Function Of Vascular Endothelial Growth Factor Research Articles

Complementary interplay between matrix metalloproteinase-9, vascular endothelial growth factor and osteoclast function drives endochondral bone formation

Long bone development depends on endochondral bone formation, a complex process requiring exquisite balance between hypertrophic cartilage (HC) formation and its ossification. Dysregulation of this process may result in skeletal dysplasias and heterotopic ossification. Endochondral ossification requires the precise orchestration of HC vascularization, extracellular matrix remodeling, and the recruitment of osteoclasts and osteoblasts. Matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and osteoclasts have all been shown to regulate endochondral ossification, but how their function interrelates is not known. We have investigated the functional relationship among these regulators of endochondral ossification, demonstrating that they have complementary but non-overlapping functions. MMP-9, VEGF and osteoclast deficiency all cause impaired growth plate ossification resulting in the accumulation of HC. VEGF mRNA and protein expression are increased at the MMP-9-/- growth plate, and VEGF activity contributes to endochondral ossification since sequestration of VEGF by soluble receptors results in further inhibition of growth plate vascularization and ossification. However, VEGF bioavailability is still limited in MMP-9 deficiency, as exogenous VEGF is able to rescue the MMP-9-/- phenotype, demonstrating that MMP-9 may partially, but not fully, regulate VEGF bioavailability. The organization of the HC extracellular matrix at the MMP-9-/- growth plate is altered, supporting a role for MMP-9 in HC remodeling. Inhibition of VEGF impairs osteoclast recruitment, whereas MMP-9 deficiency leads to an accumulation of osteoclasts at the chondro-osseous junction. Growth plate ossification in osteoclast-deficient mice is impaired in the presence of normal MMP-9 expression, indicating that other osteoclastic functions are also necessary. Our data delineate the complementary interplay between MMP-9, VEGF and osteoclast function that is necessary for normal endochondral bone formation and provide a molecular framework for investigating the molecular defects contributing to disorders of endochondral bone formation.

Cutting Edge: Vascular Endothelial Growth Factor-Mediated Signaling in Human CD45RO+ CD4+ T Cells Promotes Akt and ERK Activation and Costimulates IFN-γ Production

In this study, we find that CD45RO+ memory populations of CD4+ T lymphocytes express the vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 at both the mRNA and protein levels. Furthermore, by Western blot analysis, we find that VEGF increases the phosphorylation and activation of ERK and Akt within CD4+CD45RO+ T cells. These VEGF-mediated signaling responses were inhibited by a KDR-specific small interfering RNA in a VEGF receptor-expressing Jurkat T cell line and by SU5416, a pharmacological KDR inhibitor, in CD4+CD45RO+ T cells. We also find that VEGF augments mitogen-induced production of IFN-gamma in a dose-dependent manner (p < 0.001) and significantly (p < 0.05) increases directed chemotaxis of this T cell subset. Collectively, our results for the first time define a novel function for VEGF and KDR in CD45RO+ memory T cell responses that are likely of great pathophysiological importance in immunity.

The Suppression of Hypoxia-inducible Factor and Vascular Endothelial Growth Factor by siRNA Does not Affect the Radiation Sensitivity of Multicellular Tumor Spheroids

