 36 cytokines in the array. The production of these inflammatory cytokines by VEGF was much stronger than the induction of cell adhesion molecule in ECs. We further found that the cytokine production by VEGF was essentially mediated by the Gö-6976-sensitive protein kinase D (PKD) family kinases. Importantly, the VEGF-induced production of IL-6, IL-8, and GRO-alpha was inhibited approximately 70%, 40%, or 37% by PKD1 silencing (more than 90% knockdown) with three small interference RNAs that target different PKD1 regions. Moreover, silencing PKD2 downregulated VEGFR2 and markedly inhibited the cytokine production by VEGF in ECs. Our results indicate that VEGF, via VEGFR2-PKD1 axis, induces the production of proinflammatory cytokine IL-6, IL-8, and GRO-alpha in ECs but not in leukocytes, which may offer new insights into the mechanism of the proinflammatory activity of VEGF.

CD40-Induced Signaling in Human Endothelial Cells Results in mTORC2- and Akt-Dependent Expression of Vascular Endothelial Growth Factor In Vitro and In Vivo

We have examined CD40-dependent signals in endothelial cells (EC) mediating the expression of vascular endothelial growth factor (VEGF) and VEGF-induced angiogenesis. We treated confluent cultures of EC with soluble CD40L (sCD40L), and by Western blot found a marked increase in the phosphorylation of Akt, 4EBP-1, and S6K1, compared with untreated cells. EC were transfected with a full-length VEGF promoter-luciferase construct and cultured in the absence or presence of rapamycin and sCD40L. We found that rapamycin, which blocks mTORC1 and mTORC2 signaling, inhibited sCD40L-mediated transactivation of VEGF. In addition, by Western blot, we found that the transfection of EC with small interfering RNA (siRNA) to rictor (to inhibit mTORC2), and not raptor (to inhibit mTORC1), inhibited sCD40L-dependent protein expression of VEGF. In additions, we found that basal levels of phosphorylated Akt as well as VEGF were increased in EC transfected with the raptor siRNA. Also, rapamycin failed to inhibit VEGF promoter activation, as well as VEGF protein expression in EC transfected with a constitutively active construct of Akt, further demonstrating that mTORC1 is not necessary for CD40- and Akt-induced expression of VEGF. Finally, we injected human CD40L-transfected fibroblasts or mock transfectants into human skin on SCID mice. We found that the injection of CD40L transfectants, but not mock cells, resulted in VEGF expression and mediated a marked angiogenesis reaction, and this response was reduced in mice treated with rapamycin. Together, these observations indicate that mTORC2 and Akt facilitate CD40-inducible expression of VEGF in EC, which is of clinical importance in tumor growth and the progression of chronic inflammatory diseases.

Clinical Use of Anti‐Vascular Endothelial Growth Factor Monoclonal Antibodies in Metastatic Colorectal Cancer

Abstract Vascular endothelial growth factor (VEGF) is the most potent proangiogenic factor and has been identified as an important target of cancer therapy. Blocking endothelial cell VEGF activity inhibits tumor angiogenesis; normalizes tumor vasculature, facilitating improved chemotherapy delivery; and prevents the recruitment of progenitor cells from the bone marrow. Bevacizumab, the only United States Food and Drug Administration (FDA)-approved anti-VEGF agent, is a monoclonal antibody that inhibits the binding of VEGF to VEGF receptors. The addition of bevacizumab to standard first- and second-line chemotherapy regimens for the treatment of metastatic colorectal cancer improves overall and progression-free survival times and increases the time to disease progression. Studies are evaluating bevacizumab as adjuvant therapy. The optimal bevacizumab dosage is unknown, but 5 mg/kg every 2 weeks is currently recommended for initial therapy. A surrogate efficacy marker is needed to optimize bevacizumab use, both for dose and patient selection; the clinical applicability of several surrogate efficacy markers is being evaluated. Generally, bevacizumab is well tolerated; however, several serious adverse effects that may occur (e.g., hypertensive crisis) can usually be appropriately prevented or managed. Although current recommendations suggest the administration of the first bevacizumab dose over 90 minutes to prevent infusion-related hypersensitivity reactions, recent study results show that 5 and 10 mg/kg can safely be administered over 10 and 20 minutes, respectively. Whether the addition of bevacizumab to metastatic colorectal cancer treatment regimens is a cost-effective treatment option is unknown; health economic studies are needed. When used for FDA-approved indications or for off-label indications being evaluated in select clinical trials, Medicare reimburses for bevacizumab therapy.

