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Cold Spring Harbor Symposia on Quantitative Biology 02/2002; 67:217-21.
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ABSTRACT: A mouse model of non-necrotic vascular deficiency in the adult heart was studied using cine-magnetic resonance imaging (MRI) and other techniques. The mice lacked cardiomyocyte-derived vascular endothelial growth factor (VEGF) following a targeted knockout in the ventricular cardiomyocytes. Quantitative endothelial labeling showed that the capillary density was significantly reduced in the hearts of knockout mice. Gene expression patterns suggested that they were hypoxic. Semiautomated MR image analysis was employed to obtain both global and regional measurements of left ventricular function at 10 or more time points through the cardiac cycle. MRI measurements showed a marked reduction in ejection fraction both at rest and under low- and high-dose dobutamine stress. Regional wall thickness, thickening, and displacement were all attenuated in the knockout mice. A prolonged high-dose dobutamine challenge was monitored by MRI. A maximal response was sustained for 90 minutes, suggesting that it did not depend on endogenous glycogen stores.
Journal of Magnetic Resonance Imaging 11/2001; 14(4):374-82. · 2.70 Impact Factor
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J LeCouter,
J Kowalski,
J Foster,
P Hass,
Z Zhang,
L Dillard-Telm,
G Frantz,
L Rangell,
L DeGuzman,
G A Keller,
F Peale,
A Gurney,
K J Hillan, N Ferrara
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ABSTRACT: The known endothelial mitogens stimulate growth of vascular endothelial cells without regard to their tissue of origin. Here we report a growth factor that is expressed largely in one type of tissue and acts selectively on one type of endothelium. This molecule, called endocrine-gland-derived vascular endothelial growth factor (EG-VEGF), induced proliferation, migration and fenestration (the formation of membrane discontinuities) in capillary endothelial cells derived from endocrine glands. However, EG-VEGF had little or no effect on a variety of other endothelial and non-endothelial cell types tested. Similar to VEGF, EG-VEGF possesses a HIF-1 binding site, and its expression is induced by hypoxia. Both EG-VEGF and VEGF resulted in extensive angiogenesis and cyst formation when delivered in the ovary. However, unlike VEGF, EG-VEGF failed to promote angiogenesis in the cornea or skeletal muscle. Expression of human EG-VEGF messenger RNA is restricted to the steroidogenic glands, ovary, testis, adrenal and placenta and is often complementary to the expression of VEGF, suggesting that these molecules function in a coordinated manner. EG-VEGF is an example of a class of highly specific mitogens that act to regulate proliferation and differentiation of the vascular endothelium in a tissue-specific manner.
Nature 09/2001; 412(6850):877-84. · 36.28 Impact Factor
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N Ferrara
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ABSTRACT: 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.
AJP Cell Physiology 07/2001; 280(6):C1358-66. · 3.54 Impact Factor
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ABSTRACT: Lysophosphatidic acid (LPA) stimulates ovarian tumor growth at concentrations present in ascitic fluid. Vascular endothelial growth factor (VEGF) stimulates angiogenesis and plays a pivotal role in the formation of ovarian cancer-associated ascites. We examined whether LPA promotes ovarian tumor growth by increasing angiogenesis via VEGF.
VEGF expression was examined in a simian virus 40 T-antigen-immortalized ovarian surface epithelial cell line (IOSE-29) and in ovarian cancer cell lines (OVCAR-3, SKOV-3, and CAOV-3) treated with LPA. VEGF promoter activity was measured in OVCAR-3 cells after transfection or cotransfection with c-Fos and c-Jun, components of AP1 transcription factor, potential binding sites for which are present in the VEGF promoter. The expression of the LPA receptors Edg2 and Edg4 was also assessed. All statistical tests were two-sided.
