Abstract: Osteoporotic bones have reduced spongy bone mass, altered bone architecture, and increased marrow fat. Bone marrow stem cells from osteoporotic patients are more likely to differentiate into adipocytes than control cells, suggesting that adipocyte differentiation may play a role in osteoporosis. VEGF is highly expressed in osteoblastic precursor cells and is known to stimulate bone formation. Here we tested the hypothesis that VEGF is also an important regulator of cell fate, determining whether differentiation gives rise to osteoblasts or adipocytes. Mice with conditional VEGF deficiency in osteoblastic precursor cells exhibited an osteoporosis-like phenotype characterized by reduced bone mass and increased bone marrow fat. In addition, reduced VEGF expression in mesenchymal stem cells resulted in reduced osteoblast and increased adipocyte differentiation. Osteoblast differentiation was reduced when VEGF receptor 1 or 2 was knocked down but was unaffected by treatment with recombinant VEGF or neutralizing antibodies against VEGF. Our results suggested that VEGF controls differentiation in mesenchymal stem cells by regulating the transcription factors RUNX2 and PPARγ2 as well as through a reciprocal interaction with nuclear envelope proteins lamin A/C. Importantly, our data support a model whereby VEGF regulates differentiation through an intracrine mechanism that is distinct from the role of secreted VEGF and its receptors.
Abstract: The vascular beds supplying the retina may sustain injury as a result of underlying disease such as diabetes, and/or the interaction of genetic predisposition, environmental insults, and age. The vascular pathologic features observed in different intraocular vascular diseases can be categorized broadly as proliferation, exemplified by proliferative diabetic retinopathy, leakage such as macular edema secondary to retinal vein occlusion, or a combination of proliferation and leakage, as seen in neovascular age-related macular degeneration (AMD). The World Health Organization has identified diabetic retinopathy and AMD as priority eye diseases for the prevention of vision loss in developed countries. The pathologic transformations of the retinal vasculature seen in intraocular vascular disease are associated with increased expression of vascular endothelial growth factor A (VEGF), a potent endothelial-specific mitogen. Furthermore, in model systems, VEGF alone is sufficient to trigger intraocular neovascularization, and its inhibition is associated with functional and anatomic improvements in the affected eye. Therapeutic interventions with effect on VEGF include intraocular capture and neutralization by engineered antibodies or chimeric receptors, downregulation of its expression with steroids, or alleviation of retinal ischemia, a major stimulus for VEGF expression, with retinal ablation by laser treatment. Data from prospective randomized clinical trials indicate that VEGF inhibition is a potent therapeutic strategy for intraocular vascular disease. These findings are changing clinical practice and are stimuli for further study of the basic mechanisms controlling intraocular angiogenesis. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references.
Abstract: Disappointing results from most late-stage clinical trials of cancer therapeutics indicate a need for improved and more-predictive animal tumor models. This insufficiency of models, combined with the advent of a class of drugs that target the tumor microenvironment rather than the tumor cell, presents new challenges for designing and interpreting preclinical efficacy studies. A comparison of the clinical efficacy of anti-angiogenic drugs with their corresponding preclinical studies over the past two decades offers many lessons that can inform and improve the design of experiments in existing mouse models. In addition, technological and logistical advances in mouse models of human cancer over the past five years have the potential to increase the clinical translatability of animal studies.
Abstract: Angiogenesis plays a crucial role during tumorigenesis and much progress has been recently made in elucidating the role of VEGF and other growth factors in the regulation of angiogenesis. Recently, microRNAs (miRNAs) have been shown to modulate a variety of physiogical and pathological processes. We identified a set of differentially expressed miRNAs in microvascular endothelial cells co-cultured with tumour cells. Unexpectedly, most miRNAs were derived from tumour cells, packaged into microvesicles (MVs), and then directly delivered to endothelial cells. Among these miRNAs, we focused on miR-9 due to the strong morphological changes induced in cultured endothelial cells. We found that exogenous miR-9 effectively reduced SOCS5 levels, leading to activated JAK-STAT pathway. This signalling cascade promoted endothelial cell migration and tumour angiogenesis. Remarkably, administration of anti-miR-9 or JAK inhibitors suppressed MV-induced cell migration in vitro and decreased tumour burden in vivo. Collectively, these observations suggest that tumour-secreted miRNAs participate in intercellular communication and function as a novel pro-angiogenic mechanism.
Abstract: Inhibiting angiogenesis has become an important therapeutic strategy for cancer treatment but, like other current targeted therapies, benefits experienced for late-stage cancers can be curtailed by inherent refractoriness or by acquired drug resistance, requiring a need for better mechanistic understanding of such effects. Numerous preclinical studies have demonstrated that VEGF pathway inhibitors suppress primary tumour growth and metastasis. However, it has been recently reported that short-term VEGF and VEGFR inhibition can paradoxically accelerate tumour invasiveness and metastasis in certain models. Here we comprehensively compare the effects of both antibody and small molecule receptor tyrosine kinase (RTK) inhibitors targeting the VEGF-VEGFR pathway, using short-term therapy in various mouse models of metastasis. Our findings demonstrate that antibody inhibition of VEGF pathway molecules does not promote metastasis, in contrast to selected small molecule RTK inhibitors at elevated-therapeutic drug dosages. In particular, a multi-targeted RTK inhibitor, sunitinib, which most profoundly potentiated metastasis, also increased lung vascular permeability and promoted tumour cell extravasation. Mechanistically, sunitinib, but not anti-VEGF treatment, attenuated endothelial barrier function in culture and caused a global inhibition of protein tyrosine phosphorylation, including molecules important for maintaining endothelial cell-cell junctions. Together these findings indicate that, rather than a specific consequence of inhibiting the VEGF signalling pathway, pharmacological inhibitors of the VEGF pathway can have dose- and drug class-dependent side-effects on the host vasculature. These findings also advocate for the continued identification of mechanisms of resistance to anti-angiogenics and for therapy development to overcome it.
Abstract: Resistance to anti-angiogenic therapy can occur via several potential mechanisms. Unexpectedly, recent studies showed that short-term inhibition of either VEGF or VEGFR enhanced tumour invasiveness and metastatic spread in preclinical models. In an effort to evaluate the translational relevance of these findings, we examined the consequences of long-term anti-VEGF monoclonal antibody therapy in several well-validated genetically engineered mouse tumour models of either neuroendocrine or epithelial origin. Anti-VEGF therapy decreased tumour burden and increased overall survival, either as a single agent or in combination with chemotherapy, in all four models examined. Importantly, neither short- nor long-term exposure to anti-VEGF therapy altered the incidence of metastasis in any of these autochthonous models, consistent with retrospective analyses of clinical trials. In contrast, we observed that sunitinib treatment recapitulated previously reported effects on tumour invasiveness and metastasis in a pancreatic neuroendocrine tumour (PNET) model. Consistent with these results, sunitinib treatment resulted in an up-regulation of the hypoxia marker GLUT1 in PNETs, whereas anti-VEGF did not. These results indicate that anti-VEGF mediates anti-tumour effects and therapeutic benefits without a paradoxical increase in metastasis. Moreover, these data underscore the concept that drugs targeting VEGF ligands and receptors may affect tumour metastasis in a context-dependent manner and are mechanistically distinct from one another.
Abstract: In inflammatory CNS conditions such as multiple sclerosis (MS), current options to treat clinical relapse are limited, and more selective agents are needed. Disruption of the blood-brain barrier (BBB) is an early feature of lesion formation that correlates with clinical exacerbation, leading to edema, excitotoxicity, and entry of serum proteins and inflammatory cells. Here, we identify astrocytic expression of VEGF-A as a key driver of BBB permeability in mice. Inactivation of astrocytic Vegfa expression reduced BBB breakdown, decreased lymphocyte infiltration and neuropathology in inflammatory and demyelinating lesions, and reduced paralysis in a mouse model of MS. Knockdown studies in CNS endothelium indicated activation of the downstream effector eNOS as the principal mechanism underlying the effects of VEGF-A on the BBB. Systemic administration of the selective eNOS inhibitor cavtratin in mice abrogated VEGF-A-induced BBB disruption and pathology and protected against neurologic deficit in the MS model system. Collectively, these data identify blockade of VEGF-A signaling as a protective strategy to treat inflammatory CNS disease.
Abstract: Vascular endothelial growth factor and its receptors, FLK1/KDR and FLT1, are key regulators of angiogenesis. Unlike FLK1/KDR, the role of FLT1 has remained elusive. FLT1 is produced as soluble (sFLT1) and full-length isoforms. Here, we show that pericytes from multiple tissues produce sFLT1. To define the biologic role of sFLT1, we chose the glomerular microvasculature as a model system. Deletion of Flt1 from specialized glomerular pericytes, known as podocytes, causes reorganization of their cytoskeleton with massive proteinuria and kidney failure, characteristic features of nephrotic syndrome in humans. The kinase-deficient allele of Flt1 rescues this phenotype, demonstrating dispensability of the full-length isoform. Using cell imaging, proteomics, and lipidomics, we show that sFLT1 binds to the glycosphingolipid GM3 in lipid rafts on the surface of podocytes, promoting adhesion and rapid actin reorganization. sFLT1 also regulates pericyte function in vessels outside of the kidney. Our findings demonstrate an autocrine function for sFLT1 to control pericyte behavior.
Abstract: Coordination between the vascular system and forming organs is essential for proper embryonic development. The vasculature expands by sprouting angiogenesis, during which tip cells form filopodia that incorporate into capillary loops. Although several molecules, such as vascular endothelial growth factor A (Vegfa), are known to induce sprouting, the mechanism that terminates this process to ensure neovessel stability is still unknown. Sphingosine-1-phosphate receptor 1 (S1P(1)) has been shown to mediate interaction between endothelial and mural cells during vascular maturation. In vitro studies have identified S1P(1) as a pro-angiogenic factor. Here, we show that S1P(1) acts as an endothelial cell (EC)-autonomous negative regulator of sprouting angiogenesis during vascular development. Severe aberrations in vessel size and excessive sprouting found in limbs of S1P(1)-null mouse embryos before vessel maturation imply a previously unknown, mural cell-independent role for S1P(1) as an anti-angiogenic factor. A similar phenotype observed when S1P(1) expression was blocked specifically in ECs indicates that the effect of S1P(1) on sprouting is EC-autonomous. Comparable vascular abnormalities in S1p(1) knockdown zebrafish embryos suggest cross-species evolutionary conservation of this mechanism. Finally, genetic interaction between S1P(1) and Vegfa suggests that these factors interplay to regulate vascular development, as Vegfa promotes sprouting whereas S1P(1) inhibits it to prevent excessive sprouting and fusion of neovessels. More broadly, because S1P, the ligand of S1P(1), is blood-borne, our findings suggest a new mode of regulation of angiogenesis, whereby blood flow closes a negative feedback loop that inhibits sprouting angiogenesis once the vascular bed is established and functional.
Abstract: Monocytes/macrophages are critical in orchestrating the tissue-repair response. However, the mechanisms that govern macrophage regenerative activities during the sequential phases of repair are largely unknown. In the present study, we examined the dynamics and functions of diverse monocyte/macrophage phenotypes during the sequential stages of skin repair. By combining the analysis of a new CCR2-eGFP reporter mouse model with conditional mouse mutants defective in myeloid cell-restricted CCR2 signaling or VEGF-A synthesis, we show herein that among the large number of inflammatory CCR2(+)Ly6C(+) macrophages that dominate the early stage of repair, only a small fraction strongly expresses VEGF-A that has nonredundant functions for the induction of vascular sprouts. The switch of macrophage-derived VEGF-A during the early stage of tissue growth toward epidermal-derived VEGF-A during the late stage of tissue maturation was critical to achieving physiologic tissue vascularization and healing progression. The results of the present study provide new mechanistic insights into CCR2-mediated recruitment of blood monocyte subsets into damaged tissue, the dynamics and functional consequences of macrophage plasticity during the sequential repair phases, and the complementary role of macrophage-derived VEGF-A in coordinating effective tissue growth and vascularization in the context of tissue-resident wound cells. Our findings may be relevant for novel monocyte-based therapies to promote tissue vascularization.
Abstract: Bv8, also known as prokineticin 2, has been characterized as an important mediator of myeloid cell mobilization and myeloid cell-dependent tumor angiogenesis. Bv8 expression is dramatically enhanced by G-CSF, both in vitro and in vivo. The mechanisms involved in such up-regulation remain unknown. Using pharmacological inhibitors that interfere with multiple signaling pathways known to be activated by G-CSF, we show that signal transducer and activator of transcription 3 (Stat3) activation is required for Bv8 up-regulation in mouse bone marrow cells, whereas other Stat family members and extracellular signal-regulated kinase (ERK) activation are not involved. We further identified CD11b(+) Gr1(+) myeloid cells as the primary cell population in which Stat3 signaling is activated by G-CSF. Bv8 expression induced by G-CSF was also significantly reduced by siRNA-mediated Stat3 knockdown. Moreover, chromatin immunoprecipitation studies indicate that G-CSF significantly induces binding of phospho-Stat3 to the Bv8 promoter, which was abolished by pretreatment with the Stat3 inhibitor WP1066. Luciferase assay confirmed that the phospho-Stat3 binding site is a functional enhancer of the Bv8 promoter. The key role of Stat3 signaling in regulating G-CSF-induced Bv8 expression was further confirmed by in vivo studies. We show that the regulation of Bv8 expression in human bone marrow cells is also Stat3 signaling-dependent. Stat3 is recognized as a key regulator of inflammation-dependent tumorigenesis. We propose that such a role of Stat3 reflects at least in part its ability to regulate Bv8 expression.
Abstract: The formation of the vascular network is an intricate and complex process that is an obligate requirement during vertebrate development. The cardiovascular system is the first organ to develop and reach a functional state, which underscores the crucial role of the vasculature in the developing embryo. The development of the vasculature into highly branched conduits needs to occur in numerous sites and in precise patterns to supply oxygen and nutrients to the rapidly expanding tissue of the embryo. This process is mediated by the coordinated response of vascular endothelial and mural cells to the heterogeneous angiogenic cues provided by tissues and organs, whereas aberrant regulation and coordination of angiogenic signals during development result in lethality, impaired organ development, or disease states. This article reviews the essential signaling pathways required for establishment of the vertebrate vasculature with a major focus on a key regulatory factor, vascular endothelial growth factor (VEGF). We also discuss current knowledge of physiological angiogenic processes as well as their disruptions in pathological processes, particularly tumorigenesis.