Protein Kinase D-dependent Phosphorylation and Nuclear Export of Histone Deacetylase 5 Mediates Vascular Endothelial Growth Factor-induced Gene Expression and Angiogenesis

Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEGF-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and histone deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase Cgamma-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis.

Induction of Hypoxia-inducible Factor 1α Gene Expression by Vascular Endothelial Growth Factor

Transcriptional regulation of vascular endothelial growth factor (VEGF) is critically dependent on hypoxia-inducible factor 1 (HIF-1). However, not only hypoxia, but selected growth factors can induce HIF-1. High levels of both VEGF and HIF-1 coexist in certain conditions, e.g. tumors. Nonetheless, the possibility that the stimulatory relationship between HIF-1 and VEGF may be bi-directional has not been addressed up to date. The present study in endothelial cells analyzed whether HIF-1 is regulated by a product of its own transcriptionally activated genes, namely, VEGF. As a main finding, VEGF-A(165) induced the increase of HIF-1alpha mRNA and HIF-1alpha protein and nuclear translocation. Autologous endothelial cell VEGF mRNA and protein were also increased upon exposure to exogenous VEGF. The signaling implication of reactive oxygen species was examined by comparison with H(2)O(2) and hypoxanthine/xanthine oxidase and by the superoxide dismutase mimetic, MnTMPyP, the Rac1-NAD(P)H oxidase complex inhibitor, apocynin, transfection of a dominant negative Rac1 mutant, and transfection of a p67phox antisense oligonucleotide. Superoxide anion, largely dependent on Rac1-NAD(P)H oxidase complex activity, was the critical signaling element. The transductional functionality of the pathway was confirmed by means of a reporter gene flanked by a transcription site-related VEGF sequence and by quantitative PCR. In summary, the present results reveal a previously undescribed action of VEGF on the expression of its own transcription factor, HIF-1, and on VEGF itself. This effect is principally mediated by superoxide anion, therefore identifying a new, potentially relevant role of reactive oxygen species in VEGF signaling.

Simvastatin Stimulates Vascular Endothelial Growth Factor Production by Hypoxia-inducible Factor-1α Upregulation in Endothelial Cells

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and plays an important pathophysiological role in the maintenance of tissue structure as well as regeneration after ischemic injury. Three-hydroxy-3methylglutaryl-CoA reductase inhibitors reduce vascular inflammation and induce angiogenesis. This study examined whether simvastatin stimulates VEGF expression in endothelial cells as well as the nature of its underlying mechanism. Simvastatin induced mRNA expression and protein secretion of VEGF in endothelial cells that were reversed by pretreatment with mevalonate and geranylgeranylpyrophosphate but not by farnesylpyrophosphate. Adenovirus-mediated expression of the dominant-negative mutant of RhoA induced VEGF mRNA and protein. Simvastatin increased hypoxia-inducible factor-1alpha (HIF-1alpha) protein level without changing its mRNA expression. Inhibition of RhoA had similar effects to simvastatin on VEGF expression. Inhibition of RhoA caused the translocation of HIF-1alpha to the nuclear fraction. Depletion of HIF-1alpha by RNA interference blocked simvastatin-induced VEGF mRNA expression. Simvastatin stimulates VEGF expression by RhoA downregulation and HIF-1alpha upregulation in endothelial cells. These data indicate a novel role for RhoA as a negative regulator of HIF-1alpha.