LPA treatment increased steady-state VEGF messenger RNA (mRNA) levels in OVCAR-3 cells in a time- and dose-dependent fashion and stimulated VEGF promoter activity without prolonging mRNA half-life in these cells, but LPA had little effect on IOSE-29 cells. Forced overexpression of c-Jun and c-Fos in OVCAR-3 cells stimulated VEGF promoter activity fourfold. LPA also elevated VEGF protein levels by 1.5-fold in SKOV-3 cells (P =.0148), 1.9-fold in CAOV-3 cells (P<.001), and threefold in OVCAR-3 cells (P<.0001). Both Edg2 and Edg4 were detected in ovarian cancer cells; however, only Edg2 was present in normal ovarian surface epithelial cells and IOSE-29 cells.
LPA stimulates ovarian tumor growth, at least in part, via induction of VEGF expression through transcriptional activation. However, this LPA response is not evident in normal ovarian surface epithelial cells. Our data suggest that Edg4, but not Edg2, plays a role in LPA stimulation of ovarian tumor growth.
JNCI Journal of the National Cancer Institute 06/2001; 93(10):762-8. · 13.76 Impact Factor
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F J Giordano,
H P Gerber,
S P Williams,
N VanBruggen,
S Bunting,
P Ruiz-Lozano,
Y Gu,
A K Nath,
Y Huang,
R Hickey,
N Dalton,
K L Peterson,
J Ross,
K R Chien, N Ferrara
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ABSTRACT: 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 beta-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.
Proceedings of the National Academy of Sciences 06/2001; 98(10):5780-5. · 9.68 Impact Factor
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ABSTRACT: 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.
American Journal Of Pathology 04/2001; 158(3):1111-20. · 4.89 Impact Factor
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ABSTRACT: 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 Cgamma 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.
Journal of Biological Chemistry 03/2001; 276(5):3222-30. · 4.77 Impact Factor
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ABSTRACT: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and an angiogenic inducer as well as a mediator of vascular permeability. The biological effects of VEGF are mediated by two tyrosine kinase receptors, Flt-1 (VEGFr-1) and KDR (VEGFR-2). 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 inhibition of VEGF activity may be beneficial for these conditions.
Acta Haematologica 02/2001; 106(4):148-56. · 1.35 Impact Factor
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N Ferrara
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ABSTRACT: 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.
Current Opinion in Biotechnology 01/2001; 11(6):617-24. · 7.71 Impact Factor
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ABSTRACT: Growth of the human rhabdomyosarcoma A673 cell line in nude mice is substantially reduced but not completely suppressed after systemic administration of the antihuman vascular endothelial growth factor (VEGF) monoclonal antibody (Mab) A.4.6.1. Potentially, such escape might be attributable to incomplete local penetration of the antibody because of a diffusion barrier associated with tumor growth. Alternatively, it might reflect a compensatory up-regulation of murine VEGF, produced by the stroma of the host, or of other angiogenic factor genes. To test these potential mechanisms, systemic administration of Mab A.4.6.1, was performed in conjunction with intratumoral administration of an irrelevant antibody, an antihuman VEGF Fab or mFlt(1-3)-IgG that neutralizes both human and murine VEGF. Tumor growth in the systemic-plus-intratumoral anti-VEGF group was not different from that in the systemic anti-VEGF-plus-intratumoral-control antibody group, arguing against the possibility that bioavailability is the factor that limits the antitumor efficacy of Mab A.4.6.1. However, intratumoral mFlt(l-3)-IgG administration dramatically enhanced the activity of systemic anti-VEGF Mab and resulted in complete suppression of tumor growth, which indicated that host VEGF significantly contributes to tumor growth. Systemic administration of mFlt(1-3)-IgG alone replicated these findings. Histological analysis of residual tumor tissues revealed an almost complete absence of host-derived vasculature and massive tumor-cell necrosis in the mFlt(1-3)-IgG groups. Such extensive necrotic areas were not present in the other groups. Real-time reverse transcription-PCR analysis of total RNA derived from tumor tissues indicated strong up-regulation of both human and murine VEGF as well as other genes regulated by hypoxia. Our findings emphasize the need to completely block VEGF for maximal inhibition of tumor growth.
Cancer Research 12/2000; 60(22):6253-8. · 7.86 Impact Factor
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ABSTRACT: Magnetic resonance imaging enhanced with macromolecular contrast medium was used to monitor effects of angiogenesis inhibition on tumor microvascular permeability and ascites volume in an athymic rat model of human ovarian cancer.
