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Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) has been recently identified as a mitogen specific for the endothelium of steroidogenic glands. Here we report a characterization of the signal transduction of EG-VEGF in a responsive cell type, bovine adrenal cortex-derived endothelial (ACE) cells. EG-VEGF led to a time- and dose-dependent phosphorylation of p44/42 MAPK. This effect was blocked by pretreatment with pertussis toxin, suggesting that G alpha(i) plays an important role in mediating EG-VEGF-induced activation of MAPK signaling. The inhibitor of p44/42 MAPK phosphorylation PD 98059 resulted in suppression of both proliferation and migration in response to EG-VEGF. EG-VEGF also increased the phosphorylation of Akt in a phosphatidylinositol 3-kinase-dependent manner. Consistent with such an effect, EG-VEGF was a potent survival factor for ACE cells. We also identified endothelial nitric-oxide synthase as one of the downstream targets of Akt activation. Phosphorylation of endothelial nitric-oxide synthase in ACE cells was stimulated by EG-VEGF with a time course correlated to the Akt phosphorylation. Our data demonstrate that EG-VEGF, possibly through binding to a G-protein coupled receptor, results in the activation of MAPK p44/42 and phosphatidylinositol 3-kinase signaling pathways, leading to proliferation, migration, and survival of responsive endothelial cells.

The endothelium of the vascular beds is extremely diverse and exquisitely distinct with respect to the specific tissue compartment served by the vessels. The molecular identity and function of the instructive signals that tailor the tissue-specific endothelial phenotype have been largely undefined. Presumably, a complex, integrated network of signals derived from the tissue parenchyma and/or stromal compartments is responsible. Recently, we identified a novel angiogenic mitogen, endocrine-gland-derived vascular endothelial growth factor, EG-VEGF, with a selective activity and very distinct expression pattern. Human EG-VEGF is expressed by steroid producing cells in the adrenal gland, placenta, testis and ovary, and is a mitogen for endothelial cells derived from these microvascular beds. EG-VEGF may represent the first of a novel class of tissue-specific angiogenic factors that function to regulate and fine-tune endothelial cell growth, structural and functional properties. The identification of other selective angiogenic molecules will allow insight into exciting, basic developmental issues and increase our armamentarium of factors for therapeutic angiogenic and anti-angiogenic strategies.

Cardiac and craniofacial developmental programs are intricately linked during early embryogenesis, which is also reflected by a high frequency of birth defects affecting both regions. The molecular nature of the crosstalk between mesoderm and neural crest progenitors and the involvement of endothelial cells within the cardio–craniofacial field are largely unclear. Here we show in the mouse that genetic ablation of vascular endothelial growth factor receptor 2 (Flk1) in the mesoderm results in early embryonic lethality, severe deformation of the cardio–craniofacial field, lack of endothelial cells and a poorly formed vascular system. We provide evidence that endothelial cells are required for migration and survival of cranial neural crest cells and consequently for the deployment of second heart field progenitors into the cardiac outflow tract. Insights into the molecular mechanisms reveal marked reduction in Transforming growth factor beta 1 (Tgfb1) along with changes in the extracellular matrix (ECM) composition. Our collective findings in both mouse and avian models suggest that endothelial cells coordinate cardio–craniofacial morphogenesis, in part via a conserved signaling circuit regulating ECM remodeling by Tgfb1.

Although vascular complications are a hallmark of diabetes, the molecular mechanisms that underlie endothelial dysfunction are unclear. We showed that reactive oxygen species generated from hyperglycemia promoted ligand-independent phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2). This VEGFR2 signaling occurred within the Golgi compartment and resulted in progressively decreased availability of VEGFR2 at the cell surface. Consequently, the responses of endothelial cells to exogenous VEGF in a mouse model of diabetes were impaired because of a specific deficiency of VEGFR2 at the cell surface, despite a lack of change in transcript abundance. Hyperglycemia-induced phosphorylation of VEGFR2 did not require intrinsic receptor kinase activity, and was instead mediated by Src family kinases. The reduced cell surface abundance of VEGFR2 in diabetic mice was reversed by treatment with the antioxidant N-acetyl-L-cysteine, suggesting a causative role for oxidative stress. These findings uncover a mode of ligand-independent VEGFR2 signaling that can progressively lead to continuously muted responses to exogenous VEGF and limit angiogenic events.

Evidence accumulating over the last decade has established the fundamental role of vascular endothelial growth factor (VEGF) as a key regulator of normal and abnormal angiogenesis. The biological effects of VEGF are mediated by two tyrosine kinase receptors, Flt-1 (VEGFR-1) and KDR (VEGFR-2). The signaling and biological properties of these two receptors are strikingly different. VEGF is essential for early development of the vasculature to the extent that inactivation of even a single allele of the VEGF gene results in embryonic lethality. VEGF is also required for female reproductive functions and endochondral bone formation. Substantial evidence also implicates VEGF as an angiogenic mediator in tumors and intraocular neovascular syndromes, and numerous clinical trials are presently testing the hypothesis that inhibition of VEGF may have therapeutic value.

The role of the cardiac myocyte as a mediator of paracrine signaling in the heart has remained unclear. To address this issue, we generated mice with cardiac myocyte-specific deletion of the vascular endothelial growth factor gene, thereby producing a cardiomyocyte-specific knockout of a secreted factor. The hearts of these mice had fewer coronary microvessels, thinned ventricular walls, depressed basal contractile function, induction of hypoxia-responsive genes involved in energy metabolism, and an abnormal response to β-adrenergic stimulation. These findings establish the critical importance of cardiac myocyte-derived vascular endothelial growth factor in cardiac morphogenesis and determination of heart function. Further, they establish an adult murine model of hypovascular nonnecrotic cardiac contractile dysfunction.

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis in both physiological and pathological processes. Hepatocyte growth factor (HGF) is a mesenchyme-derived mitogen that also stimulates cell migration, and branching and/or tubular morphogenesis of epithelial and endothelial cells. In the present study, we tested the hypothesis that simultaneous administration of HGF and VEGF would synergistically promote new blood vessel formation. HGF acted in concert with VEGF to promote human endothelial cell survival and tubulogenesis in 3-D type I collagen gels, a response that did not occur with either growth factor alone. The synergistic effects of VEGF and HGF on endothelial survival correlated with greatly augmented mRNA levels for the anti-apoptotic genes Bcl-2 and A1. Co-culture experiments with human neonatal dermal fibroblasts and human umbilical vein endothelial cells demonstrated that neonatal dermal fibroblasts, in combination with VEGF, stimulated human umbilical vein endothelial cells tubulogenesis through the paracrine secretion of HGF. Finally, in vivo experiments demonstrated that the combination of HGF and VEGF increased neovascularization in the rat corneal assay greater than either growth factor alone. We suggest that combination therapy using HGF and VEGF co-administration may provide a more effective strategy to achieve therapeutic angiogenesis.