Abstract: The embryonic heart is composed of two cell layers: the myocardium, which contributes to cardiac muscle tissue, and the endocardium, which covers the inner lumen of the heart. Whereas significant progress has been made toward elucidating the embryonic origins of the myocardium, the origins of the endocardium remain unclear. Here, we have identified an endocardium-forming field medial to the cardiac crescent, in a continuum with the endothelial plexus. In vivo live imaging of quail embryos revealed that endothelial progenitors, like second/anterior heart field progenitors, migrate to, and enter, the heart from the arterial pole. Furthermore, embryonic endothelial cells implanted into the cardiac crescent contribute to the endocardium, but not to the myocardium. In mouse, lineage analysis focusing on endocardial cells revealed an unexpected heterogeneity in the origins of the endocardium. To gain deeper insight into this heterogeneity, we conditionally ablated Flk1 in distinct cardiovascular progenitor populations; FLK1 is required in vivo for formation of the endocardium in the Mesp1 and Tie2 lineages, but not in the Isl1 lineage. Ablation of Flk1 coupled with lineage analysis in the Isl1 lineage revealed that endothelium-derived Isl1(-) endocardial cells were significantly increased, whereas Isl1(+) endocardial cells were reduced, suggesting that the endocardium is capable of undergoing regulative compensatory growth. Collectively, our findings demonstrate that the second heart field contains distinct myocardial and endocardial progenitor populations. We suggest that the endocardium derives, at least in part, from vascular endothelial cells.
Abstract: PlGF, one of the ligands for VEGFR-1, has been implicated in tumor angiogenesis. However, more recent studies indicate that genetic or pharmacological inhibition of PlGF signaling does not result in reduction of microvascular density in a variety of tumor models. Here we screened 12 human tumor cell lines and identified 3 that are growth inhibited by anti-PlGF antibodies in vivo. We found that efficacy of anti-PlGF treatment strongly correlates with VEGFR-1 expression in tumor cells, but not with antiangiogenesis. In addition, PlGF induced VEGFR-1 signaling and biological responses in tumor cell lines sensitive to anti-PlGF, but not in refractory tumor cell lines or in endothelial cells. Also, genetic ablation of VEGFR-1 signaling in the host did not affect the efficacy of PlGF blockade. Collectively, these findings suggest that the role of PlGF in tumorigenesis largely consists of promoting autocrine/paracrine growth of tumor cells expressing a functional VEGFR-1 rather than stimulation of angiogenesis.
Abstract: Napoleone Ferrara and his colleagues at Genentech were the first to isolate and clone vascular endothelial growth factor (VEGF) in 1989. His laboratory has investigated many aspects of VEGF biochemistry and molecular biology. In 1993, Ferrara reported that inhibition of VEGF-induced angiogenesis by specific monoclonal antibodies resulted in dramatic suppression of the growth of a variety of tumors in vivo. These findings provided an important evidence that inhibition of angiogenesis may suppress tumor growth and blocking VEGF action could have therapeutic value for a variety of malignancies. A further development was the design in a rational fashion in 1997 of a humanized anti-VEGF monoclonal antibody (Avastin), now in clinical trials as a treatment for several solid tumors and also outside of cancer, in the treatment of age-related macular degeneration (AMD). Ferrara's work is revolutionizing quality of life for many of the estimated 1.2 million individuals in the US who have wet AMD. Upwards of a million AMD patients worldwide have already received anti-VEGF antibody therapy.
Abstract: In this issue of Cancer Cell, Mazzieri, Pucci, and colleagues describe the marked effects of inhibiting the proangiogenic cytokine, Angiopoietin-2, on tumor angiogenesis and progression in spontaneous tumor models, as well as the proangiogenic functions of TIE2-expressing macrophages.
Abstract: VEGF inhibitors are widely used as a therapy for tumors and intravascular neovascular disorders, but limited and conflicting data regarding their relative biological potencies are available. The purpose of the study is to compare different protein VEGF inhibitors for their ability to inhibit VEGF-stimulated activities. We tested ranibizumab, the full-length variant of ranibizumab (Mab Y0317), bevacizumab, the VEGF-TrapR1R2 and Flt(1-3)-IgG in bioassays measuring VEGF-stimulated proliferation of bovine retinal microvascular endothelial cells or chemotaxis of human umbilical vein endothelial cells (HUVEC). The inhibitors were also compared for their ability to inhibit MAP kinase activation in HUVECs following VEGF addition. Ranibizumab, VEGF-TrapR1R2 and Flt(1-3)-IgG had very similar potencies in the bioassays tested. Bevacizumab was over 10-fold less potent than these molecules. Mab Y0317 was over 30-fold more potent than bevacizumab. The findings reported in this manuscript describe important intrinsic characteristics of several VEGF inhibitors that may be useful to design and interpret preclinical or clinical studies.
Abstract: Myeloid cells are a feature of most tissues. Here we show that during development, retinal myeloid cells (RMCs) produce Wnt ligands to regulate blood vessel branching. In the mouse retina, where angiogenesis occurs postnatally, somatic deletion in RMCs of the Wnt ligand transporter Wntless results in increased angiogenesis in the deeper layers. We also show that mutation of Wnt5a and Wnt11 results in increased angiogenesis and that these ligands elicit RMC responses via a non-canonical Wnt pathway. Using cultured myeloid-like cells and RMC somatic deletion of Flt1, we show that an effector of Wnt-dependent suppression of angiogenesis by RMCs is Flt1, a naturally occurring inhibitor of vascular endothelial growth factor (VEGF). These findings indicate that resident myeloid cells can use a non-canonical, Wnt-Flt1 pathway to suppress angiogenic branching.
Abstract: Vascular endothelial growth factor-A (VEGF) is a potent regulator of vascular permeability, inflammatory response, and cell survival in the lung. To explore the functions of VEGF produced locally in type II pneumocytes, we generated mice with a conditional deletion of VEGF-A using Cre recombinase driven by the human surfactant protein C (SPC) promoter. In 7- to 10-week-old VEGF-knockout (SPC-VEGF-KO) mice, lung histology and physiology were essentially normal, except for higher dynamic lung compliance and lower pulmonary vascular permeability. Emphysema was seen in 28- to 32-week-old animals. To investigate the role of type II pneumocyte-derived VEGF in acute lung injury, we challenged 7- to 10-week-old SPC-VEGF-KO mice and their wild-type littermates with intestinal ischemia-reperfusion. Bronchoalveolar lavage fluid total cell count, pulmonary permeability, and lung injury score were significantly attenuated, and total lung VEGF levels were significantly lower in SPC-VEGF-KO mice compared with wild-type controls. In SPC-VEGF-KO mice, activated caspase 3-positive type II epithelial cells were increased after intestinal ischemia-reperfusion, even though there was no significant difference in the total number of cells positive for terminal deoxynucleotidyl transferase dUTP nick-end labeling. We conclude that VEGF in type II cells helps protect alveolar epithelial cells from caspase-dependent apoptosis. However, VEGF produced from type II cells may contribute to increased vascular permeability during acute lung injury.
Abstract: It is now established that bone marrow-derived myeloid cells regulate tumor angiogenesis. This was originally inferred from studies of human tumor biopsies in which a positive correlation was seen between the number of tumor-infiltrating myeloid cells, such as macrophages and neutrophils, and tumor microvessel density. However, unequivocal evidence was only provided once mouse models were used to examine the effects on tumor angiogenesis by genetically or pharmacologically targeting myeloid cells. Since then, identifying the exact myeloid cell types involved in this process has proved challenging because of myeloid cell heterogeneity and the expression of overlapping phenotypic markers in tumors. As a result, investigators often simply refer to them now as "bone marrow-derived myeloid cells." Here we review the findings of various attempts to phenotype the myeloid cells involved and discuss the therapeutic implications of correctly identifying-and thus being able to target-this proangiogenic force in tumors.
Abstract: It is established that tumor cell-derived VEGF acts on endothelial cells to promote angiogenesis and tumor growth. Here, we demonstrate that in K5-SOS-dependent mouse skin tumors, autocrine VEGF is required for tumor cell proliferation in a cell-autonomous and angiogenesis-independent manner. VEGF is upregulated in SOS-expressing tumors, and its deletion in epidermal cells delays tumorigenesis by suppressing angiogenesis and tumor cell proliferation. Epidermis-specific Flt1 deletion also impairs tumorigenesis and proliferation. Surprisingly, complete tumor inhibition occurs in the absence of VEGF in EGFR mutant mice, demonstrating that VEGFR and EGFR synergize in neoplastic cells to promote tumor growth. Mechanistically, K5-SOS upregulates VEGF, Flt1, and Neuropilin-1 in an Erk-dependent manner, thereby activating an autocrine proliferation loop, whereas EGFR prevents tumor cells from apoptosis. Moreover, Flt1 is upregulated in human SCC, and its inhibition in SCC cells impairs proliferation. Thus, in addition to regulating angiogenesis, VEGF has to be considered as a potent growth factor for epidermal tumors.
Abstract: FDA approval of several inhibitors of the VEGF pathway has enabled significant advances in the therapy of cancer and neovascular age-related macular degeneration. However, similar to other therapies, inherent/acquired resistance to anti-angiogenic drugs may occur in patients, leading to disease progression. So far the lack of predictive biomarkers has precluded identification of patients most likely to respond to such treatments. Recent suggest that both tumor and non-tumor (stromal) cell types are involved in the reduced responsiveness to the treatments. The present review examines the role of tumor- as well as stromal cell-derived pathways involved in tumor growth and in refractoriness to anti-VEGF therapies.
Abstract: Vascular endothelial growth factor (VEGF, VEGF-A) is a major regulator of physiological and pathological angiogenesis. One feature of VEGF is the existence of multiple isoforms arising from alternative exon splicing. Our initial biochemical and biological studies indicated that such isoforms are uniquely suited to generate angiogenic gradients by virtue of their differential ability to interact with the extracellular matrix (ECM). Although ECM-bound VEGF was bioactive, processing by physiologically relevant proteases such as plasmin was identified as a key mechanism to convert ECM-bound VEGF into freely diffusible forms. This retrospective article examines the early studies and also emphasizes the subsequent progress in our understanding of these processes in health and disease.
Abstract: Histological examinations of MMTV-Wnt1 tumors reveal drastic differences in the tumor vasculature when compared to MMTV-Her2 tumors. However, these differences have not been formally described, nor have any angiogenic factors been implicated to be involved in the Wnt1 tumors.
Abstract: 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.
Abstract: Long bone development depends on endochondral bone formation, a complex process requiring exquisite balance between hypertrophic cartilage (HC) formation and its ossification. Dysregulation of this process may result in skeletal dysplasias and heterotopic ossification. Endochondral ossification requires the precise orchestration of HC vascularization, extracellular matrix remodeling, and the recruitment of osteoclasts and osteoblasts. Matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and osteoclasts have all been shown to regulate endochondral ossification, but how their function interrelates is not known. We have investigated the functional relationship among these regulators of endochondral ossification, demonstrating that they have complementary but non-overlapping functions. MMP-9, VEGF and osteoclast deficiency all cause impaired growth plate ossification resulting in the accumulation of HC. VEGF mRNA and protein expression are increased at the MMP-9-/- growth plate, and VEGF activity contributes to endochondral ossification since sequestration of VEGF by soluble receptors results in further inhibition of growth plate vascularization and ossification. However, VEGF bioavailability is still limited in MMP-9 deficiency, as exogenous VEGF is able to rescue the MMP-9-/- phenotype, demonstrating that MMP-9 may partially, but not fully, regulate VEGF bioavailability. The organization of the HC extracellular matrix at the MMP-9-/- growth plate is altered, supporting a role for MMP-9 in HC remodeling. Inhibition of VEGF impairs osteoclast recruitment, whereas MMP-9 deficiency leads to an accumulation of osteoclasts at the chondro-osseous junction. Growth plate ossification in osteoclast-deficient mice is impaired in the presence of normal MMP-9 expression, indicating that other osteoclastic functions are also necessary. Our data delineate the complementary interplay between MMP-9, VEGF and osteoclast function that is necessary for normal endochondral bone formation and provide a molecular framework for investigating the molecular defects contributing to disorders of endochondral bone formation.
Abstract: Vascular endothelial growth factor (VEGF) plays a critical role in normal development as well as retinal vasculature disease. During retinal vascularization, VEGF is most strongly expressed by not yet vascularized retinal astrocytes, but also by retinal astrocytes within the developing vascular plexus, suggesting a role for retinal astrocyte-derived VEGF in angiogenesis and vessel network maturation. To test the role of astrocyte-derived VEGF, we used Cre-lox technology in mice to delete VEGF in retinal astrocytes during development. Surprisingly, this only had a minor impact on retinal vasculature development, with only small decreases in plexus spreading, endothelial cell proliferation and survival observed. In contrast, astrocyte VEGF deletion had more pronounced effects on hyperoxia-induced vaso-obliteration and led to the regression of smooth muscle cell-coated radial arteries and veins, which are usually resistant to the vessel-collapsing effects of hyperoxia. These results suggest that VEGF production from retinal astrocytes is relatively dispensable during development, but performs vessel stabilizing functions in the retinal vasculature and might be relevant for retinopathy of prematurity in humans.
Abstract: Targeting the vascular endothelial growth factor (VEGF) pathway has had a significant impact in the therapy of cancer and intraocular neovascular disorders. Similar to other therapies, inherent/acquired resistance to anti-VEGF drugs may occur in cancer patients, leading to disease recurrence. This review describes recent findings on the role of myeloid cells in refractoriness or/and acquired resistance to such therapies.