The hypoxic microenvironment is closely associated with the radiation resistance of tumor cells. Hypoxia induces several genes such as hypoxia-inducible factor (HIF-1) and vascular endothelial growth factor (VEGF) to promote tumor cell growth and survival. The up-regulated expression levels of HIF-1 and VEGF in tumor cells also correlate with their resistance to radiation, suggesting that these genes are potential therapeutic targets for strategies designed to enhance radiation effects. To further investigate this possibility, we investigated the effects of suppressing these genes upon the radiation sensitivity of cancer cells. We conducted these experiments using multicellular spheroids as a three-dimensional in vitro tumor model and RNA interference as the method of gene suppression. SQ5 human lung carcinoma cells were treated with HIF-1/VEGF siRNA and/or radiation. Reversed transfection methods were employed for the spheroids. Gene expression was analyzed using quantitative RT-PCR and western blotting. Cell toxicity was qualified by colony formation assay. Compared with monolayer cells, spheroids showed up-regulated expression of HIF-1 and increased radiation resistance. Hypoxic conditions elevated the expression of HIF-1 and VEGF and enhanced the surviving fraction of spheroids after exposure to radiation. However, when the expression of HIF-1 and VEGF was down-regulated by transfection of targeting siRNA, this did not influence the cytotoxic effects of the radiation under either normoxic or hypoxic conditions. We have established a method to transfect siRNA into spheroid cells. Our current data indicate that the functions of HIF-1 or VEGF are independent of radiation sensitivity in spheroids under either normoxic or hypoxic conditions.

Ultrastructural alterations in endothelial mitochondria are associated with enhanced nitrotyrosine accumulation and progressive reduction of VEGF expression in sequential protocol renal allograft biopsies with calcineurin inhibitor toxicity

Calcineurin inhibitors (cyclosporine and tacrolimus; CNIs) continue to be used as constituents of post-transplant immunosuppression in most centers. However, renal toxicity associated with the use of these drugs remains a problem adversely affecting the long-term graft survival. Fifteen adequate protocol renal allograft biopsies, with histological features of CNI toxicity among 140 protocol biopsies performed at 1-, 6-, and 12-month post-transplant, were included. Mitochondrial alterations in the tubular epithelial cells and endothelia of glomerular, peritubular capillaries and arterioles were graded semiquantitatively and further ultrastructural morphometric evaluation of numerical density and area of the mitochondria was performed. Immunohistochemical staining for nitrotyrosine (marker of peroxynitrite formation) and vascular endothelial growth factor (VEGF) was performed and expression graded semiquantitatively. Higher grades of alterations were seen in endothelial mitochondria as compared with tubular mitochondria in biopsies with calcineurin inhibitor toxicity (CNIT). Endothelial mitochondrial numerical density showed progressive decline over 1-, 6- and 12-month biopsies while area showed progressive increase in biopsies with CNIT as compared with controls. Upregulation of nitrotyrosine was seen even at 1-month post-transplant, persisted at 6 and 12 months, and was significantly greater than that in control biopsies. Intense VEGF expression was noted in early CNIT while progressive reduction was seen in 6- and 12-month protocol biopsies. This study shows a relatively high incidence of CNIT in protocol renal allograft biopsies, indicating that this might be an important mechanism of background damage to the allograft. Structural alterations in endothelial mitochondria are consistent findings in protocol biopsies with CNIT and this relatively specific mitochondrial damage may stem from the peroxynitrite-mediated damage associated with progressive loss of protective function of VEGF.

Vascular Endothelial Growth Factor (VEGF) Receptor-2 Tyrosine 1175 Signaling Controls VEGF-induced von Willebrand Factor Release from Endothelial Cells via Phospholipase C-γ1- and Protein Kinase A-dependent Pathways

There is increasing evidence that vascular endothelial growth factor (VEGF) contributes to inflammation independent of its angiogenic functions. Targeting some of the components in endothelial Weibel-Palade bodies (WPBs) effectively inhibits VEGF-induced inflammation, but little is known about how VEGF regulates WPB exocytosis. In this study, we showed that VEGF receptor-2 (VEGFR2), but not VEGFR1, is responsible for VEGF-induced release of von Willebrand factor (vWF), a major marker of WPBs. This is in good contrast to VEGF-stimulated interleukin-6 release from endothelium, which is selectively mediated through VEGFR1. We further demonstrated that VEGFR2-initiated phospholipase C-gamma1 (PLCgamma1)/calcium signaling is important but insufficient for full vWF release, suggesting the possible participation of another effector pathway. We found that cAMP/protein kinase A (PKA) signaling is required for full vWF release. Importantly, a single mutation of Tyr(1175) in the C terminus of VEGFR2, a tyrosine residue crucial for embryonic vasculogenesis, abolished vWF release, concomitant with defective activations of both PLCgamma1 and PKA. These data suggest that Tyr(1175) mediates both PLCgamma1-dependent and PKA-dependent signaling pathways. Taken together, our results not only reveal a novel Tyr(1175)-mediated signaling pathway but also highlight a potentially new therapeutic target for the management of vascular inflammation.