Groups of 6 athymic rats implanted intraperitoneally with SKOV-3, a human ovarian cancer cell line, were treated through a 14-day course with antibody to vascular endothelial growth factor or with saline solution for control animals. Dynamic magnetic resonance imaging was performed with a 92,000-d contrast agent, albumin-(gadolinium-diethylenetriaminepentaacetic acid)(30). Vascular permeability was estimated from dynamic enhancement data that were analyzed with a unidirectional 2-compartment kinetic model.
Animals treated with vascular endothelial growth factor antibody accumulated significantly smaller volumes of peritoneal ascites (P <.05) and showed significantly lower magnetic resonance imaging-assayed tumor microvascular permeabilities (P <.05) than did control animals.
Magnetic resonance imaging enhanced with a macromolecular contrast agent in an athymic rat model of human ovarian cancer treated with anti-vascular endothelial growth factor antibody can be used to measure a reduction in tumor microvascular permeability, corresponding to a reduction in ascites production.
American Journal of Obstetrics and Gynecology 10/2000; 183(4):956-63. · 3.47 Impact Factor
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ABSTRACT: Vascular endothelial growth factor (VEGF) has two highly homologous tyrosine kinase receptors: Flt-1 (VEGFR-1) and KDR (VEGFR-2). KDR is strongly phosphorylated on tyrosines and can transmit mitogenic and motogenic signals following VEGF binding, while Flt-1 is markedly less effective in mediating such functions. To dissect the regions that account for the differences between the two receptors, we generated a series of chimeric Flt-1-KDR molecules. We found that the juxtamembrane region of Flt-1 prevents key signaling functions. When the juxtamembrane region of Flt-1 is replaced by that of KDR, Flt-1 becomes competent to mediate endothelial cell migration and phosphatidylinositol 3'-kinase activation in response to VEGF. Further mutational analysis shows that a short divergent sequence is responsible for such repressor function. However, mutant Flt-1 receptors lacking this sequence do not transmit effective proliferative signals, suggesting that this receptor function is regulated separately. These results define a novel functional domain that serves to repress Flt-1 activity in endothelial cells.
The EMBO Journal 09/2000; 19(15):4064-73. · 9.20 Impact Factor
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ABSTRACT: Vascularization of the growth plate region represents a key mechanism for the coupling of two fundamental processes determining the rate of bone growth, chondrogenesis (cartilage production) and osteogenesis (bone formation). Precise coupling is crucial during periods of rapid bone growth or fracture repair in adults, and changes in the balance might induce pathologic conditions such as osteoarthritis and ectopic bone formation. During the formation of the growth plates of long bones, there is a close and dynamic interaction between developing vascular structures and the cartilage, which is one of the least vascular tissues in the body. Recent experimental findings provide an explanation why the close proximity of cartilage and vasculature is mutually exclusive: vascular invasion of cartilage is associated with chondrocyte apoptosis and consequently, inhibition of angiogenesis in the growth plate delays chondrocyte cell death, resulting in a massive expansion in the number of hypertrophic cartilage cells in the growth plate. The fundamental importance of chondrocytes in the growth, development and repair of the skeleton has led to intense investigation of the mechanisms that regulate chondrocyte maturation and apoptosis.
Trends in Cardiovascular Medicine 08/2000; 10(5):223-8. · 2.49 Impact Factor
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ABSTRACT: Promoting the formation of new collateral vessels in ischemic tissues using angiogenic growth factors (therapeutic angiogenesis) is a an exciting frontier of cardiovascular medicine. Conversely, inhibition of the action of key regulators of angiogenesis, such as VEGF, constitutes a promising approach for the treatment of solid tumors and intraocular neovascular syndromes. These concepts are being tested now in clinical trials.