The zinc-finger transcription factor, early growth response 3, mediates VEGF-induced angiogenesis

Early growth response 3 (Egr3) is a member of a zinc-finger transcription factor subfamily, which we previously found to be strongly upregulated by vascular endothelial growth factor (VEGF)-A in an oligonucleotide microarray screen of endothelial cells. Here, we show that Egr3 is the predominant Egr family member upregulated by VEGF in endothelial cells at 45 min, and that VEGF induced a rapid increase in Egr-dependent transcriptional activation mediated via its major signalling receptor, VEGFR2/KDR, and the protein kinase C (PKC) pathway. VEGF-induced Egr3 gene expression was also mediated in part via a PKC-dependent activation of protein kinase D. Inhibition of Egr3 gene expression by RNA interference was effective in inhibiting basal and VEGF-induced Egr3 gene expression, and it also inhibited VEGF-mediated endothelial cell proliferation, migration and tubulogenesis. These findings indicate that Egr3 has an essential downstream role in VEGF-mediated endothelial functions leading to angiogenesis and may have particular relevance for adult angiogenic processes involved in vascular repair and neovascular disease.

NADPH Oxidase Activity Selectively Modulates Vascular Endothelial Growth Factor Signaling Pathways

Vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) play critical roles in vascular physiology and pathophysiology. We have demonstrated previously that NADPH oxidase-derived ROS are required for VEGF-mediated migration and proliferation of endothelial cells. The goal of this study was to determine the extent to which VEGF signaling is coupled to NADPH oxidase activity. Human umbilical vein endothelial cells and/or human coronary artery endothelial cells were transfected with short interfering RNA against the p47(phox) subunit of NADPH oxidase, treated in the absence or presence of VEGF, and assayed for signaling, gene expression, and function. We show that NADPH oxidase activity is required for VEGF activation of phosphoinositide 3-kinase-Akt-forkhead, and p38 MAPK, but not ERK1/2 or JNK. The permissive role of NADPH oxidase on phosphoinositide 3-kinase-Akt-forkhead signaling is mediated at post-VEGF receptor levels and involves the nonreceptor tyrosine kinase Src. DNA microarrays revealed the existence of two distinct classes of VEGF-responsive genes, one that is ROS-dependent and another that is independent of ROS levels. VEGF-induced, thrombomodulin-dependent activation of protein C was dependent on NADPH oxidase activity, whereas VEGF-induced decay-accelerating factor-mediated protection of endothelial cells against complement-mediated lysis was not. Taken together, these findings suggest that NADPH oxidase-derived ROS selectively modulate some but not all the effects of VEGF on endothelial cell phenotypes.

Regulation of nicotine-induced apoptosis of pulmonary artery endothelial cells by treatment of N-acetylcysteine and vitamin E

This study investigated the frequency of apoptosis in rat pulmonary artery endothelial cells after intraperitoneal nicotine injection, examining the roles of the inflammatory markers myeloperoxidase (MPO), tumour necrosis factor alpha (TNF-alpha), and vascular endothelial growth factor (VEGF) in nicotine-induced vascular damage and the protective effects of two known antioxidant agents, N-acetylcysteine (NAC) and vitamin E. Female Wistar rats were divided into four groups, each composed of nine rats: negative control group, positive control group, NAC-treated group (500 mg/kg), and vitamin E-treated group (500 mg/kg). Nicotine was intraperitoneally injected at a dosage of 0.6 mg/kg for 21 days. Following nicotine injection, the antioxidants were administered orally; treatment was continued until the rats were killed. Lung tissue samples were stained with hematoxylin-eosin (H&E) for histopathological assessments. Apoptosis level in endothelial cells was determined by using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling) method. Staining of cytoplasmic TNF-alpha and VEGF in endothelial cells, and perivascular MPO activity were evaluated by immunohistochemistry. The treatments with NAC and vitamin E significantly reduced the rate of nicotine-induced endothelial cell apoptosis. NAC and vitamin E significantly reduced the increases in the local production of TNF-alpha and VEGF, and perivascular MPO activity. This findings suggest that NAC can be as effective as vitamin E in protecting against nicotine-induced endothelial cell apoptosis.