Induction of Neuronal Vascular Endothelial Growth Factor Expression by cAMP in the Dentate Gyrus of the Hippocampus Is Required for Antidepressant-Like Behaviors

The cAMP cascade and vascular endothelial growth factor (VEGF) are critical modulators of depression. Here we have tested whether the antidepressive effect of the cAMP cascade is mediated by VEGF in the adult hippocampus. We used a conditional genetic system in which the Aplysia octopamine receptor (Ap oa(1)), a G(s)-coupled receptor, is transgenically expressed in the forebrain neurons of mice. Chronic activation of the heterologous Ap oa(1) by its natural ligand evoked antidepressant-like behaviors, accompanied by enhanced phosphorylation of cAMP response element-binding protein and transcription of VEGF in hippocampal dentate gyrus (DG) neurons. Selective knockdown of VEGF in these cells during the period of cAMP elevation inhibited the antidepressant-like behaviors. These findings reveal a molecular interaction between the cAMP cascade and VEGF expression, and the pronounced behavioral consequences of this interaction shed light on the mechanism underlying neuronal VEGF functions in antidepression.

Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) Functions to Promote Uterine Decidual Angiogenesis during Early Pregnancy in the Mouse

Implantation of an embryo induces rapid proliferation and differentiation of uterine stromal cells, forming a new structure, the decidua. One salient feature of decidua formation is a marked increase in maternal angiogenesis. Vascular endothelial growth factor (VEGF)-dependent pathways are active in the ovary, uterus, and embryo, and inactivation of VEGF function in any of these structures might prevent normal pregnancy development. We hypothesized that decidual angiogenesis is regulated by VEGF acting through specific VEGF receptors (VEGFRs). To test this hypothesis, we developed a murine pregnancy model in which systemic administration of a receptor-blocking antibody would act specifically on uterine angiogenesis and not on ovarian or embryonic angiogenesis. In our model, ovarian function was replaced with exogenous progesterone, and blocking antibodies were administered prior to embryonic expression of VEGFRs. After administration of a single dose of the anti-VEGFR-2 antibody during the peri-implantation period, no embryos were detected on embryonic d 10.5. The pregnancy was disrupted because of a significant reduction in decidual angiogenesis, which under physiological conditions peaks on embryonic d 5.5 and 6.5. Inactivation of VEGFR-3 reduced angiogenesis in the primary decidual zone, whereas administration of VEGFR-1 blocking antibodies had no effect. Pregnancy was not disrupted after administration of anti-VEGFR-3 or anti-VEGFR-1 antibodies. Thus, the VEGF/VEGFR-2 pathway plays a key role in the maintenance of early pregnancy through its regulation of peri-implantation angiogenesis in the uterine decidua. This newly formed decidual vasculature serves as the first exchange apparatus for the developing embryo until the placenta becomes functionally active.