Endothelial cells express two related vascular endothelial growth factor (VEGF) receptor tyrosine kinases, KDR (kinase-insert domain containing receptor, or VEGFR-2) and Flt-1 (fms-like tyrosine kinase, or VEGFR-1). Although considerable experimental evidence links KDR activation to endothelial cell mitogenesis, there is still significant uncertainty concerning the role of individual VEGF receptors for other biological effects such as vascular permeability. VEGF mutants that bind to either KDR or Flt-1 with high selectivity were used to determine which of the two receptors serves to mediate different VEGF functions. In addition to mediating mitogenic signaling, selective KDR activation was sufficient for the activation of intracellular signaling pathways implicated in cell migration. KDR stimulation caused tyrosine phosphorylation of both phosphatidylinositol 3-kinase and phospholipase Cγ in primary endothelial cells and stimulated cell migration. KDR-selective VEGF was also able to induce angiogenesis in the rat cornea to an extent indistinguishable from wild type VEGF. We also demonstrate that KDR, but not Flt-1, stimulation is responsible for the induction of vascular permeability by VEGF.

Although most cancer therapies are directed against tumor cells, an emerging area of cancer therapeutics focuses on targeting cells of the tumor microenvironment. Inhibiting the Src family kinase with dasatinib decreases tumor growth through inhibiting growth of tumor-associated endothelial and myeloid cells. Clin Cancer Res; 16(3); 775–7

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and an angiogenic inducer as well as a mediator of vascular permeability. VEGF is essential for developmental angiogenesis and is also required for female reproductive functions and endochondral bone formation. Substantial evidence also implicates VEGF in tumors and intraocular neovascular syndromes. Currently, several clinical trials are ongoing to test the hypothesis that the inhibition of VEGF activity may be beneficial for these conditions.

Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.

We employed two independent approaches to inactivate the angiogenic protein VEGF in newborn mice: inducible, Cre-loxP-mediated gene targeting, or administration of mFlt(1-3)-IgG, a soluble VEGF receptor chimeric protein. Partial inhibition of VEGF achieved by inducible gene targeting resulted in increased mortality, stunted body growth and impaired organ development, most notably of the liver. Administration of mFlt(1-3)-IgG, which achieves a higher degree of VEGF inhibition, resulted in nearly complete growth arrest and lethality. Ultrastructural analysis documented alterations in endothelial and other cell types. Histological and biochemical changes consistent with liver and renal failure were observed. Endothelial cells isolated from the liver of mFlt(1-3)-IgG-treated neonates demonstrated an increased apoptotic index, indicating that VEGF is required not only for proliferation but also for survival of endothelial cells. However, such treatment resulted in less significant alterations as the animal matured, and the dependence on VEGF was eventually lost some time after the fourth postnatal week. Administration of mFlt(1-3)-IgG to juvenile mice failed to induce apoptosis in liver endothelial cells. Thus, VEGF is essential for growth and survival in early postnatal life. However, in the fully developed animal, VEGF is likely to be involved primarily in active angiogenesis processes such as corpus luteum development.

Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis. Previous studies have shown that the ability of murine embryonic stem (ES) cells to form teratocarcinomas in nude mice is substantially reduced following targeted inactivation of the VEGF gene. We sought to determine whether VEGF-/- ES cells' tumorigenic phenotype can be rescued by transfection with a mutant H-ras. VEGF-/- ES cells were transfected with expression vector which directs the constitutive synthesis of oncogenic Val-12 ras. Expression of ras protein was documented by Western blot analysis. We injected several clones with different levels of Val-12 ras expression in nude mice. In agreement with our earlier report, VEGF-/- ES cells formed much smaller tumors than control ES cells. However, none of the ras-expressing clones tested formed tumors larger than those derived from parental VEGF-/- cells. Thus, pluripotent cells such as ES cells are unable to compensate for the loss of VEGF even in the presence of a potent oncogenic stimulus such as mutant ras. These findings strengthen the hypothesis that VEGF-mediated angiogenesis is crucial for effective in vivo tumor growth.

Vascular endothelial growth factor (VEGF) has been found to have various functions on endothelial cells, the most prominent of which is the induction of proliferation and differentiation. In this report we demonstrate that VEGF or a mutant, selectively binding to the Flk-1/KDR receptor, displayed high levels of survival activity, whereas Flt-1-specific ligands failed to promote survival of serum-starved primary human endothelial cells. This activity was blocked by the phosphatidylinositol 3′-kinase (PI3-kinase)-specific inhibitors wortmannin and LY294002. Endothelial cells cultured in the presence of VEGF and the Flk-1/KDR-selective VEGF mutant induced phosphorylation of the serine-threonine kinase Akt in a PI3-kinase-dependent manner. Akt activation was not detected in response to stimulation with placenta growth factor or an Flt-1-selective VEGF mutant. Furthermore, a constitutively active Akt was sufficient to promote survival of serum-starved endothelial cells in transient transfection experiments. In contrast, overexpression of a dominant-negative form of Akt blocked the survival effect of VEGF. These findings identify the Flk-1/KDR receptor and the PI3-kinase/Akt signal transduction pathway as crucial elements in the processes leading to endothelial cell survival induced by VEGF. Inhibition of apoptosis may represent a major aspect of the regulatory activity of VEGF on the vascular endothelium.

We investigated the possibility that vascular endothelial growth factor (VEGF) treatment could regulate KDR/Flk-1 receptor expression in endothelial cells. Bovine adrenal cortex endothelial cells were incubated with 200 pM rhVEGF165 for 0-7 days. Western blot analysis showed a 3-5-fold increase in total KDR protein following 4-day VEGF treatment. Scatchard analysis revealed that VEGF induced a 2-3-fold increase in high affinity receptor number (5.0 x 10(4)/cell versus 2. 4 x 10(4)/cell) without significantly affecting receptor binding affinity (Kd 76 pM versus 72 pM). Quantitative polymerase chain reaction analysis demonstrated a 3-fold increase in KDR mRNA levels following VEGF exposure. VEGF-induced KDR expression primarily occurred at the transcriptional level as demonstrated by a luciferase reporter assay system. Receptor selective mutants with wild-type KDR binding and decreased Flt-1 binding also induced KDR up-regulation; in contrast, mutants with decreased KDR binding and wild-type Flt-1 binding did not, suggesting that KDR receptor signaling mediated the increase in KDR expression. Inhibition of tyrosine kinase, Src tyrosine kinase, protein kinase C, and mitogen-activated protein kinase activities all blocked VEGF-induced KDR up-regulation. Finally, co-incubation of nitric-oxide synthase inhibitors with VEGF had no significant effect on KDR expression, but 100 microM sodium nitroprusside, a NO donor, significantly inhibited VEGF-induced KDR up-regulation, indicating that NO negatively regulates KDR expression. In conclusion, our data demonstrate that VEGF binding to the KDR receptor tyrosine kinase results in an increase in KDR receptor gene transcription and protein expression. Thus, KDR up-regulation induced by VEGF may represent an important positive feedback mechanism for VEGF action in tumor and ischemia-induced angiogenesis.