Abstract: To determine the role of vascular endothelial growth factor (Vegf) in embryonic erythroid development we have deleted or overexpressed Vegf specifically in the erythroid lineage using the EpoR-iCre transgenic line in combination with Cre/loxP conditional gain and loss of function Vegf alleles. ROSA26 promoter-based expression of the Vegf(164) isoform in the early erythroid lineage resulted in a differentiation block of primitive erythroid progenitor (EryP) development and a partial block in definitive erythropoiesis between the erythroid burst-forming unit and erythroid colony-forming unit stages. Decreased mRNA expression levels of the key erythroid transcription factor Gata1 were causally linked to this phenotype. Conditional deletion of Vegf within the erythroid lineage was associated with increased Gata1 levels and increased erythroid differentiation. Expression of a ROSA26-based GATA2 transgene rescued Gata1 mRNA levels and target genes and restored erythroid differentiation in our Vegf gain of function model. These results demonstrate that Vegf modulates Gata1 expression levels in vivo and provides new molecular insight into Vegf's ability to modulate erythropoiesis.
Abstract: The cardiovascular system ensures the delivery of nutrients, oxygen, and blood and immune cells to all organs and tissues: it is also responsible for the removal of waste metabolites. The vascular system develops and matures through two tightly regulated processes: vasculogenesis and angiogenesis. Angiogenesis is active only under specific physiological conditions in healthy adults but the vasculature can be aberrantly activated to generate new blood vessels during pathological conditions such as cancer and chronic inflammation. In this Opinion article we discuss the parallels and differences in the angiogenic process under either a physiological or a pathological state, especially tumorigenesis.
Abstract: It has been recently reported that treatment with an anti-placenta growth factor (PlGF) antibody inhibits metastasis and primary tumor growth. Here we show that, although anti-PlGF treatment inhibited wound healing, extravasation of B16F10 cells, and growth of a tumor engineered to overexpress the PlGF receptor (VEGFR-1), neutralization of PlGF using four novel blocking antibodies had no significant effect on tumor angiogenesis in 15 models. Also, genetic ablation of the tyrosine kinase domain of VEGFR-1 in the host did not result in growth inhibition of the anti-VEGF-A sensitive or resistant tumors tested. Furthermore, combination of anti-PlGF with anti-VEGF-A antibodies did not result in greater antitumor efficacy than anti-VEGF-A monotherapy. In conclusion, our data argue against an important role of PlGF during primary tumor growth in most models and suggest that clinical evaluation of anti-PlGF antibodies may be challenging.
Abstract: Bevacizumab [Avastin; anti-vascular endothelial growth factor (VEGF) antibody] is an antiangiogenic IgG approved for treating patients with certain types of colon, breast, and lung cancer. In these indications, bevacizumab is administered every 2 to 3 weeks, prompting us to study ways to reduce the frequency of administration. Increasing affinity to neonatal Fc receptor (FcRn) may extend the pharmacokinetic half-life of an antibody, but the quantitative effect of FcRn affinity on clearance has not been clearly elucidated. To gain further insight into this relationship, we engineered a series of anti-VEGF antibody variants with minimal amino acid substitutions and showed a range of half-life improvements in primates. These results suggest that, if proven clinically safe and effective, a modified version of bevacizumab could potentially provide clinical benefit to patients on long-term anti-VEGF therapy through less-frequent dosing and improved compliance with drug therapy. Moreover, despite having half-life similar to that of wild-type in mice due to the species-specific FcRn binding effects, the variant T307Q/N434A exhibited superior in vivo potency in slowing the growth of certain human tumor lines in mouse xenograft models. These results further suggest that FcRn variants may achieve increased potency through unidentified mechanisms in addition to increased systemic exposure.
Abstract: Priming of the organ-specific premetastatic sites is thought to be an important yet incompletely understood step during metastasis. In this study, we show that the metastatic tumors we examined overexpress granulocyte-colony stimulating factor (G-CSF), which expands and mobilizes Ly6G+Ly6C+ granulocytes and facilitates their subsequent homing at distant organs even before the arrival of tumor cells. Moreover, G-CSF-mobilized Ly6G+Ly6C+ cells produce the Bv8 protein, which has been implicated in angiogenesis and mobilization of myeloid cells. Anti-G-CSF or anti-Bv8 antibodies significantly reduced lung metastasis. Transplantation of Bv8 null fetal liver cells into lethally irradiated hosts also reduced metastasis. We identified an unexpected role for Bv8: the ability to stimulate tumor cell migration through activation of one of the Bv8 receptors, prokineticin receptor (PKR)-1. Finally, we show that administration of recombinant G-CSF is sufficient to increase the numbers of Ly6G+Ly6C+ cells in organ-specific metastatic sites and results in enhanced metastatic ability of several tumors.
Abstract: Limb development constitutes a central model for the study of tissue and organ patterning; yet, the mechanisms that regulate the patterning of limb vasculature have been left understudied. Vascular patterning in the forming limb is tightly regulated in order to ensure sufficient gas exchange and nutrient supply to the developing organ. Once skeletogenesis is initiated, limb vasculature undergoes two seemingly opposing processes: vessel regression from regions that undergo mesenchymal condensation; and vessel morphogenesis. During the latter, vessels that surround the condensations undergo an extensive rearrangement, forming a stereotypical enriched network that is segregated from the skeleton. In this study, we provide evidence for the centrality of the condensing mesenchyme of the forming skeleton in regulating limb vascular patterning. Both Vegf loss- and gain-of-function experiments in limb bud mesenchyme firmly established VEGF as the signal by which the condensing mesenchyme regulates the vasculature. Normal vasculature observed in limbs where VEGF receptors Flt1, Flk1, Nrp1 and Nrp2 were blocked in limb bud mesenchyme suggested that VEGF, which is secreted by the condensing mesenchyme, regulates limb vasculature via a direct long-range mechanism. Finally, we provide evidence for the involvement of SOX9 in the regulation of Vegf expression in the condensing mesenchyme. This study establishes Vegf expression in the condensing mesenchyme as the mechanism by which the skeleton patterns limb vasculature.
Abstract: Bv8, also known as prokineticin 2, has been recently shown to be a mediator of myeloid cell-dependent tumor angiogenesis in mouse models. We wished to determine whether these findings might be potentially relevant to human disease.
Abstract: A network of capillaries branches from the hyaloid vascular system and surrounds the mammalian lens throughout much of its embryonic development. These vessels are presumed to be important for the growth and maturation of the lens, although the lenses of non-mammalian vertebrates have no comparable vessels. Over expression of VEGF-A in the lens increases the extent of these capillaries, but it is not known whether VEGF-A from the lens is necessary for their formation or survival. To address this question, we deleted Vegfa in the lens. This prevented the formation of the capillary networks adjacent to the lens capsule, but did not alter nearby hyaloid vessels at the surface of the retina. Postnatal lenses lacking Vegfa were smaller than wild type and, by 1 month of age, many had mild nuclear opacities. These opacities regressed with age. The lens is hypoxic throughout most of life and VEGF-A expression is often regulated by the transcription factor, hypoxia inducible factor-1. Lenses lacking Hif1a were of apparently normal size, had markedly reduced levels of mRNA for VEGF-A and glyceraldehyde-3-phosphate dehydrogenase, but had normal-appearing capillaries covering their surface. We conclude that VEGF-A from the lens is necessary for the formation of the normal hyaloid vascular system and that lack of these capillaries was the most likely cause of growth retardation during fetal and early postnatal lens development. In the absence of HIF-1 function, sufficient VEGF-A is produced by the lens to promote capillary formation. Further study is needed to explain the formation of the mild opacities seen in some lenses lacking Vegfa and their regression later in life.
Abstract: Recent studies suggest that tumor-associated CD11b(+)Gr1(+) myeloid cells contribute to refractoriness to antiangiogenic therapy with an anti-VEGF-A antibody. However, the mechanisms of peripheral mobilization and tumor-homing of CD11b(+)Gr1(+) cells are unclear. Here, we show that, compared with other cytokines [granulocyte-macrophage colony stimulating factor (GM-CSF), stromal derived factor 1alpha, and placenta growth factor], G-CSF and the G-CSF-induced Bv8 protein have preferential expression in refractory tumors. Treatment of refractory tumors with the combination of anti-VEGF and anti-G-CSF (or anti-Bv8) reduced tumor growth compared with anti-VEGF-A monotherapy. Anti-G-CSF treatment dramatically suppressed circulating or tumor-associated CD11b(+)Gr1(+) cells, reduced Bv8 levels, and affected the tumor vasculature. Conversely, G-CSF delivery to animals bearing anti-VEGF sensitive tumors resulted in reduced responsiveness to anti-VEGF-A treatment through induction of Bv8-dependent angiogenesis. We conclude that, at least in the models examined, G-CSF expression by tumor or stromal cells is a determinant of refractoriness to anti-VEGF-A treatment.
Abstract: Angiogenesis, the growth of new blood vessels, is required for a variety of normal proliferative processes. Furthermore, angiogenesis is well established as also playing an important role in neoplastic growth and metastasis. Numerous regulators of angiogenesis have been identified and characterized over the last few decades. Among these, vascular endothelial growth factor (VEGF)-A appears especially important in several pathophysiological processes. Several VEGF inhibitors have been approved, by the US Food and Drug Administration, for the treatment of tumors or age-releted macular degeneration. This review examines the various mouse tumor models in which VEGF inhibitors have been tested and the lessons learned from these studies.
Abstract: Tumor- or cancer-associated fibroblasts (TAFs or CAFs) from different tumors exhibit distinct angiogenic and tumorigenic properties. Unlike normal skin fibroblasts or TAFs from TIB6 tumors that are sensitive to anti-VEGF treatment (TAF-TIB6), TAFs from resistant EL4 tumors (TAF-EL4) can stimulate TIB6 tumor growth even when VEGF is inhibited. We show that platelet-derived growth factor C (PDGF-C) is upregulated in TAFs from resistant tumors. PDGF-C-neutralizing antibodies blocked the angiogenesis induced by such TAFs in vivo, slowed the growth of EL4 and admixture (TAF-EL4 + TIB6) tumors, and exhibited additive effects with anti-VEGF-A antibodies. Hence, our data reveal an additional mechanism for TAF-mediated tumorigenesis and suggest that some tumors may overcome inhibition of VEGF-mediated angiogenesis through upregulation of PDGF-C.
Abstract: Vascular endothelial growth factor (VEGF, VEGF-A) is a major regulator of physiological and pathological angiogenesis. Several VEGF inhibitors have been approved by the FDA for the treatment of advanced cancer and neovascular age-related macular degeneration. This brief review provides a historic account of the challenges associated with the discovery of VEGF and the early steps in elucidating the role of this molecule in the regulation of angiogenesis.
Abstract: Little is known concerning the onset, duration, and magnitude of direct therapeutic effects of anti-vascular endothelial growth factor (VEGF) therapies. Such knowledge would help guide the rational development of targeted therapeutics from bench to bedside and optimize use of imaging technologies that quantify tumor function in early-phase clinical trials.
Abstract: Angiogenesis is required for a variety of normal and pathological, proliferative processes. Numerous regulators of angiogenesis have been identified and characterized over the last decades. Among these, vascular endothelial growth factor (VEGF)-A appears especially important in normal development and in disease processes. Several VEGF inhibitors have been approved by the FDA for the treatment of tumors or the neovascular form of age-related macular degeneration. This article examines the molecular and biological characteristics of VEGF and also discusses preclinical and clinical studies with VEGF inhibitors and the lessons learned from these studies.
Abstract: In vertebrates, endothelial cells (ECs) form blood vessels in every tissue. Here, we investigated vascular lumen formation in the developing aorta, the first and largest arterial blood vessel in all vertebrates. Comprehensive imaging, pharmacological manipulation, and genetic approaches reveal that, in mouse embryos, the aortic lumen develops extracellularly between adjacent ECs. We show that ECs adhere to each other, and that CD34-sialomucins, Moesin, F-actin, and non-muscle Myosin II localize at the endothelial cell-cell contact to define the luminal cell surface. Resultant changes in EC shape lead to lumen formation. Importantly, VE-Cadherin and VEGF-A act at different steps. VE-Cadherin is required for localizing CD34-sialomucins to the endothelial cell-cell contact, a prerequisite to Moesin and F-actin recruitment. In contrast, VEGF-A is required for F-actin-nm-Myosin II interactions and EC shape change. Based on these data, we propose a molecular mechanism of in vivo vascular lumen formation in developing blood vessels.
Abstract: Blood vessel networks form via sprouting of endothelial cells from parent vessels. Extrinsic cues guide sprouts after they leave the initiation site, but these cues are likely insufficient to regulate initial outward movement, and many embryonic vessel networks form in the absence of a strong extrinsic gradient. We hypothesized that nascent sprouts are guided by spatial cues produced along their own vessels, and that soluble Flt-1 (sFlt-1) participates in this guidance. Analysis of developing vessels with perturbed flt-1 function revealed misguided emerging sprouts, and transgenic sFlt-1 rescued sprout guidance parameters. sflt-1 activity in endothelial cells immediately adjacent to the emerging sprout significantly improved local sprout guidance. Thus, we propose that a vessel-intrinsic system initially guides emerging sprouts away from the parent vessel, utilizing spatially regulated expression of sFlt-1 in conjunction with exogenous VEGF-A. Local sprout guidance defects are predicted to contribute to vessel dysmorphogenesis during perturbed development and disease.
Abstract: Identification and characterization of VEGF as an important regulator of angiogenesis, and FDA approval of the first anti-angiogenic drugs, has enabled significant advances in the therapy of cancer and neovascular age-related macular degeneration. However, similar to other therapies, inherent/acquired resistance to anti-angiogenic drugs may occur in patients, leading to disease recurrence. Recent studies in several experimental models suggest that tumor and non-tumor (stromal) cell types may be involved in the reduced responsiveness to the treatments. The present review examines the role of tumor- as well as stromal cell-derived pathways involved in tumor growth and in refractoriness to anti-VEGF therapies.
Abstract: The purpose of this study was to evaluate different methods of detecting and quantifying experimentally induced choroidal neovascularization (CNV) and vascular changes induced on CNV by an anti-VEGF-A monoclonal antibody.