A new mouse model resembling human diabetic nephropathy: uncoupling of VEGF with eNOS as a novel pathogenic mechanism

Diabetics develop a variety of histological abnormalities in the kidney. Early features include glomerular hypertrophy, glomerular basement membrane thickening, and mesangial expansion, whereas mesangiolysis, glomerular capillary aneurysm and nodular lesions develop in late phase. The goal of preventing diabetic nephropathy is important, but its achievement has been difficult due in part to a lack of an animal model for human diabetic nephropathy. Most animal models develop mild lesions in early phase diabetes, but not advanced lesions in late phase. Vascular endothelial growth factor (VEGF) mediates diabetic nephropathy, but its precise role remains to be determined. A complexity of VEGF function is that it is protective in nondiabetic renal diseases but is deleterious in diabetic nephropathy. Because diabetes is associated with endothelial dysfunction, we hypothesized that VEGF is deleterious in the setting of endothelial dysfunction. To test this hypothesis, we recently developed a new model of diabetic nephropathy in mice deficient in endothelial nitric oxide synthase (eNOS). Importantly, these mice developed the advanced lesions of diabetic nephropathy resembling to those in human diabetic nephropathy. In addition, these models also exhibit an uncoupling condition of VEGF with NO. In this review, we discuss our hypothesis which is that uncoupling of VEGF with NO causes advanced diabetic nephropathy.

Vascular endothelial growth factor genotypes and haplotypes are associated with pre-eclampsia but not with gestational hypertension

Vascular endothelial growth factor (VEGF) is relevant for normal pregnancy, and abnormalities in VEGF functions are associated with hypertensive disorders of pregnancy. Because there are few studies on how VEGF genetic polymorphisms affect susceptibility to pre-eclampsia (PE), and no studies on how they affect susceptibility to gestational hypertension (GH), we compared VEGF genotype and haplotype distributions in normotensive and hypertensive pregnancies. Genotypes and haplotypes for VEGF polymorphisms (C-2578A, G-1154A and G-634C) were determined in 303 pregnant women (108 healthy pregnant, HP; 101 with GH and 94 with PE). When white and non-white pregnant women were considered together, no significant differences were found in the distributions of VEGF genotypes or haplotypes (P > 0.05) in the three groups. However, with only white subjects, significant differences were found in genotypes distributions for two (C-2578A and G-634C) VEGF polymorphisms (both P < 0.05) between the HP and the PE groups. Importantly, the haplotype including the variants C-2578, G-1154 and C-634, which is associated with higher VEGF gene expression, was less common in the PE group compared with the HP group (4% versus 16%; P = 0.0047). However, we found no significant differences in VEGF haplotypes distributions when the HP and GH groups were compared (P > 0.05). These findings suggest a protective effect for the 'C-2578, G-1154 and C-634' haplotype against the development of PE, but no major effects of VEGF gene variants on susceptibility to GH.

Deletion of vascular endothelial growth factor in myeloid cells accelerates tumorigenesis

Angiogenesis and the development of a vascular network is required for tumor progression, and involves release of angiogenic factors, including vascular endothelial growth factor (VEGF), from both malignant and stromal cell types1. Infiltration by cells of the myeloid lineage is a hallmark of many tumors, and in many cases the macrophages in these infiltrates express VEGF2. Here we show that deletion of inflammatory cell-derived VEGF attenuates the formation of a typical high-density vessel network, thus blocking the angiogenic switch in solid tumors. Vasculature in tumors lacking myeloid cell-derived VEGF was less tortuous, with increased pericyte coverage and decreased vessel length, indicating vascular normalization. In addition, loss of myeloid-derived VEGF decreases VEGFR2 phosphorylation in tumors, even though overall VEGF levels in the tumors are unaffected. However, myeloid deletion of VEGF resulted in an accelerated tumor progression in multiple subcutaneous isograft models and an autochthonous transgenic model of mammary tumorigenesis, with less overall tumor cell death and decreased tumor hypoxia. Furthermore, loss of myeloid cell VEGF increased tumor susceptibility to chemotherapeutic cytotoxicity. This demonstrates that myeloid-derived VEGF is essential for tumorigenic alteration of vasculature and signaling to VEGFR2, and that these changes act to retard, not promote, tumor progression.