Nonclassic Endogenous Novel Regulators of Angiogenesis

Angiogenesis, the process through which new blood vessels arise from preexisting ones, is regulated by several "classic" factors, among which the most studied are vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). In recent years, investigations showed that, in addition to the classic factors, numerous endogenous peptides play a relevant regulatory role in angiogenesis. Such regulatory peptides, each of which exerts well-known specific biological activities, are present, along with their receptors, in the blood vessels and may take part in the control of the "angiogenic switch." An in vivo and in vitro proangiogenic effect has been demonstrated for erythropoietin, angiotensin II (ANG-II), endothelins (ETs), adrenomedullin (AM), proadrenomedullin N-terminal 20 peptide (PAMP), urotensin-II, leptin, adiponectin, resistin, neuropeptide-Y, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), and substance P. There is evidence that the angiogenic action of some of these peptides is at least partly mediated by their stimulating effect on VEGF (ANG-II, ETs, PAMP, resistin, VIP and PACAP) and/or FGF-2 systems (PAMP and leptin). AM raises the expression of VEGF in endothelial cells, but VEGF blockade does not affect the proangiogenic action of AM. Other endogenous peptides have been reported to exert an in vivo and in vitro antiangiogenic action. These include somatostatin and natriuretic peptides, which suppress the VEGF system, and ghrelin, that antagonizes FGF-2 effects. Investigations on "nonclassic" regulators of angiogenesis could open new perspectives in the therapy of diseases coupled to dysregulation of angiogenesis.

The Scaffolding Adapter Gab1 Mediates Vascular Endothelial Growth Factor Signaling and Is Required for Endothelial Cell Migration and Capillary Formation

Vascular endothelial growth factor (VEGF) is involved in the promotion of endothelial cell proliferation, migration, and capillary formation. These activities are mainly mediated by the VEGFR2 receptor tyrosine kinase that upon stimulation, promotes the activation of numerous proteins including phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase (PI3K), Akt, Src, and ERK1/2. However, the VEGFR2-proximal signaling events leading to the activation of these targets remain ill defined. We have identified the Gab1 adapter as a novel tyrosine-phosphorylated protein in VEGF-stimulated cells. In bovine aortic endothelial cells, Gab1 associates with VEGFR2, Grb2, PI3K, SHP2, Shc, and PLCgamma, and its overexpression enhances VEGF-dependent cell migration. Importantly, silencing of Gab1 using small interfering RNAs leads to the impaired activation of PLCgamma, ERK1/2, Src, and Akt; blocks VEGF-induced endothelial cell migration; and perturbs actin reorganization and capillary formation. In addition, co-expression of VEGFR2 with Gab1 mutants unable to bind SHP2 or PI3K in human embryonic kidney 293 cells and bovine aortic endothelial cells mimics the defects observed in Gab1-depleted cells. Our work thus identifies Gab1 as a novel critical regulatory component of endothelial cell migration and capillary formation and reveals its key role in the activation of VEGF-evoked signaling pathways required for angiogenesis.

AMP-activated protein kinase mediates VEGF-stimulated endothelial NO production

Vascular endothelial growth factor (VEGF) is an important regulator of endothelial cell function. VEGF stimulates NO production, proposed to be a result of phosphorylation and activation of endothelial NO synthase (eNOS) at Ser1177. Phosphorylation of eNOS at this site also occurs after activation of AMP-activated protein kinase (AMPK) in cultured endothelial cells. We therefore determined whether AMPK mediates VEGF-stimulated NO synthesis in endothelial cells. VEGF caused a rapid, dose-dependent stimulation of AMPK activity, with a concomitant increase in phosphorylation of eNOS at Ser1177. Infection of endothelial cells with an adenovirus expressing a dominant negative mutant AMPK partially inhibited both VEGF-stimulated eNOS Ser1177 phosphorylation and NO production. VEGF-stimulated AMPK activity was completely inhibited by the Ca2+/calmodulin-dependent protein kinase kinase inhibitor, STO-609. Stimulation of AMPK via Ca2+/calmodulin-dependent protein kinase kinase represents a novel signalling mechanism utilised by VEGF in endothelial cells that contributes to eNOS phosphorylation and NO production.