Ovarian cancer is characterized by the rapid growth of solid intraperitoneal tumors and large volumes of ascitic fluid. Vascular endothelial growth factor (VEGF) augments tumor growth by inducing neovascularization and may stimulate ascites formation by increasing vascular permeability. We examined the role of VEGF in ovarian carcinoma using in vivo models in which intraperitoneal or subcutaneous tumors were induced in immunodeficient mice using the human ovarian carcinoma cell line SKOV-3. After tumor engraftment (7 to 10 days), some mice were treated with a function-blocking VEGF antibody (A4.6.1) specific for human VEGF. A4.6.1 significantly (P < 0.05) inhibited subcutaneous SKOV-3 tumor growth compared with controls. However, tumor growth resumed when A4.6.1 treatment was discontinued. In mice bearing intraperitoneal tumors (IP mice), ascites production and intraperitoneal carcinomatosis were detected 3 to 7 weeks after SKOV-3 inoculation. Importantly, A4.6.1 completely inhibited ascites production in IP mice, although it only partially inhibited intraperitoneal tumor growth. Tumor burden was variable in A4.6.1-treated IP mice; some had minimal tumor, whereas in others tumor burden was similar to that of controls. When A4.6.1 treatment was stopped, IP mice rapidly (within 2 weeks) developed ascites and became cachectic. These data suggest that in ovarian cancer, tumor-derived VEGF is obligatory for ascites formation but not for intraperitoneal tumor growth. Neutralization of VEGF activity may have clinical application in inhibiting malignant ascites formation in ovarian cancer.

The human adrenal cortex has a complex vasculature that is essential for growth, organ maintenance, and access of secreted hormones to the circulation. Growth and function of the adrenal cortex are regulated by corticotropin (ACTH), the actions of which are in part mediated by locally produced growth factors. As cortical growth and vascularization must increase in a coordinated manner, we hypothesized that ACTH also influences adrenal cortical angiogenesis by stimulating the local expression of specific angiogenic factors. Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific angiogenic peptide, the expression of which has been detected in adrenal cortical cells. Therefore, we examined the localization of VEGF expression in the midgestation (16–20 weeks) human fetal adrenal cortex and determined whether VEGF expression and secretion by isolated human fetal adrenal cortical cells are regulated by ACTH. By immunohistochemical analysis, strong cytoplasmic staining for VEGF was detected in scattered clusters of fetal zone (inner cortical compartment) cells. In contrast, cells in the outer, definitive zone of the cortex stained only weakly for VEGF. The predominant staining for VEGF in the fetal zone correlated with the extensive vasculature of this zone as detected by immunohistochemical staining for von Willebrand factor, which is specific for endothelial cells. In primary cultures of human fetal adrenal cortical cells, ACTH (1 nmol/L) and forskolin (10 μmol/L) increased the abundance of messenger ribonucleic acid transcripts encoding VEGF, as assessed by Northern and slot blot analyses. The stimulatory effect of ACTH and forskolin on VEGF gene expression occurred within 2 h of agonist exposure and persisted for at least 24 h. ACTH and forskolin also increased VEGF protein secretion by fetal adrenal cortical cells, as assessed by enzyme-linked immunosorbent assay for VEGF in fetal adrenal cortical cell-conditioned medium. A significant (P< 0.05) increase in VEGF secretion was detected as early as 8 h after ACTH or forskolin treatment. By 24 h after the addition of ACTH or forskolin, VEGF secreted from isolated human fetal adrenal cells was increased 5- to 6-fold. These data demonstrate that the human fetal adrenal cortex, particularly the cells of the inner fetal zone, express VEGF and that VEGF expression and secretion by these cells are directly regulated by ACTH and the activation of adenylate cyclase. Thus, VEGF may be a local regulator of adrenal cortical angiogenesis and an important mediator of the tropic action of ACTH, ensuring the coordination of ACTH-stimulated cortical growth and vascularization.

The development and endocrine function of the ovarian corpus luteum (CL) are dependent on the growth of new capillary vessels. Although several molecules have been implicated as mediators of CL angiogenesis, at present there is no direct evidence for the involvement of any. Here we report the unexpected finding that treatment with truncated soluble Flt-1 receptors, which inhibit vascular endothelial growth factor (VEGF) bioactivity, resulted in virtually complete suppression of CL angiogenesis in a rat model of hormonally induced ovulation. This effect was associated with inhibition of CL development and progesterone release. Failure of maturation of the endometrium was also observed. Areas of ischemic necrosis were demonstrated in the corpora lutea (CLs) of treated animals. However, no effect on the preexisting ovarian vasculature was observed. These findings demonstrate that, in spite of the redundancy of potential mediators, VEGF is essential for CL angiogenesis. Furthermore, they have implications for the control of fertility and the treatment of ovarian disorders characterized by hypervascularity and hyperplasia.

Flt-1 is one of two receptor tyrosine kinases through which the angiogenic factor vascular endothelial growth factor (VEGF) functions. Placenta growth factor (PlGF) is an additional ligand for Flt-1. The second immunoglobulin-like domain in the extracellular domain of Flt-1 has previously been identified as the region containing the critical ligand-binding determinants. We analyzed the contribution of charged residues within the first three domains of Flt-1 to ligand binding by alanine-scanning mutagenesis. Domain 2 residues Arg159, Glu208 and His223-Arg224 (together) affect both VEGF and PlGF binding, while Glu137, Lys171, His223, and Arg224 affect PlGF but not VEGF. Several charged residues, especially Asp187, are important in maintaining the structural integrity of domain 2. In addition, some residues in domain 3 contribute to binding (Asp231) or provide for additional discrimination between ligands (Arg280-Asp283).

Early stage primary human cutaneous melanoma is known to remain relatively avascular and dormant for up to a decade, after which it may give rise to more rapidly growing, vascular and metastatically-competent primary tumor. Clinical dormancy of early stage human melanomas can be recapitulated experimentally by injection of cell lines established from such tumors into nude mice. For example, WM1341B cells, which were isolated from a thin vertical growth phase (VGP) human melanoma, are non-tumorigenic in nude mice even though some of the cells remain viable for at least three weeks at the site of orthotopic injection. These cells produce little or no vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), a potent stimulator of angiogenesis. In order to determine whether their in vivo dormant behaviour may therefore be related to an inability to induce tumor angiogenesis, subpopulations of WM1341B cells were engineered to constitutively overexpress the VEGF/VPF121 isoform. This apparently single modification was sufficient to induce overt and progressively growing tumors by several independent VEGF/VPF121 producing clones, which could be largely blocked by systemic treatment of mice with a monoclonal anti-VEGF neutralizing antibody (A 4.6.1). No evidence for an autocrine mechanism of growth stimulation by VEGF was found. Taken together, these results support the notion that defective angiogenesis may, at least in part, account for dormant phenotype of some early stage primary melanomas. Since the induction of an overt tumorigenic phenotype in several VEGF/VPF transfected WM1341B clones appears to depend exclusively on their expression of VEGF/VPF, such sublines should be useful for screening the activity of known or potential VEGF/VPF ligand or VEGF/VPF receptor antagonists in an in vivo context.