Abstract: The secreted Bv8 protein has been recently characterized as a regulator of myeloid cell mobilization and a neutrophil-derived mediator of tumor angiogenesis in several xenografts, but its role in tumor progression in an endogenous setting was unknown. The rat insulin promoter (RIP)-T-antigen (Tag) is a well characterized transgenic mouse model of multistage pancreatic beta-cell tumorigenesis. Also, the role of neutrophils in RIP-Tag angiogenic switching, as assessed by systemic ablation using anti-Gr1 antibodies at different stages of tumor progression, has been recently described. Here, we show that early treatment of RIP-Tag mice with anti-Bv8 antibodies resulted in a significant reduction in the number of angiogenic islets relative to control antibody-treated mice, implicating Bv8 in the angiogenic switch during neoplasia. Histological analysis showed a significant reduction in vascular surface areas in hyperplastic and angiogenic lesions in pancreatic islets from anti-Bv8-treated mice. Anti-Bv8 treatment also inhibited the mobilization and homing of CD11b+Gr1+ cells to the peripheral blood and the emerging neoplastic lesions. However, anti-Bv8 treatment had no effect on tumor vascularization or burden when initiated at later stages of tumor progression. The stage-dependent efficacy of anti-Bv8 treatment appears remarkably similar to that reported after neutrophil ablation, suggesting that Bv8 is an important mediator of neutrophil-dependent angiogenesis in this transgenic model. In summary, our studies verify a role for Bv8 in the mobilization and recruitment of myeloid cells and in the induction of tumor angiogenesis in the early stages of neoplastic progression.
Abstract: Multiple endocrine neoplasia type 1 (MEN1) is defined clinically by the combined occurrence of multiple tumors, typically of the parathyroid glands, pancreatic islet cells, and anterior pituitary gland. A mouse model with a heterozygous deletion of the Men1 gene recapitulates the tumorigenesis of MEN1. We wished to determine the role of vascular endothelial growth factor (VEGF)-A in the vascularization and growth of MEN1-associated tumors, with an emphasis on pituitary adenomas.
Abstract: The glomerular microvasculature is particularly susceptible to injury in thrombotic microangiopathy, but the mechanisms by which this occurs are unclear. We report the cases of six patients who were treated with bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), in whom glomerular disease characteristic of thrombotic microangiopathy developed. To show that local reduction of VEGF within the kidney is sufficient to trigger the pathogenesis of thrombotic microangiopathy, we used conditional gene targeting to delete VEGF from renal podocytes in adult mice; this resulted in a profound thrombotic glomerular injury. These observations provide evidence that glomerular injury in patients who are treated with bevacizumab is probably due to direct targeting of VEGF by antiangiogenic therapy.
Abstract: Bevacizumab is a humanized anti-human VEGF-A monoclonal antibody (mAb) approved by the United States Food and Drug Administration for cancer therapy and used off label to treat neovascular age-related macular degeneration. Earlier studies characterized bevacizumab as species specific and lacking the ability to neutralize murine (m) VEGF-A. However, a recent study reported that bevacizumab is a potent inhibitor of hemangiogenesis and lymphangiogenesis in murine models. The authors sought to reassess the interaction between bevacizumab and mVEGF-A.
Abstract: Tumor heterogeneity complicates the quantification of tumor microvascular characteristics assessed by dynamic contrast-enhanced MRI (DCE-MRI). To address this issue a novel approach was developed that combines DCE-MRI with diffusion-based multispectral (MS) analysis to quantify the microvascular characteristics of specific tumor tissue populations. Diffusion-based MS segmentation (feature space: apparent diffusion coefficient, T(2) and proton density) was performed to identify tumor tissue populations and the DCE-MRI characteristics were determined for each tissue class. The ability of this MS DCE-MRI technique to detect microvascular changes due to treatment with an antibody (G6-31) to vascular endothelial growth factor-A (VEGF) was evaluated in a tumor xenograft mouse model. Anti-VEGF treatment resulted in a significant reduction in K(trans) for the MS viable tumor tissue class (-0.0034 +/- 0.0022 min(-1), P < 0.01) at 24 hr posttreatment that differ significantly from the change observed in the control group (0.0002 +/- 0.0025 min(-1)). Viable tumor K(trans) for the anti-VEGF group was also reduced 62% relative to the pretreatment values (P < 0.01). Necrotic tissue classes were found to add only noise to DCE-MRI estimates. This approach provides a means to measure physiological parameters within the viable tumor and address the issue of tumor heterogeneity that complicates DCE-MRI analysis.
Abstract: Cells of the innate immune system have a key role in maintaining homeostasis by providing the first line of defense against many pathogens. Innate immunity can also modulate the activity of acquired immunity by several mechanisms. However, subsets of myeloid cells can facilitate tumor growth, because these cells produce angiogenic factors and can also prevent the immune system from attacking tumor cells. Recent studies also emphasize the role of myeloid cells in mediating refractoriness to anti-VEGF treatments. This function of myeloid cells occurs through a proangiogenic pathway that is, at least in part, driven by the secreted protein Bv8. This review summarizes recent findings on the complex role of bone marrow-derived cells in tumor growth.
Abstract: Angiogenesis is critical for growth of many tumor types and the development of anti-angiogenic agents opened a new era in cancer therapy. However, similar to other anti-cancer therapies, inherent/acquired resistance to anti-angiogenic drugs may occur in cancer patients leading to disease recurrence. Recent studies in several experimental models suggest that both tumor and non-tumor (stromal) cell types may be involved in the reduced responsiveness to the treatments. The current review focuses on the role of stromal cells in tumor growth and in refractoriness to anti-VEGF treatment.
Abstract: Angiogenesis, the growth of new blood vessels, is required for a variety of normal proliferative processes. Furthermore, it is well established that angiogenesis plays an important role also in neoplastic growth and metastasis. Numerous regulators of angiogenesis have been identified and characterized over the last decades. Among these, vascular endothelial growth factor (VEGF)-A appears especially important in several pathophysiological processes. Several VEGF inhibitors have been approved by the Food and Drug Administration for the treatment of tumors or age-related macular degeneration. This chapter examines the various mouse tumor models in which VEGF inhibitors have been tested and the lessons learned from these studies.
Abstract: CD11b+Gr1+ cells, which include neutrophils, macrophages, and myeloid-derived suppressor cells, have been shown to contribute to tumor angiogenesis. Recently, we found that accumulation of CD11b+Gr1+ in tumors renders them refractory to angiogenic blockade by vascular endothelial growth factor (VEGF) antibodies. This effect was traced to a pathway of CD11b+Gr1+-mediated angiogenesis that is, at least in part, driven by the secreted protein Bv8, which is up-regulated by the important myeloid growth factor granulocyte colony-stimulating factor (G-CSF). Thus, G-CSF may promote tumor angiogenesis through a Bv8-dependent pathway that bypasses VEGF and renders tumors refractory to anti-VEGF therapy.
Abstract: The vascular endothelium is best known for its role in oxygen and nutrient delivery to the various tissues. Growing evidence supports a far more complex role in tissue homeostasis. In particular, reciprocal interactions between endothelial cells and the local microenvironment may regulate organ development and pattern formation. Such interactions appear to be important also in the adult, in normal and pathological conditions.
Abstract: Identification and characterization of several important regulators of angiogenesis, and FDA approval of the first antiangiogenic drugs, has opened a new era in the therapy of cancer and neovascular age-related macular degeneration. This brief review focuses on the progress in targeting one of the major regulators of angiogenesis, VEGF-A, and also discusses potential cellular and molecular mechanisms underlying resistance to antiangiogenic treatments.
Abstract: Inhibiting angiogenesis is a promising strategy to treat cancer and several other disorders, including intraocular neovascular syndromes. The identification of vascular endothelial growth factor (VEGF)-A as a major regulator of normal and pathological angiogenesis has enabled significant progress toward effective treatments for such disorders. Several VEGF inhibitors have been recently approved by the U.S. Food and Drug Administration for the treatment of cancer and the neovascular form of age-related macular degeneration. This review summarizes the basic biology of VEGF-A and illustrates the clinical progress in targeting this molecule.
Abstract: Non-immune (naïve) antibody phage libraries have become an important source of human antibodies. The synthetic phage antibody library described here utilizes a single human framework with a template containing human consensus complementarity-determining regions (CDRs). Diversity of the libraries was introduced at select CDR positions using tailored degenerate and trinucleotide codons that mimic natural human antibodies. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 semaphorins, is expressed on endothelial cells and neurons. NRP1 is required for vascular development and is expressed widely in the developing vasculature. To investigate the possibility of function blocking antibodies to NRP1 as potential therapeutics, and study the consequence of targeting NRP1 in murine tumor models, panels of antibodies that cross-react with human and murine NRP1 were generated from a designed antibody phage library. Antibody (YW64.3) binds to the CUB domains (a1a2) of NRP1 and completely blocks Sema3A induced neuron collapse; antibody (YW107.4.87) binds to the coagulation factor V/VIII domains (b1b2) of NRP1 and blocks VEGF binding and VEGF induced cell migration. YW107.4.87 inhibits tumor growth in animal xenograft models. These antibodies have provided valuable tools to study the roles of NRP1 in vascular and tumor biology.
Abstract: VEGF-A is important in tumor angiogenesis, and a humanized anti-VEGF-A monoclonal antibody (bevacizumab) has been approved by the FDA as a treatment for metastatic colorectal and nonsquamous, non-small-cell lung cancer in combination with chemotherapy. However, contributions of both tumor- and stromal-cell derived VEGF-A to vascularization of human tumors grown in immunodeficient mice hindered direct comparison between the pharmacological effects of anti-VEGF antibodies with different abilities to block host VEGF. Therefore, by gene replacement technology, we engineered mice to express a humanized form of VEGF-A (hum-X VEGF) that is recognized by many anti-VEGF antibodies and has biochemical and biological properties comparable with WT mouse and human VEGF-A. The hum-X VEGF mouse model was then used to compare the activity and safety of a panel of VEGF Mabs with different affinities for VEGF-A. Although in vitro studies clearly showed a correlation between binding affinity and potency at blocking endothelial cell proliferation stimulated by VEGF, in vivo experiments failed to document any consistent correlation between antibody affinity and the ability to inhibit tumor growth and angiogenesis in most animal models. However, higher-affinity antibodies were more likely to result in glomerulosclerosis during long-term treatment.
Abstract: Anti-VEGF-A monoclonal antibodies, in combination with chemotherapy, result in a survival benefit in patients with metastatic colorectal and non-small cell lung cancer, but little is known regarding the impact of anti-VEGF-A therapy on benign or premalignant tumors. The Apc+/min mice have been widely used as a model recapitulating early intestinal adenoma formation. To investigate whether tumor growth in Apc+/min mice is mediated by VEGF-A-dependent angiogenesis, we used two independent approaches to inhibit VEGF-A: monotherapy with a monoclonal antibody (Mab) targeting VEGF-A and genetic deletion of VEGF-A selectively in intestinal epithelial cells. Short-term (3 or 6 weeks) treatment with anti-VEGF-A Mab G6-31 resulted in a nearly complete suppression of adenoma growth throughout the small intestine. Growth inhibition by Mab G6-31 was associated with a decrease in vascular density. Long-term (up to 52 weeks) treatment with Mab G6-31 led to a substantial increase in median survival. Deletion of VEGF-A in intestinal epithelial cells of Apc+/min mice yielded a significant inhibition of tumor growth, albeit of lesser magnitude than that resulting from Mab G6-31 administration. These results establish that inhibition of VEGF-A signaling is sufficient for tumor growth cessation and confers a long-term survival benefit in an intestinal adenoma model. Therefore, VEGF-A inhibition may be a previously uncharacterized strategy for the prevention of the angiogenic switch and growth in intestinal adenomas.
Abstract: Bone-marrow-derived cells facilitate tumour angiogenesis, but the molecular mechanisms of this facilitation are incompletely understood. We have previously shown that the related EG-VEGF and Bv8 proteins, also known as prokineticin 1 (Prok1) and prokineticin 2 (Prok2), promote both tissue-specific angiogenesis and haematopoietic cell mobilization. Unlike EG-VEGF, Bv8 is expressed in the bone marrow. Here we show that implantation of tumour cells in mice resulted in upregulation of Bv8 in CD11b+Gr1+ myeloid cells. We identified granulocyte colony-stimulating factor as a major positive regulator of Bv8 expression. Anti-Bv8 antibodies reduced CD11b+Gr1+ cell mobilization elicited by granulocyte colony-stimulating factor. Adenoviral delivery of Bv8 into tumours was shown to promote angiogenesis. Anti-Bv8 antibodies inhibited growth of several tumours in mice and suppressed angiogenesis. Anti-Bv8 treatment also reduced CD11b+Gr1+ cells, both in peripheral blood and in tumours. The effects of anti-Bv8 antibodies were additive to those of anti-Vegf antibodies or cytotoxic chemotherapy. Thus, Bv8 modulates mobilization of CD11b+Gr1+ cells from the bone marrow during tumour development and also promotes angiogenesis locally.
Abstract: Vascular endothelial growth factor (VEGF) is essential for developmental and pathological angiogenesis. Here we show that in the absence of any pathological insult, autocrine VEGF is required for the homeostasis of blood vessels in the adult. Genetic deletion of vegf specifically in the endothelial lineage leads to progressive endothelial degeneration and sudden death in 55% of mutant mice by 25 weeks of age. The phenotype is manifested without detectable changes in the total levels of VEGF mRNA or protein, indicating that paracrine VEGF could not compensate for the absence of endothelial VEGF. Furthermore, wild-type, but not VEGF null, endothelial cells showed phosphorylation of VEGFR2 in the absence of exogenous VEGF. Activation of the receptor in wild-type cells was suppressed by small molecule antagonists but not by extracellular blockade of VEGF. These results reveal a cell-autonomous VEGF signaling pathway that holds significance for vascular homeostasis but is dispensable for the angiogenic cascade.
Abstract: The identification and characterization of several important regulators of angiogenesis, which led to Food and Drug Administration approval of the first antiangiogenic drugs, has opened a new era in cancer therapy. This article focuses on the clinical progress in targeting one of the major regulators of angiogenesis, vascular endothelial growth factor-A and also discusses some recent advances in the elucidation of potential cellular and molecular mechanisms underlying refractoriness or resistance to antiangiogenic therapies.