Protein Kinase C-related Kinase and ROCK Are Required for Thrombin-induced Endothelial Cell Permeability Downstream from Gα12/13 and Gα11/q

Increase in vascular permeability occurs under many physiological conditions such as wound repair, inflammation, and thrombotic reactions and is central in diverse human pathologies, including tumor-induced angiogenesis, ocular diseases, and septic shock. Thrombin is a pro-coagulant serine protease, which causes the local loss of endothelial barrier integrity thereby enabling the rapid extravasation of plasma proteins and the local formation of fibrin-containing clots. Available information suggests that thrombin induces endothelial permeability by promoting actomyosin contractility through the Rho/ROCK signaling pathway. Here we took advantage of pharmacological inhibitors, knockdown approaches, and the emerging knowledge on how permeability factors affect endothelial junctions to investigate in detail the mechanism underlying thrombin-induced endothelial permeability. We show that thrombin signals through PAR-1 and its coupled G proteins Galpha(12/13) and Galpha(11/q) to induce RhoA activation and intracellular calcium elevation, and that these events are interrelated. In turn, this leads to the stimulation of ROCK, which causes actin stress-fiber formation. However, this alone is not sufficient to account for thrombin-induced permeability. Instead, we found that protein kinase C-related kinase, a Rho-dependent serine/threonine kinase, is activated in endothelial cells upon thrombin stimulation and that its expression is required for endothelial permeability and the remodeling of cell-extracellular matrix and cell-cell adhesions. Our results demonstrate that the signal initiated by thrombin bifurcates at the level of RhoA to promote changes in the cytoskeletal architecture through ROCK, and the remodeling of focal adhesion components through protein kinase C-related kinase. Ultimately, both pathways converge to cause cell-cell junction disruption and provoke vascular leakage.

Urothelial expression of neuropilins and VEGF receptors in control and interstitial cystitis patients

Interstitial cystitis (IC) is a chronic and painful bladder syndrome of unknown cause with no reliable biological marker or effective therapy. Vascular endothelial growth factor (VEGF), which plays a key role in bladder inflammation, is closely associated with the vascular alterations observed in patients with IC. However, our recent findings of VEGF receptors (VEGF-Rs) and VEGF coreceptors on nonendothelial cells in human and mouse urothelium suggest that additional VEGF targets and functions are possible in IC bladders. We report here that VEGF-Rs and coreceptors (neuropilins; NRP) are strongly expressed in both the human bladder urothelium and in the human bladder cancer cell line (J82) and that the expression of NRP2 and VEGF-R1 is significantly downregulated in IC compared with control subjects. In addition, treatment of J82 cells with bacillus Calmette-Guérin (BCG), a novel treatment strategy for IC, upregulates the messages for NRPs and VEGF-Rs. Furthermore, intravesical instillation of an internalizable VEGF fluorescent tracer (scVEGF/Cy5.5) into mouse urinary bladders results in a marked ligand accumulation in the urothelium and bladder parenchyma, indicating that urothelial VEGF-Rs are functionally active and capable of ligand interaction and internalization. Our results suggest that the VEGF pathway is altered in IC, that urinary VEGF may gain access to the bladder wall via these receptors, and that BCG treatment may replenish the missing VEGF-Rs/NRP receptors. Together, these results suggest that levels of NRPs, VEGF-Rs, and VEGF are new putative markers for the diagnosis of IC and that modulating these receptors can be exploited as therapeutic strategies.

CCN family 2/connective tissue growth factor (CCN2/CTGF) regulates the expression of Vegf through Hif-1α expression in a chondrocytic cell line, HCS-2/8, under hypoxic condition