Vascular endothelial growth factor (VEGF) and its two endothelial cell-specific receptor tyrosine kinases, Flk-1/KDR and Flt-1, play a key role in physiological and pathological angiogenesis. Hypoxia has been shown to be a major mechanism for up-regulation of VEGF and its receptors in vivo. When we exposed human umbilical vein endothelial cells to hypoxic conditions in vitro, we observed increased levels of Flt-1expression. In contrast, Flk-1/KDR mRNA levels were unchanged or slightly repressed. These findings suggest a differential transcriptional regulation of the two receptors by hypoxia. To identify regulatory elements involved in the hypoxic response, promoter regions of the mouse Flt-1 and Flk-1/KDR genes were isolated and tested in conjunction with luciferase reporter gene. In transient transfection assays, hypoxia led to strong transcriptional activation of the Flt-1 promoter, whereasFlk-1/KDR transcription was essentially unchanged. Promoter deletion analysis demonstrated a 430-bp region of the Flt-1promoter to be required for transcriptional activation in response to hypoxia. This region includes a heptamer sequence matching the hypoxia-inducible factor-1 (HIF) consensus binding site previously found in other hypoxia-inducible genes such as the VEGFgene and erythropoietin gene. We further narrowed down the element mediating the hypoxia response to a 40-base pair sequence including the putative HIF binding site. We show that this element acts like an enhancer, since it activated transcription irrespective of its location or orientation in the construct. Furthermore, mutations within the putative HIF consensus binding site lead to impaired transcriptional activation by hypoxia. These findings indicate that, unlike the KDR/Flk-1 gene, the Flt-1 receptor gene is directly up-regulated by hypoxia via a hypoxia-inducible enhancer element located at positions −976 to −937 of theFlt-1 promoter.

The establishment of a vascular supply is required for organ development and differentiation as well as for tissue repair and reproductive functions in the adult1. Neovascularization (angiogenesis) is also implicated in the pathogenesis of a number of disorders. These include: proliferative retinopathies, age-related macular degeneration, tumors, rheumatoid arthritis, and psoriasis1,2. A strong correlation has been noted between density of microvessels in primary breast cancers and their nodal metastases and patient survival3. Similarly, a correlation has been reported between vascularity and invasive behavior in several other tumors4–6.

Although it has been documented that the nervous and the vascular systems share numerous analogies and are closely intermingled during development and pathological processes, interactions between the two systems are still poorly described. In this study, we investigated whether vascular endothelial growth factor (VEGF), which is a key regulator of vascular development, also modulates neuronal developmental processes. We report that VEGF enhances the gamma-aminobutyric acid (GABA)/glycinergic but not glutamatergic synaptic activity in embryonic spinal motoneurons (MNs), without affecting MNs excitability. In response to VEGF, the frequency of these synaptic events but not their amplitude was increased. Blocking endogenous VEGF led to an opposite effect by decreasing frequency of synaptic events. We found that this effect occurred specifically at early developmental stages (E13.5 and E15.5) and vanished at the prenatal stage E17.5. Furthermore, VEGF was able to increase vesicular inhibitory amino acid transporter density at the MN membrane. Inhibition of single VEGF receptors did not modify electrophysiological parameters indicating receptor combinations or an alternative pathway. Altogether, our findings identify VEGF as a modulator of the neuronal activity during synapse formation and highlight a new ontogenic role for this angiogenic factor in the nervous system.

Vascular endothelial growth factor A (VEGFA) isoform treatment has been demonstrated to alter spermatogonial stem cell homeostasis. Therefore, we generated pDmrt1-Cre;Vegfa−/− (knockout, KO) mice by crossing pDmrt1-Cre mice to floxed Vegfa mice to test whether loss of all VEGFA isoforms in Sertoli and germ cells would impair spermatogenesis. When first mated, KO males took 14 days longer to get control females pregnant (P < .02) and tended to take longer for all subsequent parturition intervals (9 days; P < .07). Heterozygous males sired fewer pups per litter (P < .03) and after the first litter took 10 days longer (P < .05) to impregnate females, suggesting a more progressive loss of fertility. Reproductive organs were collected from 6-month-old male mice. There were fewer sperm per tubule in the corpus epididymides (P < .001) and fewer ZBTB16-stained undifferentiated spermatogonia (P < .003) in the testes of KO males. Testicular mRNA abundance for Bcl2 (P < .02), Bcl2:Bax (P < .02), Neurog3 (P < .007), and Ret was greater (P = .0005), tended to be greater for Sin3a and tended to be reduced for total Foxo1 (P < .07) in KO males. Immunofluorescence for CD31 and VE-Cadherin showed no differences in testis vasculature; however, CD31-positive staining was evident in undifferentiated spermatogonia only in KO testes. Therefore, loss of VEGFA isoforms in Sertoli and germ cells alters genes necessary for long-term maintenance of undifferentiated spermatogonia, ultimately reducing sperm numbers and resulting in subfertility.

Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models of AMD with spontaneous vascular invasion early in life. DOI: http://dx.doi.org/10.7554/eLife.00324.001, eLife digest The inner surface of the vertebrate eye is lined with a multilayered structure known as the retina. The bottom layer of the retina is composed of rods and cones—neurons that are directly sensitive to light—and is called the photoreceptor layer. Rods function primarily in dim light and provide black-and-white vision, while cones support daytime vision and are responsible for colour perception. Unlike the upper layers of the retina, the photoreceptor layer does not contain blood vessels: oxygen and nutrients are instead provided by a structure just underneath the retina called the choroid. The eye relies on the rods and cones converting light into electrical signals, and the photoreceptor layer must remain free of blood vessels for this process to work properly. If blood vessels extend into the photoreceptor layer from rest of the retina (which is above it) or the choroid (below), they can disrupt the retina and give rise to a condition called age-related macular degeneration, which is a leading cause of irreversible blindness in adults. Within the eye, the development of new blood vessels from pre-existing vessels is stimulated by a protein known as vascular endothelial growth factor A (VEGF-A), while an inhibitor protein called sFLT-1 prevents the growth of new blood vessels in the other tissues of the eye like the cornea. However, it has not been clear what keeps the photoreceptor layer (and also the cells that support the photoreceptor layer) free of blood vessels, and what happens to disrupt this process of vascular demarcation in age-related macular degeneration. Now, Luo et al. reveal that cells in the photoreceptor layer produce sFLT-1, and that the levels of this protein are indeed reduced in people with age-related macular degeneration. Using genetic and pharmacological methods, they show that a reduction in sFLT-1 triggers blood vessels to grow into the photoreceptor layer from above or below. Luo et al. also report two new genetic mouse models in which blood vessels form spontaneously in the photoreceptor layer at an early age, which should prove useful for further research into age-related macular degeneration. DOI: http://dx.doi.org/10.7554/eLife.00324.002