Abstract: Vascular endothelial growth factor (VEGF) is an essential regulator of normal and abnormal blood vessel growth. A monoclonal antibody (mAb) that targets VEGF suppresses tumor growth in murine cancer models and human patients. We investigated cellular and molecular events that mediate refractoriness of tumors to anti-angiogenic therapy. Inherent anti-VEGF refractoriness is associated with infiltration of the tumor tissue by CD11b+Gr1+ myeloid cells. Recruitment of these myeloid cells is also sufficient to confer refractoriness. Combining anti-VEGF treatment with a mAb that targets myeloid cells inhibits growth of refractory tumors more effectively than anti-VEGF alone. Gene expression analysis in CD11b+Gr1+ cells isolated from the bone marrow of mice bearing refractory tumors reveals higher expression of a distinct set of genes known to be implicated in active mobilization and recruitment of myeloid cells. These findings indicate that, in our models, refractoriness to anti-VEGF treatment is determined by the ability of tumors to prime and recruit CD11b+Gr1+ cells.
Abstract: There is increasing evidence that epithelial-vascular interactions are essential for tissue patterning. Here we identified components of the molecular cross talk between respiratory epithelial cells and pulmonary capillaries necessary for the formation of the gas exchange surface of the lung. Selective inactivation of the Vegf-A gene in respiratory epithelium results in an almost complete absence of pulmonary capillaries, demonstrating the dependence of pulmonary capillary development on epithelium-derived Vegf-A. Deficient capillary formation in Vegf-A deficient lungs is associated with a defect in primary septae formation, a morphogenetic process critical for distal lung morphogenesis, coupled with suppression of epithelial cell proliferation and decreased hepatocyte growth factor (Hgf) expression. Lung endothelial cells express Hgf, and selective deletion of the Hgf receptor gene in respiratory epithelium phenocopies the malformation of septae, confirming the requirement for epithelial Hgf signaling in normal septae formation and suggesting that Hgf serves as an endothelium-derived factor that signals to the epithelium. Our findings support a mechanism for primary septae formation dependent on reciprocal interactions between respiratory epithelium and the underlying vasculature, establishing the dependence of pulmonary capillary development on epithelium-derived Vegf-A, and identify Hgf as a putative endothelium-derived factor that mediates the reciprocal signaling from the vasculature to the respiratory epithelium.
Abstract: Endocrine pancreatic beta cells require endothelial signals for their differentiation and function. However, the molecular basis for such signals remains unknown. Here, we show that beta cells, in contrast to the exocrine pancreatic cells, do not form a basement membrane. Instead, by using VEGF-A, they attract endothelial cells, which form capillaries with a vascular basement membrane next to the beta cells. We have identified laminins, among other vascular basement membrane proteins, as endothelial signals, which promote insulin gene expression and proliferation in beta cells. We further demonstrate that beta1-integrin is required for the beta cell response to the laminins. The proposed mechanism explains why beta cells must interact with endothelial cells, and it may apply to other cellular processes in which endothelial signals are required.
Abstract: The glomerular filtration barrier separates the blood from the urinary space and consists of two major cell types: podocytes and fenestrated endothelial cells. Mesangial cells sit between the capillary loops and provide structural support. Proliferation and loss of mesangial cells both are central findings in a number of renal diseases, including diabetic nephropathy and mesangiolysis, respectively. Using cell-specific gene targeting, it was shown previously that vascular endothelial growth factor A (VEGF-A) production by podocytes is required for glomerular endothelial cell migration, differentiation, and survival. For further investigation of the effect of gene dose and VEGF-A knockdown within the glomerulus, mice that carry one hypomorphic VEGF-A allele and one podocyte-specific null VEGF-A allele (VEGFhypo/loxP,Neph-Cre+/-) were generated; in these mice, the "allelic dose" of VEGF-A is intermediate between glomerular-specific heterozygous and null states. VEGFhypo/loxP,Neph-Cre+/- mice die at 3 wk of age from renal failure. Although endothelial cell defects are observed, striking loss of mesangial cells occurs postnatally. In addition, differentiated mesangial cells cannot be found in glomeruli of podocyte-specific null VEGF-A mice (VEGFloxP/loxP,Cre+/-). Together, these results demonstrate a key role for VEGF-A production in the podocyte for mesangial cell survival and differentiation.
Abstract: Vascular endothelial growth factor-A (VEGF-A) and its 2 transmembrane tyrosine-kinase receptors, VEGFR-1 and VEGFR-2, constitute a ligand-receptor signaling system that is crucial for developmental angiogenesis. VEGF-B and placental growth factor (PlGF) activate VEGFR-1 selectively, however, mice lacking either ligand display only minor developmental defects. We hypothesized that the relative contributions of VEGF-B and PlGF to VEGFR-1 signaling may be masked in the presence of VEGF-A, which is abundantly expressed during postnatal development. To test this hypothesis, neonatal or adult mice were treated with a monoclonal antibody (G6-23-IgG) blocking murine VEGF-A or a soluble VEGFR-1 receptor IgG chimeric construct [mFlt(1-3)-IgG], which neutralizes VEGF-A, VEGF-B, and PlGF. Both compounds attenuated growth and survival of neonatal mice to similar extents and the pathophysiologic alterations, including a reduction in organ size and vascularization, changes in gene expression, and hematologic end points, were essentially indistinguishable. In adult mice, we observed only minor changes in response to treatment, which were similar between both anti-VEGF compounds. In conclusion, our findings suggest that PlGF and VEGF-B do not compensate during conditions of VEGF-A blockade, suggesting a minor role for compensatory VEGFR-1 signaling during postnatal development and vascular homeostasis in adults. The absence of compensatory VEGFR-1 signaling by VEGF-B and PlGF may have important implications for the development of anticancer strategies targeting the VEGF ligand/receptor system.
Abstract: Activated fibroblasts are thought to play important roles in the progression of many solid tumors, but little is known about the mechanisms responsible for the recruitment of fibroblasts in tumors. Using several methods, we identified platelet-derived growth factor A (PDGFA) as the major fibroblast chemoattractant and mitogen from conditioned medium generated by the Calu-6 lung carcinoma cell line. In addition, we showed that Calu-6 tumors express significant levels of PDGFC, and that the levels of expression of these two PDGFRalpha ligands correlate strongly with the degree of stromal fibroblast infiltration into the tumor mass. The most intense expression of PDGFRalpha was observed in fibroblasts in the tumor outer rim. We subsequently showed that disrupting PDGFRalpha-mediated signaling results in significant inhibition of tumor growth in vivo. Furthermore, analysis of a compendium of microarray data revealed significant expression of PDGFA, PDGFC, and PDGFRalpha in human lung tumors. We propose that therapies targeting this stromal cell type may be effective in treating certain types of solid tumors.
Abstract: To fully assess the role of VEGF-A in tumor angiogenesis, antibodies that can block all sources of vascular endothelial growth factor (VEGF) are desired. Selectively targeting tumor-derived VEGF overlooks the contribution of host stromal VEGF. Other strategies, such as targeting VEGF receptors directly or using receptor decoys, result in inhibiting not only VEGF-A but also VEGF homologues (e.g. placental growth factor, VEGF-B, and VEGF-C), which may play a role in angiogenesis. Here we report the identification of novel anti-VEGF antibodies, B20 and G6, from synthetic antibody phage libraries, which block both human and murine VEGF action in vitro. Their affinity-improved variants completely inhibit three human tumor xenografts in mice of skeletal muscle, colorectal, and pancreatic origins (A673, HM-7, and HPAC). Avastin, which only inhibits the tumor-derived human VEGF, is approximately 90% effective at inhibiting HM-7 and A673 growth but is <50% effective at inhibiting HPAC growth. Indeed, HPAC tumors contain more host stroma invasion and stroma-derived VEGF than other tumors. Thus, the functional contribution of stromal VEGF varies greatly among tumors, and systemic blockade of both tumor and stroma-derived VEGF is sufficient for inhibiting the growth of tumor xenografts.
Abstract: Corneal avascularity-the absence of blood vessels in the cornea-is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.
Abstract: The establishment of a vascular supply is one of the earliest and most important events occurring during embryonic development. Growth and maturation of a functional vascular network are complex and still incompletely understood processes involving orchestrated activation of vascular progenitors in the early stages of embryonic development followed by vasculogenesis and angiogenesis. These processes require a tightly regulated activation of several growth factors and their receptors. The role of vascular endothelial growth factors (VEGF) and their receptors has been studied extensively due to their prominent role during blood vessel formation. Mice deficient in various VEGF ligands or receptors show serious defects in vascular formation and maturation. Moreover, members of the VEGF family are involved in other significant biological processes, including lymphangiogenesis, vascular permeability, and hematopoiesis. Importantly, VEGF is released by tumor cells and induces tumor neovascularization. It is now well established that the VEGF axis represents an important target for antitumor therapy. Aberrant VEGF signaling is also a feature of several other pathologic conditions, such as age-related macular degeneration and rheumatoid arthritis.
Abstract: Ascites formation associated with neoplasms is most likely due to increased vascular permeability, a process in which vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) plays a pivotal role. We hypothesized that tumor-derived VEGF/VPF modulates ascites formation through a paracrine effect on both tumor and peritoneal vessels. We investigated human vascular endothelial permeability using a newly developed dual-chamber permeability assay by co-culturing human umbilical vein cells with and without ovarian cancer cell lines (OVCAR-3, Hey-A8, and OCC-1) in the presence or absence of a human VEGF monoclonal antibody and VE-cadherin function-blocking antibody. This method permits determination of mechanisms by which substances released from neoplasms and other sources of vascular endothelial cell secretagogues modulate vascular permeability and likely other pathologic states.
Abstract: Angiogenesis is a key aspect of the wet form of age-related neovascular (AMD), the leading cause of blindness in the elderly population. Substantial evidence indicated that vascular endothelial growth factor (VEGF)-A is a major mediator of angiogenesis and vascular leakage in wet AMD. VEGF-A is the prototype member of a gene family that includes also PlGF, VEGF-B, VEGF-C, VEGF-D and the orf virus-encoded VEGF-E. Several isoforms of VEGF-A can be generated due to alternative mRNA splicing. Various VEGF inhibitors have been clinically developed. Among these, ranibizumab is a high affinity recombinant Fab that neutralizes all isoforms of VEGF-A. The article briefly reviews the biology of VEGF and then focuses on the path that led to clinical development of ranibizumab.
Abstract: Since the discovery of vascular-specific growth factors with angiogenic activity, there has been a significant effort to develop cancer drugs that restrict tumorigenesis by targeting the blood supply. In this issue of the JCI, Mancuso et al. use mouse models to better understand the plasticity of the tumor vasculature in the face of antiangiogenic therapy (see the related article beginning on page 2610). They describe a rapid regrowth of the tumor vasculature following withdrawal of VEGFR inhibitors, emphasizing the importance of fully understanding the function of these and similar treatments used in the clinic at the cellular and molecular level.
Abstract: Preclinical models have examined the pharmacologic and pharmacodynamic activities of an anti-vascular endothelial growth factor (VEGF) humanized, monoclonal antibody, bevacizumab, and/or its murine equivalent A4.6.1. These studies found that single-agent therapy with bevacizumab/A4.6.1 resulted in tumor growth inhibition of 20 different human tumor cell lines (13 tumor types) implanted into nude mice irrespective of the route of administration or tumor location. Several of these studies also observed significant inhibition of tumor metastases. Various studies have examined the feasibility of combining anti-VEGF therapy with cytotoxic or biological agents. Combining bevacizumab/A4.6.1 with doxorubicin, topotecan, paclitaxel, docetaxel, or radiotherapy resulted in additive or synergistic tumor growth inhibition. Changes in vascular functions were frequently reported, including decreased vessel diameter, density, and permeability in response to treatment. A reduction in interstitial fluid pressure was also observed. In some studies, these improvements resulted in an increase in intratumoral uptake of chemotherapy, implying that the most effective use of anti-VEGF therapy is in combination with chemotherapy. Alternatively, combination treatment with radiation increased tumor oxygenation and tumor growth inhibition. Interestingly, anti-VEGF therapy has also been reported to reduce the development of ascites in ovarian mouse models. Finally, safety pharmacology studies with bevacizumab in cynomolgus monkeys showed that this agent is generally well tolerated with no unexpected adverse events.
Abstract: The vasculature forms an intrinsic functional component of the lung and its development must be tightly regulated and coordinated with lung epithelial morphogenesis. Vascular endothelial growth factor (VEGF) and its receptors are highly expressed in a complementary pattern in the lungs during embryonic development. VEGF is expressed by epithelium and the receptors in the surrounding mesenchyme. To determine the function of VEGF in lung formation, we inhibited its activity using a soluble receptor in lung renal capsule grafts. Inhibition of VEGF results in inhibition of vascular development and significant alteration in epithelial development. Epithelial proliferation is inhibited, sacculation is impaired, and the epithelium undergoes apoptosis. Interestingly, when VEGF is attenuated, epithelial differentiation still proceeds, as shown by acquisition of both proximal and distal markers. These data show that VEGF co-ordinates epithelial and vascular development. It is required for the development of the lung vasculature and the vasculature is necessary for epithelial proliferation and morphogenesis, but not for cell differentiation.
Abstract: In developing limb skin, peripheral nerves are required for arterial differentiation, and guide the pattern of arterial branching. In vitro experiments suggest that nerve-derived VEGF may be important for arteriogenesis, but its role in vivo remains unclear. Using a series of nerve-specific Cre lines, we show that VEGF derived from sensory neurons, motoneurons and/or Schwann cells is required for arteriogenesis in vivo. Arteriogenesis also requires endothelial expression of NRP1, an artery-specific coreceptor for VEGF(164) that is itself induced by VEGF. Our results provide the first evidence that VEGF is necessary for arteriogenesis from a primitive capillary plexus in vivo, and show that in limb skin the nerve is indeed the principal source of this signal. They also suggest a model in which a 'winner-takes-all' competition for VEGF may control arterial differentiation, with the outcome biased by a VEGF(164)-NRP1 positive-feedback loop. Our results also demonstrate that nerve-vessel alignment is a necessary, but not sufficient, condition for nerve-induced arteriogenesis. Different mechanisms therefore probably underlie these endothelial patterning and differentiation processes.
