Nucleotide sequence of the mouse VEGF 3'UTR and quantitative analysis of sites of polyadenylation.

Sequencing of rat and human vascular endothelial growth factor (VEGF) cDNA clones has previously identified a 3' untranslated region of approximately 1.9 kb, although the apparent site of polyadenylation differed in the two species, despite a high degree of sequence conservation in the region. Neither site is preceded by a canonical AAUAAA polyadenylation signal, a situation frequently found in genes that are subject to alternative polyadenylation. We have sequenced 2.25 kb of the 3' region of the mouse VEGF gene and mapped the usage of potential polyadenylation sites in fibroblasts cultured under both normoxic and hypoxic conditions. We find that two sites for polyadenylation are present in the mouse VEGF gene but the majority of transcripts contain the longer form of the 3'UTR and that their usage is not effected by environmental oxygen tension.

A permissive role for tumor necrosis factor in vascular endothelial growth factor-induced vascular permeability.

Vascular endothelial growth factor (VEGF) induces both angiogenesis and an increase in vascular permeability, 2 processes that are considered to be important for both tumor growth and the delivery of drugs to the site of tumors. This study demonstrates that transmembrane expression of tumor necrosis factor (tmTNF) is up-regulated in the endothelium of a murine methylcholanthrene (meth A)-induced sarcoma in comparison to the adjacent normal dermal vasculature and is also present on cultivated human endothelial cells. It is further shown that tmTNF is required for VEGF-mediated endothelial hyperpermeability in vitro and in vivo. This permissive activity of TNF appears to be selective, because anti-TNF antibodies ablated the VEGF-induced permeability but not proliferation of cultivated human endothelial cells. Furthermore, tnf gene-deficient mice show no obvious defects in vascularization and develop normally but failed to respond to administration of VEGF with an increase in vascular permeability. Subsequent studies indicated that the tmTNF and VEGF signaling pathways converge at the level of a secondary messenger, the "stress-activated protein kinase-2" (SAPK-2)/p38: (1) up-regulated endothelial expression of tmTNF resulted in the continuous activation of SAPK-2/p38 in vitro, and (2) an inhibitor of SAPK-2/p38 activation abolished the vascular permeability activity of VEGF in vivo. In conclusion, the study's finding that continuous autocrine signaling by tmTNF sensitizes endothelial cells to respond to VEGF by increasing their vascular permeability provides new therapeutic concepts for manipulating vascular hyperpermeability.

Differential inhibition of tumor angiogenesis by tie2 and vascular endothelial growth factor receptor-2 dominant-negative receptor mutants.

Tumor growth is angiogenesis-dependent. Current evidence suggests that vascular endothelial growth factor (VEGF), a major regulator of embryonic and hypoxia-mediated angiogenesis, is necessary for tumor angiogenesis. VEGF is expressed in tumor cells in vivo, and its tyrosine kinase receptors VEGFR-1 and VEGFR-2 are up-regulated in the tumor endothelium. A second endothelial cell-specific ligand/receptor tyrosine kinase system, consisting of the tie2 receptor, its activating ligand angiopoietin-1 and the inhibitory ligand angiopoietin-2, has been characterized. We have examined 6 human primary breast-cancer samples and 4 murine breast-cancer cell lines (M6363, M6378, M6444, M6468), transplanted into nude mice, by in situ hybridization and/or Northern analysis. Expression of angiopoietin-1, angiopoietin-2 and tie2 was compared to VEGF and VEGFR-2 expression. Human tumors expressed VEGFR-2 and tie2 but varied considerably in VEGF and angiopoietin-1/-2 expression. In the murine tumor models, we observed high heterogeneity of receptor and ligand expression. M6363 and M6378 tumors were analyzed in detail because they showed different expression of components of the tie2/angiopoietin signaling system. M6363 tumors expressed VEGF, VEGFR-2 and angiopoietin-2 but not tie2 or angiopoietin-1, suggesting activation of VEGFR-2 and inhibition of tie2 signaling pathways, whereas M6378 tumors expressed VEGF, VEGFR-2, tie2 and angiopoietin-1 but little angiopoietin-2, suggesting activation of both VEGFR-2 and tie2 signaling pathways. In vivo studies using truncated dominant-negative tie2 and VEGFR-2 mutants revealed inhibition of M6363 tumor growth by 15% (truncated tie2) and 36% (truncated VEGFR-2), respectively. In contrast, M6378 tumor growth was inhibited by 57% (truncated tie2) and 47% (truncated VEGFR-2), respectively. These findings support the hypothesis that tumor angiogenesis is dependent on VEGFR-2 but suggest that, in addition, tie2-dependent pathways of tumor angiogenesis may exist. For adequate application of angiogenesis inhibitors in tumor patients, analysis of prevailing angiogenesis pathways may be a prerequisite.