Background Platelet-derived growth factor-C (PDGF-C) is known to promote angiogenesis independently of vascular endothelial growth factor (VEGF), although its significance in postnatal angiogenesis in vivo remains poorly understood. Methods and Results We employed murine model of hind limb ischemia and found that expression of PDGF-C and its receptor PDGFR-alpha were markedly up-regulated in ischemic limbs. Treatment with a neutralizing antibody against PDGF-C significantly impaired blood flow recovery and neovascularization after ischemia almost to the same extent as treatment with a VEGF neutralizing antibody. Moreover, PDGF-C- deficient mice showed a reduced blood flow recovery after ischemia compared to wild-type mice, confirming a strong pro-angiogenic activity of PDGF-C. Next we injected an expression vector encoding the PDGF-C gene into ischemic limbs. Blood flow recovery and neovascularization after ischemia were significantly improved in the PDGF-C-treated group compared with the control group. It has been reported that angiogenic response to ischemia is attenuated in diabetes even after VEGF treatment, although a precise mechanism remains unknown. We hypothesized that PDGF-C might relate to the impaired angiogenesis of diabetes. To test this hypothesis, we created murine model of diabetes by intraperitoneal injection of streptozotocin (STZ). We found that expression levels of PDGF-C at baseline and after ischemia were both significantly lower in limb tissues of diabetic mice than in those of control mice, while expression levels of VEGF and other members of the PDGF family were not changed or even higher in diabetic mice. Unlike in non-diabetic mice, introduction of VEGF gene into ischemic limbs did not improve blood flow recovery in diabetic mice. On the other hand, these changes were effectively reversed by introduction of the PDGF-C gene. Conclusion These results indicate that down-regulation of PDGF-C expression in limb tissues of diabetic mice contribute to impaired angiogenesis and suggest that introduction of PDGF-C would be a novel strategy for therapeutic angiogenesis, especially when VEGF treatment is ineffective, such as in the diabetic state.

The vascular endothelial growth factor (VEGF) signaling pathway plays a pivotal role in normal development and also represents a major therapeutic target for tumors and intraocular neovascular disorders. The VEGF receptor tyrosine kinases promote angiogenesis by phosphorylating downstream proteins in endothelial cells. We applied a large-scale proteomic approach to define the VEGF-regulated phosphoproteome and its temporal dynamics in human umbilical vein endothelial cells and then used siRNA (small interfering RNA) screens to investigate the function of a subset of these phosphorylated proteins in VEGF responses. The PI3K (phosphatidylinositol 3-kinase)-mTORC2 (mammalian target of rapamycin complex 2) axis emerged as central in activating VEGF-regulated phosphorylation and increasing endothelial cell viability by suppressing the activity of the transcription factor FoxO1 (forkhead box protein O1), an effect that limited cellular apoptosis and feedback activation of receptor tyrosine kinases. This FoxO1-mediated feedback loop not only reduced the effectiveness of mTOR inhibitors at decreasing protein phosphorylation and cell survival but also rendered cells more susceptible to PI3K inhibition. Collectively, our study provides a global and dynamic view of VEGF-regulated phosphorylation events and implicates the mTORC2-FoxO1 axis in VEGF receptor signaling and reprogramming of receptor tyrosine kinases in human endothelial cells.

This Podcast features an interview with Guanglei Zhuang and Napoleone Ferrara, authors of a Research Article that appears in the 16 April 2013 issue of Science Signaling . Zhuang and Ferrara discuss results from their analysis of the temporal dynamics of protein phosphorylation events downstream of vascular endothelial growth factor (VEGF) receptor activation. VEGFs are secreted growth factors that promote angiogenesis, and they promote growth of blood vessels during normal development, in wound healing, and into tissues that are deprived of oxygen. VEGF-mediated vascularization of tumors is implicated in cancer growth and progression, and VEGFs play a role in pathologies that are characterized by the abnormal growth of blood vessels, such as diabetic retinopathy and age-related macular degeneration. Drugs that inhibit VEGF or block the activity of signaling proteins downstream of VEGF receptors are used clinically to block VEGF activity in the context of pathologic vascularization. Thus, understanding the intricacies of VEGF signaling is important for devising new ways to treat VEGF-dependent vascular pathologies.

The treatment of festering wounds is one of the most important aspects of medical care. Macrophages are important components of wound repair, both in fending off infection and in coordinating tissue repair. Here we show that macrophages use a Wnt-Calcineurin-Flt1 signaling pathway to suppress wound vasculature and delay repair. Conditional mutants deficient in both Wntless/GPR177, the secretory transporter of Wnt ligands, and CNB1, the essential component of the nuclear factor of activated T cells dephosporylation complex, displayed enhanced angiogenesis and accelerated repair. Furthermore, in myeloid-like cells, we show that noncanonical Wnt activates Flt1, a naturally occurring inhibitor of vascular endothelial growth factor-A–mediated angiogenesis, but only when calcineurin function is intact. Then, as expected, conditional deletion of Flt1 in macrophages resulted in enhanced wound angiogenesis and repair. These results are consistent with the published link between enhanced angiogenesis and enhanced repair, and establish novel therapeutic approaches for treatment of wounds.

Although angiogenesis inhibitors have provided substantial clinical benefit as cancer therapeutics, their use is limited by resistance to their therapeutic effects. While ample evidence indicates that such resistance can be influenced by the tumor microenvironment, the underlying mechanisms remain incompletely understood. Here, we have uncovered a paracrine signaling network between the adaptive and innate immune systems that is associated with resistance in multiple tumor models: lymphoma, lung and colon. Tumor-infiltrating T helper type 17 (T(H)17) cells and interleukin-17 (IL-17) induced the expression of granulocyte colony-stimulating factor (G-CSF) through nuclear factor κB (NF-κB) and extracellular-related kinase (ERK) signaling, leading to immature myeloid-cell mobilization and recruitment into the tumor microenvironment. The occurrence of T(H)17 cells and Bv8-positive granulocytes was also observed in clinical tumor specimens. Tumors resistant to treatment with antibodies to VEGF were rendered sensitive in IL-17 receptor (IL-17R)-knockout hosts deficient in T(H)17 effector function. Furthermore, pharmacological blockade of T(H)17 cell function sensitized resistant tumors to therapy with antibodies to VEGF. These findings indicate that IL-17 promotes tumor resistance to VEGF inhibition, suggesting that immunomodulatory strategies could improve the efficacy of anti-angiogenic therapy.