Abstract: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen in vitro and an angiogenic inducer in vivo. The tyrosine kinases Flt-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2) are high affinity VEGF receptors. VEGF plays an essential role in developmental angiogenesis and is important also for reproductive and bone angiogenesis. Substantial evidence also implicates VEGF as a mediator of pathological angiogenesis. Anti-VEGF monoclonal antibodies and other VEGF inhibitors block the growth of several tumor cell lines in nude mice. Clinical trials with VEGF inhibitors in a variety of malignancies are ongoing. Recently, a humanized anti-VEGF monoclonal antibody (bevacizumab; Avastin) has been approved by the FDA as a first-line treatment for metastatic colorectal cancer in combination with chemotherapy. Furthermore, VEGF is implicated in intraocular neovascularization associated with diabetic retinopathy and age-related macular degeneration.
Abstract: Tumors require nutrients and oxygen in order to grow, and new blood vessels, formed by the process of angiogenesis, provide these substrates. The key mediator of angiogenesis is vascular endothelial growth factor (VEGF), which is induced by many characteristics of tumors, most importantly hypoxia. Therefore, VEGF is an appealing target for anticancer therapeutics. In addition, VEGF is easy to access as it circulates in the blood and acts directly on endothelial cells. VEGF-mediated angiogenesis is rare in adult humans (except wound healing and female reproductive cycling), and so targeting the molecule should not affect other physiological processes. Tumor blood vessels, formed under the influence of VEGF, are disorganized, tortuous and leaky with high interstitial pressure, reducing access for chemotherapies. Inhibiting VEGF would reduce the vessel abnormality and increase the permeability of the tumor to chemotherapies. Several approaches to targeting VEGF have been investigated. The most common strategies have been receptor-targeted molecules and VEGF-targeting molecules. The disadvantage of receptor-targeted approaches is that the VEGF receptors also bind different members of the VEGF super-family and affect systems other than angiogenesis. The best-studied and most advanced approach to VEGF inhibition is the humanized monoclonal antibody bevacizumab (Avastin), which is the only anti-angiogenic agent approved for treatment of cancer.
Abstract: Inhibiting angiogenesis is a promising strategy for treatment of cancer and several other disorders, including age-related macular degeneration. Major progress towards a treatment has been achieved over the past few years, and the first antiangiogenic agents have been recently approved for use in several countries. Therapeutic angiogenesis (promoting new vessel growth to treat ischaemic disorders) is an exciting frontier of cardiovascular medicine, but further understanding of the mechanisms of vascular morphogenesis is needed first.
Abstract: The choroid in the eye provides vascular support for the retinal pigment epithelium (RPE) and the photoreceptors. Vascular endothelial growth factor (VEGF) derived from the RPE has been implicated in the physiological regulation of the choroidal vasculature, and overexpression of VEGF in this epithelium has been considered an important factor in the pathogenesis of choroidal neovascularization in age-related macular degeneration. Here, we demonstrate that RPE-derived VEGF is essential for choriocapillaris development. Conditional inactivation of VEGF expression in the RPE (in VEGFrpe-/- mice) results in the absence of choriocapillaris, occurrence of microphthalmia, and the loss of visual function. Severe abnormalities of RPE cells are already observed when VEGF expression in the RPE is only reduced (in VEGFrpe+/- mice), despite the formation of choroidal vessels at these VEGF levels. Finally, using Hif1arpe-/- mice we demonstrate that these roles of VEGF are not dependent on hypoxia-inducible factor-1alpha-mediated transcriptional regulation of VEGF expression in the RPE. Thus, hypoxia-inducible factor-1alpha-independent expression of VEGF is essential for choroid development.
Abstract: Angiogenesis is a multistep, complex and tightly regulated process that is necessary for tumor growth and metastasis. Based on data of preclinical models, several antiangiogenic compounds has been shown to modify activated tumor endothelium, which suggests that these compounds can improve cytotoxic drug delivery. Such agents have entered clinical trials as single agents or in combination with cytotoxic drugs, and have shown promising antitumor activity. The pharmacodynamic and pharmacokinetic characteristics of antiangiogenic drugs are reviewed here. Most of the early clinical testing of these agents was conducted in patients with advanced disease resistant to standard therapies. Phase III trials compared the efficacy of standard chemotherapy alone with standard chemotherapy in combination with an experimental angiogenesis inhibitor. Although some of these studies were negative or controversial, recent studies validated in large clinical trials with an anti-vascular endothelial growth factor antibody demonstrated significant clinical benefit and renewed enthusiasm for this therapeutic strategy. This review describes the clinical studies of antiangiogenic agents and highlights the challenges related to choosing appropriate strategies for the selection of patients, study design and choice of appropriate endpoints for the studies' development.
Abstract: Skeletal muscle angiogenesis is an important physiological adaptation to increased metabolic demand, possibly dependent on vascular endothelial growth factor (VEGF), the increased expression of which is a known early response to exercise. To test the hypothesis that VEGF is essential to muscle capillary maintenance, we evaluated the consequences of targeted skeletal muscle inhibition of VEGF expression in postnatal, cage-confined VEGFloxP(+/+) mice. To delete VEGF, cre recombinase expression was accomplished using direct intramuscular injection of a recombinant adeno-associated cre recombinase expressing viral vector. Four weeks postinfection, VEGF-inactivated regions revealed 64% decreases in capillary density and capillary-to-fiber ratio. Substantial apoptosis was also observed in VEGF-depleted regions. There was no evidence of rescue at 8 wk, with a persistent 67% reduction in capillary-to-fiber ratio and a 69% decrease in capillary density. These data implicate VEGF as an essential survival factor for muscle capillarity and also demonstrate insufficient VEGF-dependent signaling leads to apoptosis in mouse skeletal muscle.
Abstract: To directly examine the role of vascular endothelial growth factor (VEGFA) in cartilage development, we conditionally knocked out Vegfa in chondrocytes, using the Col2a1 promoter to drive expression of Cre recombinase. Our study of Vegfa conditional knockout (CKO) mice provides new in-vivo evidence for two important functions of VEGFA in bone formation. First, VEGFA plays a significant role in both early and late stages of cartilage vascularization, since Vegfa CKO mice showed delayed invasion of blood vessels into primary ossification centers and delayed removal of terminal hypertrophic chondrocytes. Second, VEGFA is crucial for chondrocyte survival, since massive cell death was seen in joint and epiphyseal regions of Vegfa CKO endochondral bones. Chondrocytes in these regions were found to upregulate expression of Vegfa in wild-type mice at the time when massive cell death occurred in the Vegfa CKO mice. The expression of the VEGFA receptors Npr1 and Npr2 in epiphyseal chondrocytes and lack of blood vessel reduction in the vicinity of the cartilaginous elements in the Vegfa CKO mice raise the possibility that the observed cell death is the result of a direct involvement of VEGFA in chondrocyte survival. Interestingly, the extensive cell death seen in Vegfa CKO null bones had a striking similarity to the cell death phenotype observed when hypoxia-inducible factor 1 alpha (Hif1a) expression was abolished in developing cartilage. This similarity of cell death phenotypes and the deficient VEGFA production in Hif1a null epiphyseal chondrocytes demonstrate that HIF1 alpha and VEGFA are components of a key pathway to support chondrocyte survival during embryonic bone development.
Abstract: Vascular endothelial growth factor (VEGF) is essential for the differentiation of the primitive embryonic vascular system and has been implicated in the vascularization of organs. Recently, VEGF has also been proposed to play a role in neural development, neuroprotection, and adult neurogenesis. Here we have investigated the function of VEGF in the developing brain by cre-lox technology. We show that VEGF produced by the embryonic neuroectoderm is required for the vascularization and the development of the brain. Both the invasion and the directed growth of capillaries were severely impaired in the fore-, mid- and hindbrain of VEGF(lox/lox)/nestin-cre mouse embryos homozygous for a VEGF mutation in the neural tube. These observations demonstrate that VEGF, via local secretion by neural progenitors, induces brain angiogenesis and guides the growth of capillaries toward the ventricular zone. VEGF deficiency led to developmental retardation and progressive destruction of neural tissue in all brain regions. The defect was most pronounced in telencephalic structures, such as the hippocampus, and caused microcephaly. Taken together, the findings establish the critical importance of neuroectoderm-derived VEGF in the morphogenesis of the brain. VEGF acts as a key regulator of brain angiogenesis and provides instructive cues for the correct spatial organization of the vasculature.
Abstract: Angiogenesis is the focus of therapeutic efforts to promote new vessel development in damaged tissues. Conversely, inhibiting endothelial cell growth and survival is a strategy to treat various proliferative diseases. Much evidence indicates that VEGF is a key mediator of angiogenesis. Recently, a novel angiogenic mitogen with tissue-specific expression and target selectivity was characterized. Human endocrine gland derived vascular endothelial growth factor (EG-VEGF) is selectively expressed in steroidogenic glands and promotes growth of endocrine gland endothelium. The identification of tissue-selective angiogenic factors raises the possibility that other secreted molecules in this class exist. The potential advantage of tissue-specific angiogenic therapeutics may be the reduction of systemic side effects. Additionally, these peptides or their receptors may be attractive targets for inhibition in several disorders.
Abstract: Recent studies show that testosterone-stimulated growth of the glandular tissue in the ventral prostate in adult castrated rats is preceded by increased epithelial VEGF synthesis, endothelial cell proliferation, vascular growth, and increased blood flow. These observations suggest that testosterone-stimulated prostate growth could be angiogenesis dependent, and that VEGF could play a central role in this process.
Abstract: A novel family of angiogenic mitogens have been recently characterized. Endocrine gland-derived vascular endothelial growth factor (EG-VEGF), and the mammalian homologue of Bombina variegata peptide 8 (Bv8), are two highly related endothelial cell mitogens and chemotactic factors with restricted expression profiles and selective endothelial cell activity. These peptides share two cognate G-protein coupled receptors. The expression of human EG-VEGF occurs predominantly in steroidogenic glands. Consistent with such an expression pattern, the human EG-VEGF gene promoter has a potential binding site for steroidogenic factor (SF)-1, a pivotal element for steroidogenic-specific transcription. In the human ovary, the expression of EG-VEGF is temporally and spatially complementary to the expression of VEGF-A, both in the follicular and in the luteal phase, suggesting complementary and coordinated roles of these molecules in ovarian angiogenesis. Also, EG-VEGF expression correlates with vascularity in the polycystic ovary syndrome, a leading cause of infertility. Bv8 expression is mainly restricted to the testis. The identification of these tissue-selective angiogenic factors raises the possibility that other secreted molecules with selectivity for the endothelium of other organs exist.
Abstract: We conducted a genome-wide analysis of genes that are regulated by vascular endothelial growth factor (VEGF) in endothelial cells and identified DSCR1 to be most significantly induced. Consistent with an antagonistic function on calcineurin (CnA) signaling, expression of DSCR1 in endothelial cells blocked dephosphorylation, nuclear translocation, and activity of nuclear factor of activated T cell (NFAT), a transcription factor involved in mediating CnA signaling. DSCR1 was not only induced by VEGF, but also by other compounds activating CnA signaling, suggesting a more general role for DSCR1 in activated endothelial cells. Transient expression of DSCR1 attenuated inflammatory marker genes such as tissue factor (TF), E-selectin, and Cox-2, identifying a previously unknown regulatory role for DSCR1 in activated endothelial cells. In contrast, knock-down of endogenous DSCR1 increased NFAT activity and stimulated expression of inflammatory genes on activated endothelial cells. Thus, the negative regulatory feedback loop between DSCR1 and CnA signaling in endothelial cells identified may represent a potential molecular mechanism underlying the frequently transient expression of inflammatory genes following activation of endothelial cells.
Abstract: The development of a vascular supply is a critical factor in the growth and metastatic spread of malignant tumors. Of the multitude of growth factors that regulate physiological and pathological angiogenesis, vascular endothelial growth factor (VEGF) is believed to be the most important. There is evidence that overexpression of VEGF is correlated with an adverse prognosis, at least in some tumors. Tumor-expressed VEGF is particularly attractive as a target for anticancer therapy because its angiogenesis-promoting activity is at the level of the endothelial cell and, compared with agents that directly target tumor cells, tumor penetration is less critical for VEGF inhibitors. Moreover, recent work has shown that inhibiting tumor angiogenesis increases the effectiveness of coadministered chemotherapy and radiotherapy. This suggests that drugs that target VEGF or its receptors can be combined with traditional treatment modalities to ensure maximum effectiveness. A variety of agents aimed at blocking VEGF or its receptor-signaling system are currently being developed for the treatment of cancer. Of these, bevacizumab, a humanized monoclonal antibody directed at VEGF, is the most advanced in clinical development and has shown promising results in clinical trials.
Abstract: Neointimal vascular smooth muscle cell (VSMC) proliferation is a primary cause of occlusive vascular disease, including atherosclerosis, restenosis after percutaneous interventions, and bypass graft stenosis. Angiogenesis is implicated in the progression of early atheromatous lesions in animal models, but its role in neointimal VSMC proliferation is undefined. Because percutaneous coronary interventions result in induction of periadventitial angiogenesis, we analyzed the role of this process in neointima formation.
Abstract: Bv8 and endocrine-gland-derived VEGF (EG-VEGF), or prokineticins, are two highly related, secreted proteins that we previously described as selective angiogenic mitogens. Here we describe the expression and functional characterization of Bv8 in peripheral blood cells, notably monocytes, neutrophils, and dendritic cells, and in the bone marrow. In human and mouse, the two Bv8 G protein-coupled receptors are expressed in hematopoietic stem cells and specific mature blood cells, including lymphocytes. Bv8 is highly expressed by neutrophils at sites of inflammation and can stimulate migration of monocytes, in a pertussis toxin-sensitive manner. Bv8, or EG-VEGF that shares the same receptors, increased numbers of colony-forming units granulocytic and monocytic in cultures of human or mouse hematopoietic stem cells. Systemic in vivo exposure to Bv8 or EG-VEGF resulted in significant increases in total leukocyte, neutrophil, and monocyte counts. Additionally, adenovirus (Av)Bv8 or AvEG-VEGF delivered just before 5-fluorouracil injury promoted the survival of hematopoietic cells and enhanced progenitor mobilization. In conclusion, Bv8 can promote survival and differentiation of the granulocytic and monocytic lineages. Bv8 potentially modulates growth, survival, and function of cells of the innate and adaptive immune systems, possibly through autocrine or paracrine signaling mechanisms.