Regulation of sepsis-induced apoptosis of pulmonary cells by posttreatment of erdosteine and N-aceylcysteine

This study investigated the frequency of apoptosis in rat pulmonary epithelial cells after intraperitoneal endotoxin (LPS) injection, the effects of LPS on inflammatory markers [myeloperoxidase (MPO), tumor necrosis factor alpha (TNF-α), and vascular endothelial growth factor (VEGF)] in lung damage and the protective effects of two known antioxidant agents, erdosteine and N-acetylcysteine (NAC). Male Wistar rats were divided into six groups, each composed of nine rats: two control groups, two LPS-treated groups, one erdosteine-treated group (150 mg/kg), and one NAC-treated group. LPS was intraperitoneally injected at a dosage of 20 mg/kg. Following LPS injection, the antioxidants were administered orally. The rats were killed 24 h after LPS administration. Lung tissue samples were stained with hematoxylin–eosin (H&E) for histopathological assessments. Apoptosis level in epithelial cells was determined by using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick endlabelling) method. Staining of cytoplasmic TNF-α in epithelial cells and VEGF in endothelial cells, and epithelial MPO activity were evaluated by immunohistochemistry. Posttreatment with erdosteine and NAC significantly reduced the rate of LPS-induced epithelial cell apoptosis. Posttreatment with erdosteine and NAC significantly reduced the increases in the local production of TNF-α and VEGF, and epithelial MPO activity. The effects of NAC on apoptosis, the increases in the local production of TNF-α and VEGF, were weaker than the effects of erdosteine. This finding suggests that the effects of erdosteine at the administered dose on apoptosis regulation are stronger than that of NAC.

Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling

Neuropilin-1 (NRP1) is a co-receptor for vascular endothelial growth factor (VEGF) that enhances the angiogenic signals cooperatively with VEGFR2. VEGF signaling is essential for physiological and pathological angiogenesis through its effects on vascular endothelial cells (ECs) and smooth muscle cells (SMCs), but the mechanisms coordinating this response are not well understood. Here we show that a substantial fraction of NRP1 is proteoglycan modified with either heparan sulfate or chondroitin sulfate on a single conserved Ser. The composition of the NRP1 glycosaminoglycan (GAG) chains differs between ECs and SMCs. Glycosylation increased VEGF binding in both cell types, but the differential GAG composition of NRP1 mediates opposite responsiveness to VEGF in ECs and SMCs. Finally, NRP1 expression and its GAG modification post-transcriptionally regulate VEGFR2 protein expression. These findings indicate that GAG modification of NRP1 plays a critical role in modulating VEGF signaling, and may provide new insights into physiological and pathological angiogenesis.

Protein Kinase C-dependent Protein Kinase D Activation Modulates ERK Signal Pathway and Endothelial Cell Proliferation by Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is essential for many angiogenic processes both in normal conditions and in pathological conditions. However, the signaling pathways involved in VEGF-induced angiogenesis are not well defined. Protein kinase D (PKD), a newly described serine/threonine protein kinase, has been implicated in many signal transduction pathways and in cell proliferation. We hypothesized that PKD would mediate VEGF signaling and function in endothelial cells. Here we found that VEGF rapidly and strongly stimulated PKD phosphorylation and activation in endothelial cells via VEGF receptor 2 (VEGFR2). The pharmacological inhibitors for phospholipase Cgamma (PLCgamma) and protein kinase C (PKC) significantly inhibited VEGF-induced PKD activation, suggesting the involvement of the PLCgamma/PKC pathway. In particular, PKCalpha was critical for VEGF-induced PKD activation since both overexpression of adenovirus PKCalpha dominant negative mutant and reduction of PKCalpha expression by small interfering RNA markedly inhibited VEGF-induced PKD activation. Importantly, we found that small interfering RNA knockdown of PKD and PKCalpha expression significantly attenuated ERK activation and DNA synthesis in endothelial cells by VEGF. Taken together, our results demonstrated for the first time that VEGF activates PKD via the VEGFR2/PLCgamma/PKCalpha pathway and revealed a critical role of PKD in VEGF-induced ERK signaling and endothelial cell proliferation.