During retinal vascular development there is simultaneous regression of the hyaloid vasculature and formation of the retinal vasculature; here it is demonstrated that regression of developing vasculature is light dependent and acts via the photoreceptor melanopsin. In the developing eye, as the retinal vasculature forms the blood vessels supplying the transparent hyaloid membrane surrounding the vitreous body regress. This process is clinically important as excessive vascular growth is a major cause of blindness in premature infants. Surprisingly, this study reports that light is involved in this major change in tissue architecture. Richard Lang and colleagues find that light stimulates regression of developing hyaloid vasculature in mice, acting through the photoreceptor melanopsin. In the absence of either light or melanopsin, vascular endothelial growth factor A is upregulated and abnormal retinal angiogenesis results. Whether the same processes are involved in human eye development remains to be determined. Vascular patterning is critical for organ function. In the eye, there is simultaneous regression of embryonic hyaloid vasculature1 (important to clear the optical path) and formation of the retinal vasculature2 (important for the high metabolic demands of retinal neurons). These events occur postnatally in the mouse. Here we have identified a light-response pathway that regulates both processes. We show that when mice are mutated in the gene (Opn4) for the atypical opsin melanopsin3,4,5, or are dark-reared from late gestation, the hyaloid vessels are persistent at 8 days post-partum and the retinal vasculature overgrows. We provide evidence that these vascular anomalies are explained by a light-response pathway that suppresses retinal neuron number, limits hypoxia and, as a consequence, holds local expression of vascular endothelial growth factor (VEGFA) in check. We also show that the light response for this pathway occurs in late gestation at about embryonic day 16 and requires the photopigment in the fetus and not the mother. Measurements show that visceral cavity photon flux is probably sufficient to activate melanopsin-expressing retinal ganglion cells in the mouse fetus. These data thus show that light—the stimulus for function of the mature eye—is also critical in preparing the eye for vision by regulating retinal neuron number and initiating a series of events that ultimately pattern the ocular blood vessels.

The features and regulation of uterine angiogenesis and vascular remodelling during pregnancy are poorly defined. Here we show that dynamic and variable decidual angiogenesis (sprouting, intussusception and networking), and active vigorous vascular remodelling such as enlargement and elongation of 'vascular sinus folding' (VSF) and mural cell drop-out occur distinctly in a spatiotemporal manner in the rapidly growing mouse uterus during early pregnancy - just after implantation but before placentation. Decidual angiogenesis is mainly regulated through VEGF-A secreted from the progesterone receptor (PR)-expressing decidual stromal cells which are largely distributed in the anti-mesometrial region (AMR). In comparison, P4 -PR-regulated VEGF-A-VEGFR2 signalling, ligand-independent VEGFR3 signalling and uterine natural killer (uNK) cells positively and coordinately regulate enlargement and elongation of VSF. During the postpartum period, Tie2 signalling could be involved in vascular maturation at the endometrium in a ligand-independent manner, with marked reduction of VEGF-A, VEGFR2 and PR expressions. Overall, we show that two key vascular growth factor receptors - VEGFR2 and Tie2 - strikingly but differentially regulate decidual angiogenesis and vascular remodelling in rapidly growing and regressing uteri in an organotypic manner.

The hyperpermeability of tumor vessels to macromolecules, compared with normal vessels, is presumably due to vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) released by neoplastic and/or host cells. In addition, VEGF/VPF is a potent angiogenic factor. Removal of this growth factor may reduce the permeability and inhibit tumor angiogenesis. To test these hypotheses, we transplanted a human glioblastoma (U87), a human colon adenocarcinoma (LS174T), and a human melanoma (P-MEL) into two locations in immunodeficient mice: the cranial window and the dorsal skinfold chamber. The mice bearing vascularized tumors were treated with a bolus (0.2 ml) of either a neutralizing antibody (A4.6.1) (492 μg/ml) against VEGF/VPF or PBS (control). We found that tumor vascular permeability to albumin in antibody-treated groups was lower than in the matched controls and that the effect of the antibody was time-dependent and influenced by the mode of injection. Tumor vascular permeability did not respond to i.p. injection of the antibody until 4 days posttreatment. However, the permeability was reduced within 6 h after i.v. injection of the same amount of antibody. In addition to the reduction in vascular permeability, the tumor vessels became smaller in diameter and less tortuous after antibody injections and eventually disappeared from the surface after four consecutive treatments in U87 tumors. These results demonstrate that tumor vascular permeability can be reduced by neutralization of endogenous VEGF/VPF and suggest that angiogenesis and the maintenance of integrity of tumor vessels require the presence of VEGF/VPF in the tissue microenvironment. The latter finding reveals a new mechanism of tumor vessel regression—i.e., blocking the interactions between VEGF/VPF and endothelial cells or inhibiting VEGF/VPF synthesis in solid tumors causes dramatic reduction in vessel diameter, which may block the passage of blood elements and thus lead to vascular regression.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor with a unique specificity for vascular endothelial cells. In addition to its role in vasculogenesis and embryonic angiogenesis, VEGF is implicated in pathologic neovascularization associated with tumors and diabetic retinopathy. Four different constructs of a short variant of VEGF sufficient for receptor binding were overexpressed in Escherichia coli, refolded, purified, and crystallized in five different space groups. In order to facilitate the product on of heavy atom derivatives, single cysteine mutants were designed based on the crystal structure of platelet-derived growth factor. A construct consisting of residues 8 to 109 was crystallized in space group P21, with cell parameters a = 55.6 A, b = 60.4 A, c = 77.7 A, β = 90.0°, and four monomers in the asymmetric unit. Native and derivative data were collected for two of the cysteine mutants as well as for wild-type VEGF. © 1996 Wiley-Liss, Inc.

Objective. Angiogenesis is an integral component of the vasculoproliferative phase of rheumatoid arthritis (RA). Recently, a heparin-binding cytokine termed hepatocyte growth factor (HGF), or scatter factor (due to its ability to disperse cohesive epithelial colonies), was described. We conducted this study to investigate the hypothesis that this cytokine was present in the milieu of the inflamed joint, and that it contributed to the chemotaxis of endothelial cells in the synovial tissue. Methods. We examined synovial fluid, synovial tissue, and peripheral blood from 91 patients with RA and other arthritides. We used 83 total samples in an enzyme-linked immunosorbent assay to quantitate the HGF in synovial fluids and peripheral blood. To determine whether the HGF was biologically active, an epithelial scatter factor assay was performed. Immunohistochemical analysis was used to determine localization in synovial tissues. To define a function for synovial HGF, we preincubated rheumatoid synovial fluids with neutralizing anti-HGF and measured the ability of these synovial fluids to induce endothelial chemotaxis. Results. Synovial fluid from patients with RA contained a mean ± SEM HGF concentration of 2.0 ± 0.3 ng/ml, while synovial fluid from patients with other arthritides (including inflammatory arthritis) contained 2.4 ± 0.7 ng/ml HGF. Osteoarthritis (OA) patient samples contained the smallest quantities of synovial fluid HGF at 0.9 ± 0.1 ng/ml. RA synovial fluid contained significantly more HGF than did RA peripheral blood (1.1 ± 0.2 ng/ml) (P < 0.05). Rheumatoid synovial fluids induced more scattering of cells than did OA synovial fluids, suggesting a role for this cytokine in rheumatoid joint destruction. Interleukin-1β induced expression of rheumatoid synovial tissue fibroblast antigenic HGF and scatter factor activity. Immunohistochemically, HGF, as well as the HGF receptor (the met gene product), localized to significantly more rheumatoid synovial tissue lining cells than normal lining cells (P < 0.05). Both HGF and its receptor immunolocalized to subsynovial macrophages as well. Levels of synovial tissue immunoreactive HGF correlated positively with the number of synovial tissue blood vessels. Anti-HGF neutralized a mean of 24% of the chemotactic activity for endothelial cells found in 10 rheumatoid synovial fluid samples. Conclusion. These results indicate that synovial HGF may contribute to the vasculoproliferative phase of inflammatory arthritides such as RA, by inducing HGF-mediated synovial neovascularization. These findings point to a newly described role for HGF in the fibroproliferative phase of RA-associated synovitis.