Vascular endothelial growth factor (VEGF) is essential for establishing vascularization and regulating chondrocyte development and survival. We have demonstrated that VEGF regulates the expression of CCN2/connective tissue growth factor (CCN2/CTGF) an essential mediator of cartilage development and angiogenesis, suggesting that CCN2 functions in down-stream of VEGF, and that VEGF function is mediated in part by CCN2. On the other hand, the phenotype of Ccn2 mutant growth plates, which exhibit decreased expression of VEGF in the hypertrophic zone, indicates that Vegf expression is dependent on Ccn2 expression as well. Therefore, we investigated the molecular mechanisms underlying the induction of VEGF by CCN2 using a human chondrocytic cell line, HCS-2/8. Hypoxic stimulation (5% O 2) of HCS-2/8 cells increased VEGF mRNA levels by ∼ 8 fold within 6 h as compared with the cells cultured under normoxia. In addition, VEGF expression was further up-regulated under hypoxia in HCS-2/8 cells transfected with a Ccn2 expression plasmid. Hypoxia-inducible factor (HIF)-1α mRNA and protein levels were increased by stimulation with recombinant CCN2 (rCCN2). Furthermore, the activity of a VEGF promoter that contained a HIF-1 binding site was increased in HCS-2/8, when the cells were stimulated by rCCN2. These results suggest that CCN2 regulates the expression of VEGF at a transcriptional level by promoting HIF-1α activity. In fact, HIF-1α was detected in the nuclei of proliferative and pre-hypertrophic chondrocytes of wild-type mice, whereas it was not detected in Ccn2 mutant chondrocytes in vivo. This activation cascade from CCN2 to VEGF may therefore play a critical role in chondrocyte development and survival.

Role of Cell and Matrix-Bound VEGF Isoforms in Lens Development

To determine the role of vascular endothelial growth factor (VEGF) in embryonic eye development and lens differentiation. Expression of components of the VEGF signaling pathway during lens development and in adults was characterized by beta-galactosidase staining of VEGF-LacZ mice, immunohistochemistry, and real-time (q) PCR. Embryonic eyes from wild-type mice and VEGF120/120 mice were analyzed by light microscopy and immunohistochemistry. VEGF function during lens development was analyzed using eye explants treated with VEGF-neutralizing antibody. Direct function of VEGF was demonstrated on the human lens epithelial cell line, HLE-B3. Embryonic lens epithelium and posterior lens fibers expressed VEGF and VEGFR2. qPCR revealed VEGF164 as the major isoform in embryonic lens. Transgenic mice expressing only VEGF120 (VEGF120/120 mice) showed major defects in eye development, including microphthalmia, failed lens differentiation, and hyperplastic hyaloid vessels. The lens displayed abnormal cell patterning and differentiation associated with altered c-Maf, Prox1, and p57 expression pattern in the anterior epithelium. The number of proliferating epithelial cells was drastically reduced in VEGF120/120 lenses. Altered MIP26 cellular localization and reduced E-cadherin expression in the lens epithelium were observed. VEGF-neutralization led to reduced fiber elongation of eye explants. Exogenous VEGF increased survival and proliferation of HLE-B3 cell in a dose-dependent manner. Abnormalities in ocular development in VEGF120/120 mice suggest a role for VEGF not only in the formation of ocular vascular beds but also in the differentiation of the lens itself.

Lethality in an anti‐angiogenic tumor gene therapy model upon constitutive but not inducible expression of the soluble vascular endothelial growth factor receptor 1

Neoangiogenesis is essential for tumor growth. The present study aimed to test the hypothesis that vector-mediated expression of sflt1 at high levels would result in the blockade of vascular endothelial growth factor (VEGF) function and therefore the inhibition of tumor growth. To sequester VEGF, we tested, in a subcutaneous LLC tumor model, 'gutless' high-capacity adenovirus vectors expressing the soluble VEGF receptor 1 (sflt1) in a liver-specific manner, either in a constitutive or in a RU486 induced manner. High serum levels of sflt1 were observed upon in vivo injection of both vectors. Despite the differences in expression kinetics, both modes of sflt1 expression resulted in significant though transient suppression of tumor growth. Unexpectedly, constitutive but not intermittent sflt1 expression resulted in ascites and death of all animals. Morphological analyses by light and electron microscopy indicated that the animals had died from a nephropathy, which apparently was due to the blockade of VEGF function. Although confirming earlier results of toxic effects of prolonged VEGF sequestration, the present study suggests that therapeutic anti-tumor effects can be achieved without side-effects with intermittent VEGF blockade or the use of drugs with short half-lives, as shown by the use of an inducible gene expression system.