Down Syndrome Candidate Region 1,a Downstream Target of VEGF, Participa tes in Endothelial Cell Migration and Angiogenesis

Vascular endothelial growth factor (VEGF) is a principal stimulator of angiogenesis. However, the downstream targets of VEGF in endothelial cells (ECs) are not entirely clarified. Survey of downstream targets of VEGF in human ECs identified a number of genes, including Down syndrome candidate region 1 (DSCR1). Here, we confirmed the inducible expression of DSCR1 in ECs by Northern and Western blottings. Moreover, VEGF-stimulated induction of DSCR1 was blocked by anti-VEGF receptor-2 monoclonal antibody (mAb), or the specific calcineurin inhibitors cyclosporin A and FK506. The expression of DSCR1 in ECs of neovessels was further shown by immunohistochemical analysis. We therefore examined whether DSCR1 played any roles in angiogenesis. The specific downregulation of DSCR1 expression by antisense oligonucleotide (AS-ODN) inhibited VEGF-stimulated migration of ECs as well as angiogenesis in vivo. AS-ODN inhibited the spreading of ECs on vitronectin, as well as on the immobilized anti-αvβ3 mAb, but not on anti-αvβ5 mAb. Moreover, AS-ODN inhibited tyrosine phosphorylation of focal adhesion kinase when ECs were plated on a vitronectin-coated dish. Immunoprecipitation followed by Western blotting showed the coimmunoprecipitation of DSCR1 and integrin αvβ3. These results suggest that DSCR1 is involved in angiogenesis by regulating adhesion and migration of ECs via the interaction with integrin αvβ3.

Expression of vascular endothelial growth factor receptor-3 by lymphatic endothelial cells is associated with lymph node metastasis in prostate cancer.

The molecular mechanisms underlying lymph node metastasis are poorly understood, despite the well-established clinical importance of lymph node status in many human cancers. Recently, vascular endothelial growth factor (VEGF)-C and VEGF-D have been implicated in the regulation of tumor lymphangiogenesis and enhancement of lymphatic invasion via activation of VEGF receptor-3. The purpose of this study was to determine the expression pattern of the VEGF-C/VEGF-D/VEGF receptor-3 axis in prostate cancer and its relationship with lymph node metastasis. The expression pattern of VEGF-C, VEGF-D, and VEGF receptor-3 in localized prostate cancer specimens (n = 37) was determined using immunohistochemistry. Widespread, heterogeneous staining for VEGF-C and VEGF-D was observed in all cancer specimens. Intensity of VEGF-C staining was lower in benign prostate epithelium than in adjacent carcinoma, whereas no difference between benign epithelium and carcinoma was observed for VEGF-D staining. VEGF receptor-3 immunostaining was detected in endothelial cells of lymphatic vessels in 18 of 37 tissue samples. The presence of VEGF receptor-3-positive vessels was associated with lymph node metastasis (P = 0.0002), Gleason grade (P < 0.0001), extracapsular extension (P = 0.0382), and surgical margin status (P = 0.0069). In addition, VEGF receptor-3 staining highlighted lymphatic invasion by VEGF-C-positive/VEGF-D-positive carcinoma cells. Together, these results suggest that paracrine activation of lymphatic endothelial cell VEGF receptor-3 by VEGF-C and/or VEGF-D may be involved in lymphatic metastasis. Thus the VEGF-C/VEGF-D/VEGF receptor-3 signaling pathway may provide a target for antilymphangiogenic therapy in prostate cancer.