Vascular endothelial growth factor (VEGF) is an angiogenic inducer that mediates its effects through two high affinity receptor tyrosine kinases, Flt-1 and KDR. Flt-1 is required for endothelial cell morphogenesis whereas KDR is involved primarily in mitogenesis. Flt-1 has an alternative ligand, placenta growth factor (PlGF). Both Flt-1 and KDR have seven immunoglobulin (Ig)-like domains in the extracellular domain. The significance and function of these domains for ligand binding and receptor activation are unknown. Here we show that deletion of the second domain of Flt-1 completely abolishes the binding of VEGF. Introduction of the second domain of KDR into an Flt-1 mutant lacking the homologous domain restored VEGF binding. However, the ligand specificity was characteristic of the KDR receptor. We then created chimeric receptors where the first three or just the second Ig-like domains of Flt-1 replaced the corresponding domains in Flt-4, a receptor that does not bind VEGF, and analyzed their ability to bind VEGF. Both swaps conferred upon Flt-4 the ability to bind VEGF with an affinity nearly identical to that of wild-type Flt-1. Furthermore, transfected cells expressing these chimeric Flt-4 receptors exhibited increased DNA synthesis in response to VEGF or PlGF. These results demonstrate that a single Ig-like domain is the major determinant for VEGF-PlGF interaction and that binding to this domain may initiate a signal transduction cascade.

Vascular endothelial growth factor (VEGF), a major regulator of angiogenesis, has therapeutic benefit in animal models of coronary or limb ischemia. However, the hemodynamic effects of VEGF have not been investigated. We examined the effects of VEGF on hemodynamics and cardiac performance. Mean arterial pressure (MAP), heart rate (HR), cardiac output, stroke volume, left ventricular (LV) dP/dt, and hematocrit were measured before and after intravenous injection of VEGF in conscious, instrumented rats. VEGF caused a dose-dependent reduction in MAP and an associated increase in HR. VEGF (250 micrograms/kg) significantly decreased cardiac output and stroke volume without affecting the inotropic state of the left ventricle, as determined by dP/dt. VEGF significantly increased hematocrit. Furthermore, VEGF did not affect contractility or HR in the isolated rat heart in vitro. The data suggest that the VEGF-induced decrease in cardiac output is due to reduced stroke volume, which may be caused by a decrease in venous return rather than a direct effect on myocardial contractility. In addition, pretreatment with N omega-nitro-L-arginine methyl-ester (L-NAME), a nitric oxide (NO) synthase inhibitor, significantly attenuated the depressor and tachycardic responses to VEGF, suggesting that VEGF-induced hypotension may be mediated by NO.

ANGIOGENESIS is required for a wide variety of physiological and pathological processes1. The endothelial cell-specific mitogen vascular endothelial growth factor (VEGF)2,3 is a major mediator of pathological angiogenesis4–6. Also, the expression of VEGF and its two receptors, Flt-1 and Flk-1/KDR, is related to the formation of blood vessels in mouse and rat embryos7–10. Mice homozygous for mutations that inactivate either receptor die in utero between days 8.5 and 9.5 (refs 11,12). However, ligand(s) other than VEGF might activate such receptors13,14. To assess the role of VEGF directly, we disrupted the VEGF gene in embryonic stem cells. Here we report the unexpected finding that loss of a single VEGF allele is lethal in the mouse embryo between days 11 and 12. Angiogenesis and blood-island formation were impaired, resulting in several developmental anomalies. Furthermore, VEGF-null embryonic stem cells exhibit a dramatically reduced ability to form tumours in nude mice.

Vascular endothelial growth factor (VEGF) expression in various cell types is induced by hypoxia and other stimuli. VEGF mediates endothelial cell proliferation, angiogenesis, vascular growth, and vascular permeability via the endothelial cell receptors, kinase insert domain-containing receptor (KDR)/fetal liver kinase 1 (Flk-1) and FLT-1. Alanine-scanning mutagenesis was used to identify a positively charged surface in VEGF that mediates binding to KDR/Flk-1. Arg82, Lys84 and His86, located in a hairpin loop, were found to be critical for binding KDR/Flk-1, while negatively charged residues, Asp63, Glu64, and Glu67, were associated with FLT-1 binding. A VEGF model based on PDGFb indicated these positively and negatively charged regions are distal in the monomer but are spatially close in the dimer. Mutations within the KDR site had minimal effect on FLT-1 binding, and mutants deficient in FLT-1 binding did not affect KDR binding. Endothelial cell mitogenesis was abolished in mutants lacking KDR affinity; however, FLT-1 deficient mutants induced normal proliferation. These results suggest dual sets of determinants in the VEGF dimer that cross-link cell surface receptors, triggering endothelial cell growth and angiogenesis. Furthermore, this mutational analysis implicates KDR, but not FLT-1, in VEGF induction of endothelial cell proliferation.

Vascular endothelial growth factor is a diffusible endothelial cell-specific mitogen and angiogenic factor that can also increase vascular permeability. The vascular endothelial growth factor receptors are specifically expressed on the cell surface of vascular endothelial cells. Recent studies point to vascular endothelial growth factor as a major regulator of physiological angiogenesis, such as developmental and reproductive angiogenesis. In addition vascular endothelial growth factor appears to be a crucial mediator of blood vessel growth associated with tumors and proliferative retinopathies. Antivascular endothelial growth factor antibodies have the ability to suppress the growth of a variety of tumor cell lines in nude mice and can also inhibit angiogenesis in animal models of intra-ocular neovascularization. Furthermore vascular endothelial growth factor administration promotes collateral vessel growth and results in functional improvement in animal models of coronary or limb ischemia.

Animal studies have shown that angiogenic factors can increase vascularity and improve blood pressure (BP) in an ischemic limb. Whether changes in these parameters are indicators of significant improvement in muscle function has not been demonstrated. In a rabbit model of hind limb ischemia, we measured blood flow in the extensor digitorum longus muscle (EDL) both at rest and during electrical stimulation. Ablation of the femoral artery caused significant reductions in resting and stimulated EDL blood flow. The chronic reduction in perfusion caused impairment of muscle function (p < 0.01). At 28 days after a single administration of vascular endothelial growth factor (VEGF), stimulated muscle blood flow (3 mg/kg intravenously, i.v.) and muscle function [1 mg intrarterially (i.a.) or 3 mg/kg i.v.] were significantly improved as compared with that of vehicle-treated controls. Simultaneous measurement of the hemodynamic responses in the contralateral limb and in the kidneys confirmed that the effects of VEGF were confined to the ischemic limb. The data agree with findings that angiogenic factors increase perfusion through angiogenesis. We hypothesized that neovascularization allows work-associated muscle hyperemia, resulting in a significant improvement in muscle function. Similar clinical improvements in muscle function would signify a substantial advance in the treatment of peripheral vascular disease.