The Molecular Basis of Class Side Effects Due to Treatment with Inhibitors of the VEGF/VEGFR Pathway

Vascular Endothelial Growth Factor (VEGF) is considered to be one of the most important regulators of angiogenesis and a new key target in anti-cancer treatment. Various clinical trials have validated the clinical importance of anti-VEGF or anti-VEGF receptor (VEGFR) therapy. Currently the humanized monoclonal antibody bevacizumab (blocks VEGF-A), and the tyrosine kinase inhibitors sunitinib and sorafenib (inhibit VEGFRs) are approved for patients with various malignancies and several others are expected in the coming years. Unfortunately, anti-VEGF/VEGFR treatment is not void of side effects. An array of unexpected side effects is now seen in clinical practice. Management of these side effects is extremely important in the development of the various anti-VEGF/VEGFR therapies and their optimal use. This review provides an overview of the toxicity profile of this class of agents, the molecular basis behind these side effects and indicates potential options for management. VEGF and its receptors play an important role in normal tissues and are widely expressed. It is likely that interference with this pathway induces an array of side effects due to the lack of normal function of VEGF. A consistent pattern of side effects is now emerging. Hypertension, gastro-intestinal toxicity, hypothyroidism, proteinuria, coagulation disorders and neurotoxicity are side effects observed with both anti-VEGF and anti-VEGFR inhibitors. For these side effects the role of VEGF/VEGFR pathway in normal tissue was reviewed in order to provide a molecular mechanism that linked side effect with physiological activity of VEGF/VEGFR. Insight into the molecular basis may aid specific supportive care measures to ensure optimal use of this class of agents. Inhibiting the VEGF/VEGFR pathway is an effective approach to treat cancer. It has also provided new insight into the physiological role of this pathway in various organs. Integrating the knowledge in daily oncological practice will be a challenge for the future.

The potential of anti-vascular endothelial growth factor therapy in metastatic breast cancer: Clinical experience with anti-angiogenic agents, focusing on bevacizumab

The importance of angiogenesis in tumour growth and development is well known. Overexpression of vascular endothelial growth factor (VEGF), the key mediator of angiogenesis, is associated with poor prognosis in breast cancer. As a result, several therapeutic agents that inhibit the actions of VEGF or its receptors are currently in development for use in metastatic breast cancer (MBC). This review describes the function of VEGF in normal and tumour angiogenesis, explores the rationale behind the use of anti-VEGF therapy in MBC and details the therapeutic impact of such agents on tumour vasculature. Clinical data from trials of anti-VEGF agents in MBC are discussed, with a particular focus on the efficacy and safety of bevacizumab, the anti-VEGF agent at the most advanced stage of development in this tumour type. Future potential uses of bevacizumab in breast cancer are introduced.