Nature Medicine 01/2000; 5(12):1359-64. · 22.46 Impact Factor
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N van Bruggen,
H Thibodeaux,
J T Palmer,
W P Lee,
L Fu,
B Cairns,
D Tumas,
R Gerlai,
S P Williams,
M van Lookeren Campagne, N Ferrara
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ABSTRACT: VEGF is mitogenic, angiogenic, and a potent mediator of vascular permeability. VEGF causes extravasation of plasma protein in skin bioassays and increases hydraulic conductivity in isolated perfused microvessels. Reduced tissue oxygen tension triggers VEGF expression, and increased protein and mRNA levels for VEGF and its receptors (Flt-1, Flk-1/KDR) occur in the ischemic rat brain. Brain edema, provoked in part by enhanced cerebrovascular permeability, is a major complication in central nervous system pathologies, including head trauma and stroke. The role of VEGF in this pathology has remained elusive because of the lack of a suitable experimental antagonist. We used a novel fusion protein, mFlt(1-3)-IgG, which sequesters murine VEGF, to treat mice exposed to transient cortical ischemia followed by reperfusion. Using high-resolution magnetic resonance imaging, we found a significant reduction in volume of the edematous tissue 1 day after onset of ischemia in mice that received mFlt(1-3)-IgG. 8-12 weeks after treatment, measurements of the resultant infarct size revealed a significant sparing of cortical tissue. Regional cerebral blood flow was unaffected by the administration of mFlt(1-3)-IgG. These results demonstrate that antagonism of VEGF reduces ischemia/reperfusion-related brain edema and injury, implicating VEGF in the pathogenesis of stroke and related disorders.
Journal of Clinical Investigation 01/2000; 104(11):1613-20. · 15.39 Impact Factor
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N Ferrara
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ABSTRACT: Compelling evidence indicates that vascular endothelial growth factor (VEGF) is a fundamental regulator of normal and abnormal angiogenesis. The loss of a single VEGF allele results in defective vascularization and early embryonic lethality. VEGF plays also a critical role in kidney development, and its inactivation during early postnatal life results in the suppression of glomerular development and kidney failure. Recent evidence indicates that VEGF is also essential for angiogenesis in the female reproductive tract and for morphogenesis of the epiphyseal growth plate and endochondral bone formation. Substantial experimental evidence also implicates VEGF in pathological angiogenesis. Anti-VEGF monoclonal antibodies or other VEGF inhibitors block the growth of several human tumor cell lines in nude mice. Furthermore, the concentrations of VEGF are elevated in the aqueous and vitreous humors of patients with proliferative retinopathies such as the diabetic retinopathy. In addition, VEGF-induced angiogenesis results in a therapeutic benefit in several animal models of myocardial or limb ischemia. Currently, both therapeutic angiogenesis using recombinant VEGF or VEGF gene transfer and inhibition of VEGF-mediated pathological angiogenesis are being pursued clinically.
Kidney International 10/1999; 56(3):794-814. · 6.61 Impact Factor
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N Ferrara
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ABSTRACT: Vascular endothelial growth factor (VEGF) is a fundamental regulator of normal and abnormal angiogenesis. Recent evidence indicates that VEGF is essential for embryonic vasculogenesis and angiogenesis. Furthermore, VEGF is required for the cyclical blood vessel proliferation in the female reproductive tract and for longitudinal bone growth and endochondral bone formation. Substantial experimental evidence also implicates VEGF in pathological angiogenesis. Anti-VEGF monoclonal antibodies or other VEGF inhibitors block the growth of many tumor cell lines in nude mice. Furthermore, the concentrations of VEGF are elevated in the aqueous and vitreous humors of patients with proliferative retinopathies such as the diabetic retinopathy. In addition, VEGF-induced angiogenesis results in a therapeutic benefit in several animal models of myocardial or limb ischemia. Currently, both therapeutic angiogenesis using recombinant VEGF or VEGF gene transfer and inhibition of VEGF-mediated pathological angiogenesis are being pursued.
Journal of Molecular Medicine 08/1999; 77(7):527-43. · 4.67 Impact Factor
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ABSTRACT: 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.
Nature Medicine 07/1999; 5(6):623-8. · 22.46 Impact Factor
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ABSTRACT: 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.
Development 04/1999; 126(6):1149-59. · 6.60 Impact Factor