Endothelium-specific replacement of the connexin43 coding region by a lacZ reporter gene.

The murine gap junction protein connexin43 (Cx43) is expressed in blood vessels, with vastly different contribution by endothelial and smooth muscle cells. We have used the Cre recombinase under control of TIE2 transcriptional elements to inactivate a floxed Cx43 gene specifically in endothelial cells. Cre-mediated deletion led to replacement of the Cx43 coding region by a lacZ reporter gene. This allowed us to monitor the extent of deletion and to visualize the endothelial expression pattern of Cx43. We found widespread endothelial expression of the Cx43 gene during embryonic development, which became restricted largely to capillaries and small vessels in all adult organs examined. Mice lacking Cx43 in endothelium did not exhibit altered blood pressure, in contrast to mice deficient in Cx40. Our results show that lacZ activation after deletion of the target gene allows us to determine the extent of cell type-specific deletion after phenotypical investigation of the same animal.

Role of SCL/Tal-1, GATA, and ets transcription factor binding sites for the regulation of flk-1 expression during murine vascular development.

The receptor tyrosine kinase Flk-1 is essential for embryonic blood vessel development and for tumor angiogenesis. To identify upstream transcriptional regulators of Flk-1, the gene regulatory elements that mediate endothelium-specific expression in mouse embryos were characterized. By mutational analysis, binding sites for SCL/Tal-1, GATA, and Ets transcription factors located in the Flk-1 enhancer were identified as critical elements for the endothelium-specific Flk-1 gene expression in transgenic mice. c-Ets1, a transcription factor that is coexpressed with Flk-1 during embryonic development and tumor angiogenesis, activated the Flk-1 promoter via 2 binding sites. One of these sites was required for Flk-1 promoter function in the embryonic vasculature. These results provide the first evidence that SCL/Tal-1, GATA, and Ets transcription factors act upstream of Flk-1 in a combinatorial fashion to determine embryonic blood vessel formation and are key regulators not only of the hematopoietic program, but also of vascular development.

Disruption of epithelial tight junctions is prevented by cyclic nucleotide-dependent protein kinase inhibitors.

Tight junctions (TJs), the most apical of the intercellular junctions, prevent the passage of ions and molecules through the paracellular pathway. Intracellular signalling molecules are likely to be involved in the regulation of TJ integrity. In order to specifically investigate the role of protein kinase A (PKA) in the maintenance of epithelial TJ integrity, calcium-switch experiments were performed, in which calcium was removed from EpH4 and MDCK culture medium, in the absence or presence of the PKA inhibitors H-89 or HA-1004. Removal of calcium from the culture media of the epithelial cells resulted in disruption of the TJs, characterised by a loss of membrane association of the TJ-associated proteins occludin, ZO-1 and ZO-2, by a loss of TJ strands, by a marked decrease in the transepithelial electrical resistance and by a dramatic increase in the transepithelial permeability to tracers. The association of occludin, ZO-1 and ZO-2 with the actin cytoskeleton is not affected. In contrast, when the removal of calcium was performed in the presence of either the PKA inhibitor H-89 or HA-1004, all barrier characteristics were preserved. Our data indicate that following the removal of calcium from the culture medium of epithelial cells in vitro, PKA is activated and subsequently is involved in the disruption of TJs.

Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia.

We investigated the hypothesis that hypoxia induces angiogenesis and thereby may counteract the detrimental neurological effects associated with stroke. Forty-eight to seventy-two hours after permanent middle cerebral artery occlusion we found a strong increase in the number of newly formed vessels at the border of the infarction. Using the hypoxia marker nitroimidazole EF5, we detected hypoxic cells in the ischemic border of the neocortex. Expression of vascular endothelial growth factor (VEGF), which is the main regulator of angiogenesis and is inducible by hypoxia, was strongly up-regulated in the ischemic border, at times between 6 and 24 hours after occlusion. In addition, both VEGF receptors (VEGFRs) were up-regulated at the border after 48 hours and later in the ischemic core. Finally, the two transcription factors, hypoxia-inducible factor-1 (HIF-1) and HIF-2, known to be involved in the regulation of VEGF and VEGFR gene expression, were increased in the ischemic border after 72 hours, suggesting a regulatory function for these factors. These results strongly suggest that the VEGF/VEGFR system, induced by hypoxia, leads to the growth of new vessels after cerebral ischemia. Exogenous support of this natural protective mechanism might lead to enhanced survival after stroke.