Regulation of CXCR4 by the Notch Ligand Delta-like 4 in Endothelial Cells

Gene-targeting studies have shown that Delta-like 4 (Dll4) is required for normal embryonic vascular remodeling, but the mechanisms underlying Dll4 regulatory functions are not well defined. We generated primary human umbilical vascular endothelial cells that express Dll4 protein to study Dll4 function and previously showed that Dll4 down-regulates vascular endothelial growth factor (VEGF) receptor 2 and NRP1 expression and inhibits VEGF function. We now report that expression of Dll4 in endothelial cells inhibited attachment and migration to stromal-derived growth factor 1 (SDF1) chemokine. Cell surface, total protein, and mRNA levels of CXCR4, principal signaling receptor for SDF1, were significantly decreased in Dll4-transduced endothelial cells, attributable to a significant reduction of CXCR4 promoter activity. An immobilized recombinant extracellular portion of Dll4 (rhDLL4) was sufficient to down-regulate CXCR4 mRNA and protein, whereas protein levels of SDF1, VEGF, and RDC1 were unchanged. The gamma-secretase inhibitor L-685,458 significantly reconstituted CXCR4 mRNA in rhDLL4-stimulated endothelial cells. CXCR4 mRNA levels were significantly reduced in mouse xenografts of Dll4-transduced human gliomas compared with control gliomas, and vascular CXCR4 was not detected by immunohistochemistry in the enlarged vessels within the Dll4 gliomas. Thus, Dll4 may contribute to vascular differentiation and inhibition of the angiogenic response by regulating multiple receptor pathways.

VEGF function for upregulation of endogenous PlGF expression during FGF-2-mediated therapeutic angiogenesis

Vascular endothelial growth factor (VEGF) is a major positive angiogenic factor. Using a murine hindlimb ischemia model, we previously showed that fibroblast growth factor-2 (FGF-2) enhances the expression of endogenous VEGF which highly contribute to the therapeutic effect of FGF-2 gene transfer. Recently, placental growth factor (PlGF) has been shown to play an important role in angiogenesis. However, the molecular mechanism of endogenous PlGF during FGF-2-mediated angiogenesis has not been elucidated. Severe hindlimb ischemia stimulated PlGF expression that was more strongly enhanced by FGF-2 gene transfer, and a blockade of PlGF activity diminished the recovery of blood flow by FGF-2-mediated angiogenesis. The PlGF expression in cultured endothelial cells was significantly enhanced by VEGF stimulation, but not by FGF-2. The upregulation of endogenous PlGF expression was significantly decreased by the inhibition of endogenous VEGF activity in vivo. Subsequent signal inhibition experiments revealed that the PKC, MEK, and possibly NF-κB-related pathways were essential in stimulating PlGF expression with VEGF, while p70S6K is the regulator for VEGF expression. These results indicate that the FGF-2-mediated enhancement of PlGF expression was dependent on VEGF function, and the FGF-2/VEGF axis participates in FGF-2-mediated angiogenesis indirectly via PlGF as well as directly.

Review of the role of the plasminogen activator system and vascular endothelial growth factor in subfertility

To assess the importance of the plasminogen activator (PA) system and vascular endothelial growth factor (VEGF) in subfertility. Review. Two university IVF centers. Systematic literature search (MEDLINE, Science Direct, and bibliographies of published works). The PA system in the male is involved in the passage of spermatozoa precursor cells over the blood-testis barrier, the epididymal maturation of spermatozoa, the modifications of the sperm surface for capacitation, the acrosome reaction and zona pellucida attachment, and finally the facilitation of spermatozoa to move into the fallopian tubes. In the female, the PA system plays a role during ovulation in the release of the oocyte from the follicle, the facilitation of oocyte movement into the fallopian tubes, and the extracellular matrix degradation important for angiogenesis in the ovary. The function of VEGF during fertilization is largely unknown. It has been suggested that VEGF is important for the fluid and proteins in semen and fluid secretion in the female genital tract, thereby influencing sperm motility and survival. The latter could be due to an effect of VEGF on testicular microvasculature, through which an adequate microenvironment for spermatogenesis is provided. In the female, VEGF is involved in the regulation of the cyclic ovarian angiogenesis, the development and/or selection of follicles, the accumulation of follicular fluid, and corpus luteum angiogenesis. The role of the PA system and VEGF in reproduction most likely is of great interest. However, much of the data are derived from experimental animal studies. So far, information on the importance of these systems in humans is scarcely investigated. Therefore, further research is required to elucidate the role of the PA system and VEGF in the pathogenesis and prevention of male and female subfertility. Eventually, this will contribute to the improvement of the diagnosis of subfertility and may possibly lead to targeted therapeutic management of subfertility.