Abstract: Bevacizumab, a monoclonal antibody against vascular endothelial growth factor, has shown promising preclinical and clinical activity against metastatic colorectal cancer, particularly in combination with chemotherapy.
Abstract: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen in vitro and an angiogenic inducer in a variety of in vivo models. Hypoxia has been shown to be a major inducer of VEGF gene transcription. The tyrosine kinases Flt-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2) are high-affinity VEGF receptors. The role of VEGF in developmental angiogenesis is emphasized by the finding that loss of a single VEGF allele results in defective vascularization and early embryonic lethality. VEGF is critical also for reproductive and bone angiogenesis. Substantial evidence also implicates VEGF as a mediator of pathological angiogenesis. In situ hybridization studies demonstrate expression of VEGF mRNA in the majority of human tumors. Anti-VEGF monoclonal antibodies and other VEGF inhibitors block the growth of several tumor cell lines in nude mice. Clinical trials with various VEGF inhibitors in a variety of malignancies are ongoing. Very recently, an anti-VEGF monoclonal antibody (bevacizumab; Avastin) has been approved by the Food and Drug Administration as a first-line treatment for metastatic colorectal cancer in combination with chemotherapy. Furthermore, VEGF is implicated in intraocular neovascularization associated with diabetic retinopathy and age-related macular degeneration.
Abstract: We deleted the hypoxia-responsive transcription factor HIF-1alpha in endothelial cells (EC) to determine its role during neovascularization. We found that loss of HIF-1alpha inhibits a number of important parameters of EC behavior during angiogenesis: these include proliferation, chemotaxis, extracellular matrix penetration, and wound healing. Most strikingly, loss of HIF-1alpha in EC results in a profound inhibition of blood vessel growth in solid tumors. These phenomena are all linked to a decreased level of VEGF expression and loss of autocrine response of VEGFR-2 in HIF-1alpha null EC. We thus show that a HIF-1alpha-driven, VEGF-mediated autocrine loop in EC is an essential component of solid tumor angiogenesis.
Abstract: Angiogenesis is essential for tumor growth and metastasis. A new human angiogenic mitogen, endocrine gland-derived vascular endothelial growth factor (EG-VEGF), has been recently identified; its expression pattern is restricted to endocrine glands, with the highest expression in testis. We used in situ hybridization and newly generated monoclonal antibodies to investigate the expression of EG-VEGF in normal human prenatal and adult testis and in 48 human testicular tumors of different subtypes. We found that EG-VEGF was expressed from 14 wk until birth in human fetal testis. In the adult testis, EG-VEGF was strongly expressed only in Leydig cells. In testicular tumors, EG-VEGF was expressed specifically in Leydig cell tumors, whereas germ cell-derived neoplasms, including carcinoma in situ, seminoma, and nonseminomatous germ cell tumors, were negative for this antigen. In contrast, VEGF, another powerful angiogenic factor, was expressed in seminoma, but very weakly in Leydig cell tumors. Interestingly, we found that Leydig cell tumors presented vessel surface density 3.2-fold higher than seminoma. These findings argue that human EG-VEGF may play a role in angiogenesis both during the early endocrine development of testis and in the adult testis as well as in Leydig cell tumor growth.
Abstract: We generated VEGF-null fibrosarcomas from VEGF-loxP mouse embryonic fibroblasts to investigate the mechanisms of tumor escape after VEGF inactivation. These cells were found to be tumorigenic and angiogenic in vivo in spite of the absence of tumor-derived VEGF. However, VEGF derived from host stroma was readily detected in the tumor mass and treatment with a newly developed anti-VEGF monoclonal antibody substantially inhibited tumor growth. The functional significance of stroma-derived VEGF indicates that the recruitment of stromal cells is critical for the angiogenic and tumorigenic properties of these cells. Here we identified PDGF AA as the major stromal fibroblast chemotactic factor produced by tumor cells, and demonstrated that disrupting the paracrine PDGFR alpha signaling between tumor cells and stromal fibroblasts by soluble PDGFR alpha-IgG significantly reduced tumor growth. Thus, PDGFR alpha signaling is required for the recruitment of VEGF-producing stromal fibroblasts for tumor angiogenesis and growth. Our findings highlight a novel aspect of PDGFR alpha signaling in tumorigenesis.
Abstract: New strategies for cancer therapy include the combination of angiogenesis inhibitors with cytotoxins. However, angiogenesis inhibitors may alter tumor microvessel structure and transendothelial permeability thereby reducing tumoral delivery of cytotoxic agents. The aim of this study was to estimate quantitatively the apparent permeability-surface area product (K(PS)) in tumors to a macromolecular contrast medium (MMCM), to follow changes in K(PS) induced by antibodies to vascular endothelial growth factor (anti-VEGF), and to correlate the findings with tumor accumulation of cisplatin, a highly protein-bound cytotoxin, and 5-fluorouracil (5-FU), a small unbound cytotoxin.
Abstract: Angiogenesis and lymphangiogenesis are regulated by members of the vascular endothelial growth factor (VEGF) family of cytokines, which mediate their effects via tyrosine kinase VEGF receptors -1, -2, and -3. We have used wild-type and mutant forms of VEGFs -A, -B, and -C, a pan-VEGFR tyrosine kinase inhibitor (SU5416) as well as neutralizing anti-VEGFR-2 antibodies, to determine which VEGF receptor(s) are required for bovine endothelial cell invasion and tube formation in vitro. This was compared to the ability of these cytokines to induce expression of members of the plasminogen activator (PA)-plasmin system. We found that cytokines which bind VEGFR-2 (human VEGF-A, human VFM23A, human VEGF-C(deltaNdeltaC), and rat VEGF-C(152)) induced invasion, tube formation, urokinase-type-PA, tissue-type-PA, and PA inhibitor-1, invasion and tube formation as well as signaling via the MAP kinase pathway were efficiently blocked by SU5416 and anti-VEGFR-2 antibodies. In contrast, cytokines and mutants which exclusively bind VEGFR-1 (human VFM17 and human VEGF-B) had no effect on invasion and tube formation or on the regulation of gene expression. We were unable to identify cytokines which selectively stimulate bovine VEGFR-3 in our system. Taken together, these findings point to the central role of VEGFR-2 in the angiogenic signaling pathways induced by VEGF-C(deltaNdeltaC) and VEGF-A.
Abstract: Kidney disease affects over 20 million people in the United States alone. Although the causes of renal failure are diverse, the glomerular filtration barrier is often the target of injury. Dysregulation of VEGF expression within the glomerulus has been demonstrated in a wide range of primary and acquired renal diseases, although the significance of these changes is unknown. In the glomerulus, VEGF-A is highly expressed in podocytes that make up a major portion of the barrier between the blood and urinary spaces. In this paper, we show that glomerular-selective deletion or overexpression of VEGF-A leads to glomerular disease in mice. Podocyte-specific heterozygosity for VEGF-A resulted in renal disease by 2.5 weeks of age, characterized by proteinuria and endotheliosis, the renal lesion seen in preeclampsia. Homozygous deletion of VEGF-A in glomeruli resulted in perinatal lethality. Mutant kidneys failed to develop a filtration barrier due to defects in endothelial cell migration, differentiation, and survival. In contrast, podocyte-specific overexpression of the VEGF-164 isoform led to a striking collapsing glomerulopathy, the lesion seen in HIV-associated nephropathy. Our data demonstrate that tight regulation of VEGF-A signaling is critical for establishment and maintenance of the glomerular filtration barrier and strongly supports a pivotal role for VEGF-A in renal disease.
Abstract: The vascular endothelium was once thought to function primarily in nutrient and oxygen delivery, but recent evidence suggests that it may play a broader role in tissue homeostasis. To explore the role of sinusoidal endothelial cells (LSECs) in the adult liver, we studied the effects of vascular endothelial growth factor (VEGF) receptor activation on mouse hepatocyte growth. Delivery of VEGF-A increased liver mass in mice but did not stimulate growth of hepatocytes in vitro, unless LSECs were also present in the culture. Hepatocyte growth factor (HGF) was identified as one of the LSEC-derived paracrine mediators promoting hepatocyte growth. Selective activation of VEGF receptor-1 (VEGFR-1) stimulated hepatocyte but not endothelial proliferation in vivo and reduced liver damage in mice exposed to a hepatotoxin. Thus, VEGFR-1 agonists may have therapeutic potential for preservation of organ function in certain liver disorders.
Abstract: We recently described human endocrine gland-derived vascular endothelial growth factor (EG-VEGF) as an endothelial cell mitogen with a novel selective activity and an expression pattern essentially limited to steroidogenic glands. Herein we present the identification and characterization of the mouse ortholog. The mouse cDNA and predicted amino acid sequences are, respectively, 86% and 88% identical with the human. Surprisingly, the mouse EG-VEGF transcript is predominantly expressed in liver and kidney. A comparison of human and mouse EG-VEGF promoter sequences revealed a potential binding site for NR5A1, which is known to be a pivotal element for steroidogenic-specific transcription, in the human but not mouse promoter. In situ hybridization studies localized expression of mouse EG-VEGF mRNA to hepatocytes and renal tubule cells. Interestingly, capillary endothelial cells in these sites share several common structural features with those found in steroidogenic glands. Within liver and kidney, EG-VEGF receptor expression was largely restricted to endothelial cells. Mouse EG-VEGF promoted proliferation and survival of endothelial cells. We propose that mouse EG-VEGF, like human EG-VEGF, plays a role in regulating the phenotype and growth properties of endothelial cells within distinct capillary beds.
Abstract: VEGF is a secreted growth factor that mediates its biological effects by binding to two transmembrane tyrosine kinase receptors, VEGFR-1 and VEGFR-2. The VEGF/receptor signaling system is involved in the regulation of two fundamental processes in vertebrates: the formation of blood vessels (angiogenesis) and of blood cells (hematopoiesis). Hematopoietic stem cells, capable of giving rise to all blood cell lineages, are often found in clusters with endothelial cells, the key cell type involved in the formation of blood vessels. Despite such proximity of VEGF-responsive cells, hematopoiesis occurs independently of neoangiogenesis in the adult bone marrow, suggesting that VEGF regulates the two processes by different mechanisms. In support of this hypothesis, the recently identified autocrine loop by which VEGF may control hematopoietic stem cell survival and repopulation, is fundamentally different from its paracrine effects regulating angiogenesis. Furthermore, coexpression of VEGF and its receptors, the prerequisite for autocrine loops, is frequently found in lymphomas and myelomas, suggesting that autocrine loops also play a role in hematological malignancies. Several therapeutic strategies blocking VEGF or VEGF-induced signaling are currently being investigated for the treatment of neoplastic diseases. They differ in their potential to interfere with the autocrine or paracrine effector functions of VEGF during angiogenesis, hematopoiesis, and tumor cell proliferation, properties which may ultimately determine their therapeutic potential.
Abstract: We recently identified an angiogenic mitogen, endocrine-gland-derived vascular endothelial growth factor (EG-VEGF), with selective activity for endothelial cells of endocrine tissues. Here we describe the characterization of a highly related molecule, Bv8, also known as prokineticin-2. Human Bv8 shares 60% identity and 75% similarity with EG-VEGF. The human and mouse Bv8 genes share a common structure. Like EG-VEGF, Bv8 is able to induce proliferation, survival and migration of adrenal cortical capillary endothelial cells. Bv8 gene expression is induced by hypoxic stress. Bv8 expression occurs predominantly in the testis and is largely restricted to primary spermatocytes. Adenoviral delivery of Bv8 or EG-VEGF to the mouse testis resulted in a potent angiogenic response. We have localized the expression of the Bv8EG-VEGF receptors within the testis to vascular endothelial cells. The testis exhibits relatively high turnover of endothelial cells. Therefore, Bv8 and EG-VEGF, along with other factors such as VEGF-A, may maintain the integrity and also regulate proliferation of the blood vessels in the testis.
Abstract: Granulocytes and monocytes/macrophages of the myeloid lineage are the chief cellular agents of innate immunity. Here, we have examined the inflammatory response in mice with conditional knockouts of the hypoxia responsive transcription factor HIF-1alpha, its negative regulator VHL, and a known downstream target, VEGF. We find that activation of HIF-1alpha is essential for myeloid cell infiltration and activation in vivo through a mechanism independent of VEGF. Loss of VHL leads to a large increase in acute inflammatory responses. Our results show that HIF-1alpha is essential for the regulation of glycolytic capacity in myeloid cells: when HIF-1alpha is absent, the cellular ATP pool is drastically reduced. The metabolic defect results in profound impairment of myeloid cell aggregation, motility, invasiveness, and bacterial killing. This role for HIF-1alpha demonstrates its direct regulation of survival and function in the inflammatory microenvironment.
Abstract: Angiogenic molecules are the focus of therapeutic efforts to promote new vessel development in ischemic or damaged tissue and, conversely, to inhibit endothelial cell growth and survival in proliferative disease. Two novel angiogenic mitogens have been characterized recently. Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) and the mammalian homologue of Bombina variegata peptide 8 (Bv8) are endothelial cell mitogens and chemotactic factors with restricted expression profiles and selective endothelial cell activity. These highly related peptides share two cognate G-protein-coupled receptors that are homologous to the neuropeptide Y receptor. The identification of tissue-selective angiogenic factors raises the possibility that other secreted molecules in this class exist. The potential advantage of tissue-specific angiogenic therapeutics may be the reduction of systemic side effects. Additionally, these peptides or their receptors may be attractive targets for inhibition in several disorders.