Inhibition of proteasome function induces programmed cell death in proliferating endothelial cells.

Proteolysis mediated by the ubiquitin-proteasome system has been implicated in the regulation of programmed cell death. Here we investigated the differential effects of proteasomal inhibitors on the viability of proliferating and quiescent primary endothelial cells in vitro and in vivo. Subconfluent, proliferating cells underwent carbobenzoxy-L-isoleucyl-gamma-t-butyl-L-glutamyl-L-alanyl-L-leucinal (PSI) -induced apoptosis at low concentrations (EC(50)=24 nM), whereas at least 340-fold higher concentrations of PSI were necessary to obtain the same effect in confluent, contact-inhibited cells. PSI-mediated cell death could be blocked by a caspase-3 inhibitor (Ac-DEVD-H), but not by a caspase-1 inhibitor (Ac-YVAD-H), suggesting that a caspase-3-like enzyme is activated during PSI-induced apoptosis. When applied to the embryonic chick chorioallantoic membrane, a rapidly expanding tissue, PSI induced massive apoptosis also in vivo. PSI treatment of the CAM led to the formation of areas devoid of blood flow due to the induction of apoptosis in endothelial and other cells and to the collapse of capillaries and first order vessels. Our results demonstrate that proteasomal inhibitors such as PSI may prove effective as novel anti-angiogenic and anti-neoplastic substances.

Functional interaction of vascular endothelial-protein-tyrosine phosphatase with the angiopoietin receptor Tie-2.

During development of the vertebrate vascular system essential signals are transduced via protein-tyrosine phosphorylation. Null-mutations of receptor-tyrosine kinase (RTK) genes expressed in endothelial cells (ECs) display early lethal vascular phenotypes. We aimed to identify endothelial protein-tyrosine phosphatases (PTPs), which should have similar importance in EC-biology. A murine receptor-type PTP was identified by a degenerated PCR cloning approach from endothelial cells (VE-PTP). By in situ hybridization this phosphatase was found to be specifically expressed in vascular ECs throughout mouse development. In experiments using GST-fusion proteins, as well as in transient transfections, trapping mutants of VE-PTP co-precipitated with the Angiopoietin receptor Tie-2, but not with the Vascular Endothelial Growth Factor receptor 2 (VEGFR-2/Flk-1). In addition, VE-PTP dephosphorylates Tie-2 but not VEGFR-2. We conclude that VE-PTP is a Tie-2 specific phosphatase expressed in ECs, and VE-PTP phosphatase activity serves to specifically modulate Angiopoietin/Tie-2 function. Based on its potential role as a regulator of blood vessel morphogenesis and maintainance, VE-PTP is a candidate gene for inherited vascular malformations similar to the Tie-2 gene.

Retrovirus producer cells encoding antisense VEGF prolong survival of rats with intracranial GS9L gliomas.

With increasing size tumors are continually dependent on a functional blood vessel system to guarantee the supply with oxygen and nutrients. Vascular endothelial growth factor (VEGF) is a key mediator not only of developmental but also of hypoxia-mediated and tumor-induced angiogenesis. Gene therapy using antisense VEGF with the aim to inhibit tumor angiogenesis may be a successful strategy for the treatment of highly vascular and invasive malignant gliomas. We investigated whether retrovirus producer cells encoding antisense VEGF can be used for in vivo gene transfer. The full length mouse VEGF164 cDNA was cloned in a sense and antisense direction into the retroviral expression vector pLEN. pLEN-VEGF (sense) and pLEN-FGEV (antisense) expression vectors were used to transfect the packaging cell line GP + E86 and to establish ecotropic virus producer cell lines. GP + E86:LEN-FGEV (#5) cells showed high expression of antisense VEGF mRNA, whereas GP+ E86:LEN-VEGF (#8) showed high expression of sense VEGF mRNA and active VEGF protein. Co-implantation of GS-9L cells with retrovirus producing cells containing the antisense VEGF construct into the brains of syngeneic rats showed a statistically significant inhibition of tumor growth and prolongation of survival time, while co-implantation of retrovirus producer cells containing the sense VEGF expression vector resulted in an increasing tumor growth and reduced survival time of the rats compared to control animals. Histological analysis of the tumors co-implanted with GP + E86:LEN-FGEV (#5) cells showed the suppression of angiogenesis, high degree of necrosis and no evidence of a significant immune response. Expression of antisense VEGF mRNA in these tumors was confirmed by in situ hybridization analysis. This is the first report demonstrating the potential utility of virus producer cells as in vivo gene transfer vehicles for antisense VEGF gene therapy of malignant gliomas.