Gas1 is induced by VE-cadherin and vascular endothelial growth factor and inhibits endothelial cell apoptosis

AbstractThe junctional membrane protein vascular endothelial (VE)–cadherin mediates contact inhibition of growth and inhibits apoptosis of endothelial cells. In this article we show that VE-cadherin induces expression of growth arrest–specific 1 (Gas1), an integral membrane protein up-regulated in nonproliferating cells. By comparing syngenic endothelial cell lines, we found that Gas1 mRNA was increased by 3-fold in VE-cadherin–positive cells in comparison to VE-cadherin–null cells. Ectopic expression of Gas1 in endothelial or 293 cells strongly reduced apoptosis without affecting cell growth. Addition of vascular endothelial growth factor (VEGF) also up-regulated Gas1 and this effect was augmented more so in confluent nonproliferating cells than in sparse cultures. VE-cadherin–blocking antibody partially inhibited VEGF-induced Gas1, suggesting that VE-cadherin clustering is required for an optimal response to this stimulus. Inhibition of phosphoinositole-3-OH kinase (PI3-kinase) pathway by Wortmannin prevented Gas1 synthesis and the antiapoptotic effect of VEGF, but, in cells ectopically expressing Gas1, Wortmannin was ineffective. Furthermore, inhibition of Gas1 expression by short interfering RNA (siRNA) both in vitro and in allantois organ cultures made endothelial cells refractory to the antiapoptotic effect of VEGF. Overall these data indicate that Gas1 induction by VE-cadherin and VEGF in endothelial cells requires activation of PI3-kinase. Gas1 expression positively correlates with inhibition of endothelial cell apoptosis and may contribute to the integrity of resting endothelium. (Blood. 2004;103:3005-3012)

Retrovirus-mediated human heme oxygenase-1 (HO-1) gene transfer into rat endothelial cells: the effect of HO-1 inducers on the expression of cytokines

The present study was conducted to investigate if the mechanism of human heme oxygenase-1 (HO-1) mediated angiogenesis was through the induction of vascular endothelial growth factor (VEGF). Also, the effect of HO-1 on the expression of transforming growth factor beta (TGF-β),was studied in the presence and absence of HO-1 inducers.Rat lung microvessel endothelial cell line transduced with human HO-1 gene was subjected to cell culture (six separate experiments). mRNA extraction and reverse transcriptase polymerase chain reaction (RT-PCR) experiments, were performed to evaluate the expression of HO-1, VEGF, and TGF-β in the presence and absence of HO inducers including H2O2, endotoxin and snake venoum metalloproteinase with disintegrin like activity(SnMP). ELISA technique was performed to evaluate the levels of the studied growth factors.The results of the study showed over expression of VEGF in endothelial cells transduced with HO-1 compared to control non-transduced endothelial cells. On the other hand, the expression of TGF-β and its protein level were markedly inhibited in HO-1 transduced endothelial cells compared to control non-transduced cells. Endotoxin and SnMP showed more prominent effect on the expression of VEGF and suppression of TGF-β in HO-1 transduced endothelial cells, suggesting that their effect is most probably mediated through induction of HO-1.

Soluble egg antigens from Schistosoma mansoni induce angiogenesis-related processes by up-regulating vascular endothelial growth factor in human endothelial cells.

Schistosomiasis mansoni is characterized by hepatic granuloma formation. Endothelial cell activation within these granulomas may contribute to their development and to increased vascularization in the granuloma periphery. The earliest event in granuloma formation is the lodging of schistosome eggs within presinusoidal capillaries. The eggs secrete factors that may activate endothelial cells. This study investigated the effects of Schistosoma mansoni soluble egg antigen (SEA) on angiogenic processes: proliferation, tube formation, and apoptosis of human umbilical vein endothelial cells (HUVECs). HUVECs require serum and growth factors to proliferate in vitro. Proliferation occurred when SEA or live eggs were substituted for growth factors, but not for serum. SEA increased HUVEC tube formation and decreased HUVEC apoptosis after serum and growth factor deprivation. Messenger RNA for vascular endothelial growth factor (VEGF) increased 2-fold in SEA-treated HUVECs. These findings suggest that products secreted by schistosome eggs may promote angiogenesis within hepatic granulomas by up-regulating endothelial cell VEGF.