Abstract: Angiogenesis is a key aspect of the dynamic changes occurring during the normal ovarian cycle. Hyperplasia and hypervascularity of the ovarian theca interna and stroma are also prominent features of the polycystic ovary syndrome (PCOS), a leading cause of infertility. Compelling evidence indicated that vascular endothelial growth factor (VEGF) is a key mediator of the cyclical corpus luteum angiogenesis. However, the nature of the factor(s) that mediate angiogenesis in PCOS is less clearly understood. Endocrine gland-derived (EG)-VEGF has been recently identified as an endothelial cell mitogen with selectivity for the endothelium of steroidogenic glands and is expressed in normal human ovaries. In the present study, we compared the expression of EG-VEGF and VEGF mRNA in a series of 13 human PCOS and 13 normal ovary specimens by in situ hybridization. EG-VEGF expression in normal ovaries is dynamic and generally complementary to VEGF expression in both follicles and corpora lutea. A particularly high expression of EG-VEGF was detected in the Leydig-like hilus cells found in the highly vascularized ovarian hilus. In PCOS ovaries, we found strong expression of EG-VEGF mRNA in theca interna and stroma in most of the specimens examined, thus spatially related to the new blood vessels. In contrast, VEGF mRNA expression was most consistently associated with the granulosa cell layer and sometimes the theca, but rarely with the stroma. These findings indicate that both EG-VEGF and VEGF are expressed in PCOS ovaries, but in different cell types at different stages of differentiation, thus suggesting complementary functions for the two factors in angiogenesis and possibly cyst formation.
Abstract: To reveal the functional significance of hypoxia and angiogenesis in astrocytoma progression, we created genetically engineered transformed astrocytes from murine primary astrocytes and deleted the hypoxia-responsive transcription factor HIF-1alpha or its target gene, the angiogenic factor VEGF. Growth of HIF-1alpha- and VEGF-deficient transformed astrocytes in the vessel-poor subcutaneous environment results in severe necrosis, reduced growth, and vessel density, whereas when the same cells are placed in the vascular-rich brain parenchyma, the growth of HIF-1alpha knockout, but not VEGF knockout tumors, is reversed: tumors deficient in HIF-1alpha grow faster, and penetrate the brain more rapidly and extensively. These results demonstrate that HIF-1alpha has differential roles in tumor progression, which are greatly dependent on the extant microenvironment of the tumor.
Abstract: To determine the function of VEGF-A in nervous system development, we have utilized the Nestin promoter-driven Cre recombinase transgene, in conjunction with a conditional and hypomorphic VEGF-A allele, to lower VEGF-A activity in neural progenitor cells. Mice with intermediate levels of VEGF-A activity showed decreased blood vessel branching and density in the cortex and retina, resulting in a thinner retina and aberrant structural organization of the cortex. Severe reductions in VEGF-A led to decreases in vascularity and subsequent hypoxia, resulting in the specific degeneration of the cerebral cortex and neonatal lethality. Decreased neuronal proliferation and hypoxia was evident at E11.5, leading to increased neuronal apoptosis in the cortex by E15.5. In order to address whether the observed changes in the structural organization of the nervous system were due to a direct and autocrine role of VEGF-A on the neural population, we conditionally inactivated the main VEGF-A receptor, Flk1, specifically in neuronal lineages, by using the Nestin Cre transgene. The normality of these mice ruled out the possibility that VEGF-A/Flk1 signaling has a significant autocrine role in CNS development. VEGF-A dosage is therefore a critical parameter regulating the density of the vascular plexus in the developing CNS that is in turn a key determinant in the development and architectural organization of the nervous system.
Abstract: Vascular endothelial growth factor (VEGF) is a key regulator of physiological angiogenesis during embryogenesis, skeletal growth and reproductive functions. VEGF has also been implicated in pathological angiogenesis associated with tumors, intraocular neovascular disorders and other conditions. The biological effects of VEGF are mediated by two receptor tyrosine kinases (RTKs), VEGFR-1 and VEGFR-2, which differ considerably in signaling properties. Non-signaling co-receptors also modulate VEGF RTK signaling. Currently, several VEGF inhibitors are undergoing clinical testing in several malignancies. VEGF inhibition is also being tested as a strategy for the prevention of angiogenesis, vascular leakage and visual loss in age-related macular degeneration.
Abstract: Blood vessel endothelium has been recently shown to induce endocrine pancreatic development. Because pancreatic endocrine cells or islets express high levels of vascular endothelial growth factors, VEGFs, we investigated the role of a particular VEGF, VEGF-A, on islet vascularization and islet function. By deleting VEGF-A in the mouse pancreas, we show that endocrine cells signal back to the adjacent endothelial cells to induce the formation of a dense network of fenestrated capillaries in islets. Interestingly, VEGF-A is not required for the development of all islet capillaries. However, the few remaining capillaries found in the VEGF-A-deficient islets are not fenestrated and contain an unusual number of caveolae. In addition, glucose tolerance tests reveal that the VEGF-A-induced capillary network is not strictly required for blood glucose control but is essential for fine-tuning blood glucose regulation. In conclusion, we speculate that islet formation takes place in two sequential steps: in the first step, signals from blood vessel endothelium induce islet formation next to the vessels, and in the second step, the islets signal to the endothelium. The second step involves paracrine VEGF-A signaling to elaborate the interaction of islets with the circulatory system.
Abstract: 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.
Abstract: In the RIP1-Tag2 mouse model of pancreatic islet carcinoma, angiogenesis is switched on in a discrete premalignant stage of tumor development, persisting thereafter. Signaling through VEGF receptor tyrosine kinases is a well-established component of angiogenic regulation. We show that five VEGF ligand genes are expressed in normal islets and throughout islet tumorigenesis. To begin dissecting their contributions, we produced an islet beta cell specific knockout of VEGF-A, resulting in islets with reduced vascularity but largely normal physiology. In RIP1-Tag2 mice wherein most oncogene-expressing cells had deleted the VEGF-A gene, both angiogenic switching and tumor growth were severely disrupted, as was the neovasculature. Thus, VEGF-A is crucial for angiogenesis in a prototypical model of carcinogenesis, whose loss is not readily compensated.
Abstract: The angiopoietin family of secreted factors is functionally defined by the C-terminal fibrinogen (FBN)-like domain, which mediates binding to the Tie2 receptor and thereby facilitates a cascade of events ultimately regulating blood vessel formation. By screening expressed sequence tag data bases for homologies to a consensus FBN-like motive, we have identified ANGPTL3, a liver-specific, secreted factor consisting of an N-terminal coiled-coil domain and the C-terminal FBN-like domain. Co-immunoprecipitation experiments, however, failed to detect binding of ANGPTL3 to the Tie2 receptor. A molecular model of the FBN-like domain of ANGPTL3 was generated and predicted potential binding to integrins. This hypothesis was experimentally confirmed by the finding that recombinant ANGPTL3 bound to alpha(v)beta(3) and induced integrin alpha(v)beta(3)-dependent haptotactic endothelial cell adhesion and migration and stimulated signal transduction pathways characteristic for integrin activation, including phosphorylation of Akt, mitogen-activated protein kinase, and focal adhesion kinase. When tested in the rat corneal assay, ANGPTL3 strongly induced angiogenesis with comparable magnitude as observed for vascular endothelial growth factor-A. Moreover, the C-terminal FBN-like domain alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis. Taken together, our data demonstrate that ANGPTL3 is the first member of the angiopoietin-like family of secreted factors binding to integrin alpha(v)beta(3) and suggest a possible role in the regulation of angiogenesis.
Abstract: 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.
Abstract: Vascular endothelial growth factor (VEGF) is a principal regulator of blood vessel formation and haematopoiesis, but the mechanisms by which VEGF differentially regulates these processes have been elusive. Here we describe a regulatory loop by which VEGF controls survival of haematopoietic stem cells (HSCs). We observed a reduction in survival, colony formation and in vivo repopulation rates of HSCs after ablation of the VEGF gene in mice. Intracellularly acting small-molecule inhibitors of VEGF receptor (VEGFR) tyrosine kinase dramatically reduced colony formation of HSCs, thus mimicking deletion of the VEGF gene. However, blocking VEGF by administering a soluble VEGFR-1, which acts extracellularly, induced only minor effects. These findings support the involvement in HSC survival of a VEGF-dependent internal autocrine loop mechanism (that is, the mechanism is resistant to inhibitors that fail to penetrate the intracellular compartment). Not only ligands selective for VEGF and VEGFR-2 but also VEGFR-1 agonists rescued survival and repopulation of VEGF-deficient HSCs, revealing a function for VEGFR-1 signalling during haematopoiesis.
Abstract: Anatomic remodeling and permanent closure of the newborn ductus arteriosus appears to require the development of intense hypoxia within the constricted vessel wall. Hypoxic ductus smooth muscle cells express vascular endothelial cell growth factor (VEGF). We studied premature baboons and sheep to determine the effects of VEGF inhibition (in baboons) and VEGF stimulation (in sheep) on ductus remodeling in vivo. For study of VEGF inhibition, 13 premature newborn baboons (68% gestation) were treated with inhibitors of both prostaglandin and nitric oxide production to constrict the ductus and induce ductus wall hypoxia. Six received a neutralizing monoclonal antibody against VEGF (A.4.6.1, mAbVEGF), while seven did not. Both groups developed the same degree of ductus constriction, tissue hypoxia, and VEGF expression. The mAbVEGF treatment produced a significant (P < 0.05) reduction in ductus vasa vasorum ingrowth and neointima formation (due to both a decrease in luminal endothelial cell proliferation and a decrease in smooth muscle cell migration into the neointima). For study of VEGF stimulation, nine sheep fetuses (70% gestation) had their ductus wall injected with either VEGF (n = 6) or vehicle (n = 4) in vivo. VEGF administration produced a significant (P < 0.05) increase in vasa vasorum ingrowth and neointima formation. We conclude that VEGF plays an important role in the formation of neointimal mounds and vasa vasorum ingrowth during permanent ductus closure.
Abstract: Ovarian cancer is characterized by rapid growth of solid intraperitoneal tumors and production of large volumes of ascites. Our previous studies of intraperitoneal ovarian carcinoma in an athymic mouse model demonstrated that a monoclonal antibody (mAb) to human vascular endothelial growth factor (VEGF) could prevent ascites formation. Although ascites was almost completely inhibited, tumor burden was variably reduced. To develop more effective therapy, we assessed the combination of a human VEGF mAb plus paclitaxel. Four groups of female athymic nude mice were inoculated intraperitoneally with OVCAR3 cells. Two weeks after inoculation, one group was treated with a human VEGF mAb intraperitoneally twice weekly plus paclitaxel intraperitoneally three times weekly for 6 weeks. The second group was treated with VEGF mAb alone. The third group was treated with paclitaxel alone. The remaining group was treated with vehicle only. Tumor burden in the VEGF mAb plus paclitaxel and paclitaxel alone groups was reduced by 83.3% and 85.7% and 58.5% and 59.5%, respectively, in two separate experiments, compared to controls. VEGF mAb alone caused no significant decrease in tumor burden, nor did treatment of mice inoculated intraperitoneally with HEY-A8 cells, a non-VEGF-secreting ovarian cell line. Virtually no ascites developed in the combined treatment group or the group treated with VEGF mAb alone. Paclitaxel alone reduced ascites slightly, but not significantly. Morphological studies demonstrated that VEGF immunoneutralization enhanced paclitaxel-induced apoptosis in these human ovarian cancers. Thus, combination therapy with inhibitors of VEGF plus paclitaxel may be an effective way to markedly reduce tumor growth and ascites in ovarian carcinoma.
Abstract: Angiogenesis, or formation of new blood vessels from pre-existing ones, is essential for normal development and wound healing/reproductive functions in adults. Abnormal regulation of angiogenesis has been implicated in the pathogenesis of several disorders, including cancer. Vascular endothelial growth factor (VEGF)-A is a pivotal stimulator of angiogenesis because its binding to VEGF receptors has been shown to promote endothelial cell migration and proliferation, two key features required for the development of new blood vessels. In addition, VEGF-A increases vascular permeability, which may also contribute to angiogenesis and tumor growth. Recognition of the central role of VEGF-A in angiogenesis has led to the hypothesis that its inhibition may represent a novel and effective approach to the treatment of cancer and other conditions characterized by pathologic angiogenesis. Several lines of evidence support this idea, and early clinical experience with the humanized anti-VEGF-A monoclonal antibody bevacizumab (Avastin, rhuMAb-VEGF; Genentech, South San Francisco, CA) has been encouraging. Clinical efficacy of antiangiogenic therapy with bevacizumab is being evaluated in several phase 3 trials in various types of cancer, as well as in patients with age-related macular degeneration.
Abstract: The ability of tumours to induce new blood-vessel formation has been a major focus of cancer research over the past few decades, and vascular endothelial growth factor (VEGF) is now known to be central to this process. The quest for VEGF and other factors that promote tumour angiogenesis was initiated many decades ago, and a long and complicated path has led to the development of inhibitors of these molecules as anticancer agents. How did this field begin, and how have we arrived at our present understanding of the role of VEGF in tumour progression.
Abstract: The diversity in growth and morphological characteristics among endothelial cells in different normal tissues and tumors has been long recognized. Yet there has been no clear molecular explanation for such diversity at the level of vascular endothelial growth factor A (VEGF-A) and other established regulators of angiogenesis that are expressed widely and show little tissue selectivity in their angiogenic properties. Endocrine gland-derived VEGF represents the first example of a tissue-specific angiogenic factor, likely to be followed by others.
Abstract: Several growth factors are expressed in distinct temporal and spatial patterns during fracture repair. Of these, vascular endothelial growth factor, VEGF, is of particular interest because of its ability to induce neovascularization (angiogenesis). To determine whether VEGF is required for bone repair, we inhibited VEGF activity during secondary bone healing via a cartilage intermediate (endochondral ossification) and during direct bone repair (intramembranous ossification) in a novel mouse model. Treatment of mice with a soluble, neutralizing VEGF receptor decreased angiogenesis, bone formation, and callus mineralization in femoral fractures. Inhibition of VEGF also dramatically inhibited healing of a tibial cortical bone defect, consistent with our discovery of a direct autocrine role for VEGF in osteoblast differentiation. In separate experiments, exogenous VEGF enhanced blood vessel formation, ossification, and new bone (callus) maturation in mouse femur fractures, and promoted bony bridging of a rabbit radius segmental gap defect. Our results at specific time points during the course of healing underscore the role of VEGF in endochondral vs. intramembranous ossification, as well as skeletal development vs. bone repair. The responses to exogenous VEGF observed in two distinct model systems and species indicate that a slow-release formulation of VEGF, applied locally at the site of bone damage, may prove to be an effective therapy to promote human bone repair.