Formation of transformed endothelial cells in the absence of VEGFR-2/Flk-1 by Polyoma middle T oncogene.

The middle T antigen of murine Polyomavirus (PymT) rapidly transforms endothelial cells leading to vascular malformations reminiscent of endothelial tumors or hemangiomas. Flk-1, a receptor tyrosine kinase which is activated upon binding of its ligand VEGF, is predominantly expressed in endothelial cells and essential for the formation of blood vessels since absence of Flk-1 prevents the development of mature endothelial cells in mice and in ES-cell differentiation experiments. To investigate the role of Flk-1 in PymT-induced vascular tumor formation, we studied the expression of Flk-1 and VEGF in PymT-transformed endothelial cells (Endothelioma cells, END. cells). The receptor and its ligand were both expressed in END. cells suggesting that a VEGF/Flk-1 autocrine loop might be causally involved in the formation of vascular tumors. To test this hypothesis, ES cells lacking Flk-1 were generated and the transforming potential of PymT was analysed after in vitro differentiation. Flk-1(-/-) END. cell lines were established which are morphologically identical to flk-1(+/+) END. cells and which express several markers characteristic for endothelial cells. This result suggests that PymT functionally replaces the requirement of Flk-1 in expansion and/or survival of endothelial progenitor cells. Therefore, flk-1(-/-) END. cells provide a powerful tool to dissect the downstream signaling pathways of Flk-1.

Identification of vascular endothelial growth factor (VEGF) receptor-2 (Flk-1) promoter/enhancer sequences sufficient for angioblast and endothelial cell-specific transcription in transgenic mice.

The vascular endothelial growth factor (VEGF) receptor-2 (Flk-1) is the first endothelial receptor tyrosine kinase to be expressed in angioblast precursors, and its function is essential for the differentiation of endothelial cells and hematopoietic precursors. We have identified cis-acting regulatory elements of the murine Flk-1 gene that mediate endothelium-specific expression of a LacZ reporter gene in transgenic mice. Sequences within the 5'-flanking region of the Flk-1 gene, in combination with sequences located in the first intron, specifically targeted transgene expression to angioblasts and endothelial cells of transgenic mice. The intronic regulatory sequences functioned as an autonomous endothelium-specific enhancer. Sequences of the 5'-flanking region contributed to a strong, uniform, and reproducible transgene expression and were stimulated by the transcription factor HIF-2alpha. The Flk-1 gene regulatory elements described in this study should allow the elucidation of the molecular mechanisms involved in endothelial cell differentiation and angiogenesis.

Antiangiogenic gene therapy in a rat glioma model using a dominant-negative vascular endothelial growth factor receptor 2.

Malignant gliomas are a prominent target for cancer gene therapy approaches because of their poor prognosis despite all currently available therapies. Gene therapy strategies developed to interfere with the normal function of vascular endothelial growth factor receptors have been successfully used in different experimental models to block tumor angiogenesis and to inhibit tumor growth. In this study we examined whether retroviruses encoding a mutant VEGF receptor 2 (VEGFR-2) could suppress tumor angiogenesis and thereby prolong the survival of rats bearing syngeneic intracerebral glioma tumors. Survival time of rats with intracerebral tumors was significantly prolonged in a dose-dependent manner when retroviruses carrying a VEGFR-2 mutant were cotransplanted with tumor cells. No effect on survival was observed in rats that received virus-producing cells or virus supernatant intracerebrally after 5 days of tumor injection. In established subcutaneous tumors treatment with multiple injections of virus-producing cells also inhibited tumor growth in a dose-dependent manner. After implantation of tumor cells stably transfected with a truncated form of VEGFR-2, rats exhibited a rate of survival similar to that of animals treated with high numbers of virus-producing cells encoding the truncated form of VEGFR-2. Morphologically, tumors showed signs of impaired angiogenesis, such as extensive necrosis and reduced tumor vascular density. These results suggest a dual mode of function of truncated VEGFR-2, namely dominant-negative inhibition of VEGFR-2 function and VEGF depletion by receptor binding. We further explored the safety of retrovirus-mediated gene transfer. Although virus sequences were found in different tissues after intracerebral injection of virus-producing cells, no morphological changes were observed in any tissue after a follow-up time of 6 months. Our results indicate that VEGFR-2 inhibition is useful for the treatment of malignant gliomas.