The majority of severe visual loss in the United States results from complications associated with retinal neovascularization in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Intraocular expression of the angiogenic protein vascular endothelial growth factor (VEGF) is closely correlated with neovascularization in these human disorders and with ischemia-induced retinal neovascularization in mice. In this study, we evaluated whether in vivo inhibition of VEGF action could suppress retinal neovascularization in a murine model of ischemic retinopathy. VEGF-neutralizing chimeric proteins were constructed by joining the extracellular domain of either human (Flt) or mouse (Flk) high-affinity VEGF receptors with IgG. Control chimeric proteins that did not bind VEGF were also used. VEGF-receptor chimeric proteins eliminated in vitro retinal endothelial cell growth stimulation by either VEGF (P < 0.006) or hypoxic conditioned medium (P < 0.005) without affecting growth under nonstimulated conditions. Control proteins had no effect. To assess in vivo response, animals with bilateral retinal ischemia received intravitreal injections of VEGF antagonist in one eye and control protein in the contralateral eye. Retinal neovascularization was quantitated histologically by a masked protocol. Retinal neovascularization in the eye injected with human Flt or murine Flk chimeric protein was reduced in 100% (25/25; P < 0.0001) and 95% (21/22; P < 0.0001) 0.0001) of animals, respectively, compared to the control treated eye. This response was evident after only a single intravitreal injection and was dose dependent with suppression of neovascularization noted after total delivery of 200 ng of protein (P < 0.002). Reduction of histologically evident neovascular nuclei per 6-microns section averaged 47% +/- 4% (P < 0.001) and 37% +/- 2% (P < 0.001) for Flt and Flk chimeric proteins with maximal inhibitory effects of 77% and 66%, respectively. No retinal toxicity was observed by light microscopy. These data demonstrate VEGF's causal role in retinal angiogenesis and prove the potential of VEGF inhibition as a specific therapy for ischemic retinal disease.

Background Most strategies designed to reduce restenosis by the use of pharmacological or biological reagents involve direct inhibition of vascular smooth muscle cell (SMC) proliferation. Alternatively, SMC proliferation might be indirectly inhibited if reendothelialization could be specifically facilitated at sites of balloon-induced arterial injury. Accordingly, we investigated the hypothesis that application of an endothelial cell (EC)-specific mitogen to a freshly denuded intimal surface could accelerate reendothelialization and thereby attenuate intimal hyperplasia. Methods and Results The left carotid artery of 31 Sprague-Dawley rats was subjected to balloon injury, after which 16 rats were treated with a 30-minute incubation with 100 μg of vascular endothelial growth factor (VEGF), an EC-specific mitogen. Control animals (n=15) received a 30-minute incubation with 0.9% saline. At 2 weeks after balloon injury, carotid artery reendothelialization was markedly superior in the VEGF-treated group compared with the control group (14.59±1.12 versus 7.96±0.51 mm 2 , P 2 ) versus saline (13.42±0.84 mm 2 , P Conclusions Application of VEGF, an EC-specific growth regulatory molecule, may be effectively used in vivo to promote reendothelialization and thereby indirectly attenuate neointimal thickening due to SMC proliferation.

Background Disturbed endothelium-dependent blood flow has been shown to be a feature of native collateral vessels. Recent studies have shown that recombinant angiogenic growth factors augment collateral development in animal models of hindlimb ischemia. We therefore investigated the hypothesis that the administration of an angiogenic growth factor, in this case vascular endothelial growth factor (VEGF), may promote recovery of disturbed endothelium-dependent blood flow in a rabbit model of hindlimb ischemia. Methods and Results Ischemia was induced by ligation of the external iliac artery and excision of the femoral artery in one limb of New Zealand White rabbits (day 0). Flow velocity was measured using a Doppler guide wire at rest and after administration of serotonin and acetylcholine. Blood flow (in mL/min) was calculated assuming a circular lumen geometry. In untreated control animals with an ischemic limb, serotonin administered at day 10 or 40 produced a decrease in hindlimb blood flow (71±2% and 33±6% reduction from baseline, respectively); in contrast, among animals treated with a single 500-μg bolus dose of VEGF administered selectively into the internal iliac artery at day 10 and studied at day 40, serotonin produced an increase in flow (119±8% from baseline; P P Conclusions To our knowledge, these findings constitute the first demonstration of successful pharmacological modulation of disturbed endothelium-dependent flow in the arterial circulation subserved by collateral vessels. This physiological benefit complements previously reported anatomic findings suggesting a favorable impact of angiogenic growth factors on collateral-dependent limb ischemia.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and endothelial cell-specific mitogen that stimulates urokinase-type plasminogen activator (uPA) activity in vascular endothelial cells. Here, we report that VEGF increases the high affinity binding of uPA to the same cells and that this binding is prevented by a peptide corresponding to the uPA receptor (uPAR) binding growth factor-like domain of uPA. Ligand cross-linking, ligand blotting, and uPA-Sepharose affinity chromatography revealed an increase in a cell surface uPA binding protein that corresponds to the uPAR on the basis of its affinity for uPA, M(r) of 50,000-55,000, and phosphatidylinositol-specific phospholipase C sensitivity. By Scatchard analysis, VEGF increased the number of uPAR molecules by 2.8-3.5-fold and concomitantly decreased their affinity for uPA. By northern blotting uPAR mRNA was increased in a dose- and time-dependent manner in response to VEGF. Taken together, these findings demonstrate that VEGF-induced angiogenesis is accompanied by increased uPAR expression and uPA activity on the endothelial cell surface. These observations are consistent with the notion that the uPA-uPAR interaction facilitates cellular invasion.

To investigate the relationship between angiogenesis and hepatic tumorigenesis, we examined the expression of vascular endothelial growth factor (VEGF) in 8 human colon carcinoma cell lines and in 30 human colorectal cancer liver metastases. Abundant message for VEGF was found in all tumors, localized to the malignant cells within each neoplasm. Two receptors for VEGF, KDR and flt1, were also demonstrated in most of the tumors examined. KDR and flt1 mRNA were limited to tumor endothelial cells and were more strongly expressed in the hepatic metastases than in the sinusoidal endothelium of the surrounding liver parenchyma. VEGF monoclonal antibody administration in tumor-bearing athymic mice led to a dose- and time-dependent inhibition of growth of subcutaneous xenografts and to a marked reduction in the number and size of experimental liver metastases. In hepatic metastases of VEGF antibody-treated mice, neither blood vessels nor expression of the mouse KDR homologue flk-1 could be demonstrated. These data indicate that VEGF is a commonly expressed angiogenic factor in human colorectal cancer metastases, that VEGF receptors are up-regulated as a concomitant of hepatic tumorigenesis, and that modulation of VEGF gene expression or activity may represent a potentially effective antineoplastic therapy in colorectal cancer.