A dominant mutant of occludin disrupts tight junction structure and function.

The tight junction is the most apical intercellular junction of epithelial cells and forms a diffusion barrier between individual cells. Occludin is an integral membrane protein specifically associated with the tight junction which may contribute to the function or regulation of this intercellular seal. In order to elucidate the role of occludin at the tight junction, a full length and an N-terminally truncated murine occludin construct, both FLAG-tagged at the N terminus, were stably introduced into the murine epithelial cell line CSG 120/7. Both constructs were correctly targeted to the tight junction, as defined by colocalization with another tight junction protein, ZO-1. The construct lacking the N terminus and extracellular domains of occludin was found to exert a dramatic effect on tight junction integrity. Cell monolayers failed to develop an efficient permeability barrier, as demonstrated by low transcellular electrical resistance values and an increased paracellular flux to small molecular mass tracers. Furthermore, gaps were found to have been induced in the P-face associated tight junction strands, as visualized by freeze-fracture electron microscopy. These findings demonstrate an important role for the N-terminal half of occludin in tight junction assembly and maintaining the barrier function of the tight junction.

Differential downregulation of vascular endothelial growth factor by dexamethasone in normoxic and hypoxic rat glioma cells.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a mitogen and chemotactic factor for endothelial cells in vitro and an angiogenesis and vascular permeability factor in vivo. Due to its properties, VEGF is a candidate for both angiogenesis and vascular permeability/oedema induction which typically occur in glioblastomas. In this study we test the hypothesis that the antioedema effect of dexamethasone is mediated by downregulation of VEGF or VEGF receptor expression. VEGF mRNA and protein levels of two rat glioma cells lines, C6 and GS-9L, were determined after incubation with dexamethasone under normoxic and hypoxic conditions. In normoxic C6 and GS9L cells, we observed 50-60% downregulation of VEGF mRNA by dexamethasone (P=0.015 and P=0. 01, respectively). This effect was dependent on glucocorticoid-receptor (GR) function. The inhibitory effect of dexamethasone on VEGF gene expression by tumour cells was markedly reduced by hypoxia which suggests that the upregulation of VEGF driven by hypoxia overcomes the effect of the dexamethasone. Dexamethasone did not alter VEGFR-2 mRNA levels in human umbilical endothelial cells. In a subcutaneous glioma tumour model, we observed only a 15% decrease in VEGF mRNA expression in dexamethasone treated animals (n = 12) compared with controls animals (P = 0.24). We conclude that dexamethasone may decrease brain tumour-associated oedema by reduction of VEGF expression in tumour cells. However, the highly reduced activity on hypoxic tumour cells suggests that dexamethasone efficacy may be limited by hypoxia in rapidly growing tumours.

Hypoxic regulation of vascular endothelial growth factor mRNA stability requires the cooperation of multiple RNA elements.

Vascular endothelial growth factor (VEGF) is a key regulator of developmental, physiological, and tumor angiogenesis. Upregulation of VEGF expression by hypoxia appears to be a critical step in the neovascularization of solid cancers. The VEGF mRNA is intrinsically labile, but in response to hypoxia the mRNA is stabilized. We have systematically analyzed the regions in the VEGF mRNA that are responsible for its lability under normoxic conditions and for stabilization in response to hypoxia. We find that the VEGF mRNA not only contains destabilizing elements in its 3' untranslated region (3'UTR), but also contains destabilizing elements in the 5'UTR and coding region. Each region can independently promote mRNA degradation, and together they act additively to effect rapid degradation under normoxic conditions. Stabilization of the mRNA in response to hypoxia is completely dependent on the cooperation of elements in each of the 5'UTR, coding region, and 3'UTR. Combinations of any of two of these three regions were completely ineffective in responding to hypoxia, whereas combining all three regions allowed recapitulation of the hypoxic stabilization seen with the endogenous VEGF mRNA. We conclude that multiple regions in the VEGF mRNA cooperate both to ensure the rapid degradation of the mRNA under normoxic conditions and to allow stabilization of the mRNA in response to hypoxia. Our findings highlight the complexity of VEGF gene expression and also reveal a mechanism of gene regulation that could become the target for strategies of therapeutic intervention.

Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis.

Eph receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins, regulate axon guidance and bundling in the developing brain, control cell migration and adhesion, and help patterning the embryo. Here we report that two ephrinB ligands and three EphB receptors are expressed in and regulate the formation of the vascular network. Mice lacking ephrinB2 and a proportion of double mutants deficient in EphB2 and EphB3 receptor signaling die in utero before embryonic day 11.5 (E11.5) because of defects in the remodeling of the embryonic vascular system. Our phenotypic analysis suggests complex interactions and multiple functions of Eph receptors and ephrins in the embryonic vasculature. Interaction between ephrinB2 on arteries and its EphB receptors on veins suggests a role in defining boundaries between arterial and venous domains. Expression of ephrinB1 by arterial and venous endothelial cells and EphB3 by veins and some arteries indicates that endothelial cell-to-cell interactions between ephrins and Eph receptors are not restricted to the border between arteries and veins. Furthermore, expression of ephrinB2 and EphB2 in mesenchyme adjacent to vessels and vascular defects in ephB2/ephB3 double mutants indicate a requirement for ephrin-Eph signaling between endothelial cells and surrounding mesenchymal cells. Finally, ephrinB ligands induce capillary sprouting in vitro with a similar efficiency as angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF), demonstrating a stimulatory role of ephrins in the remodeling of the developing vascular system.

SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types.

SU5416, a novel synthetic compound, is a potent and selective inhibitor of the Flk-1/KDR receptor tyrosine kinase that is presently under evaluation in Phase I clinical studies for the treatment of human cancers. SU5416 was shown to inhibit vascular endothelial growth factor-dependent mitogenesis of human endothelial cells without inhibiting the growth of a variety of tumor cells in vitro. In contrast, systemic administration of SU5416 at nontoxic doses in mice resulted in inhibition of subcutaneous tumor growth of cells derived from various tissue origins. The antitumor effect of SU5416 was accompanied by the appearance of pale white tumors that were resected from drug-treated animals, supporting the antiangiogenic property of this agent. These findings support that pharmacological inhibition of the enzymatic activity of the vascular endothelial growth factor receptor represents a novel strategy for limiting the growth of a wide variety of tumor types.

Angiogenesis in ischemic disease.

Angiogenic growth factors and their endothelial receptors function as major regulators of blood vessel formation. The VEGF/VEGFR and the Angiopoietin/Tie2 receptor systems represent key signal transduction pathways involved in the regulation of embryonic vascular development. Inactivation of any of the genes encoding these molecules results in defective vascular development and lethality between embryonic day 8.5 and 12.5. In addition, VEGF and its receptors are also critically involved in the regulation of pathological blood vessel growth in the adult during various angiogenesis-dependent diseases that are associated with tissue hypoxia, such as solid tumor growth and ischemic diseases. It is now well established that therapeutic angiogenesis can be achieved in animal models of hind limb and myocardial ischemia by exogenously adding VEGF and/or other angiogenic growth factors. Available clinical data from human trials also suggests that patients with severe cardiovascular diseases could potentially benefit from such therapies. However, much more work needs to be done to compare the potency of different angiogenic factors or the combination thereof, as well as the best way of delivery, either as recombinant proteins, as naked DNA or via adenoviral vectors. Nevertheless, the therapeutic efficacy of simply injecting naked plasmid DNA or proteins into ischemic tissue to deliver secreted angiogenic factors is an encouraging finding. Time will show whether the adverse side effects of therapeutic angiogenesis, mainly vascular permeability and edema formation, can be minimized and angiogenic factors can be used as an effective therapy in patients for the treatment of ischemic diseases such as arterial occlusive disease, myocardial infarction, and, eventually, also stroke.

Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors.

Vascular endothelial growth factor (VEGF) plays a key role in physiological blood vessel formation and pathological angiogenesis such as tumor growth and ischemic diseases. Hypoxia is a potent inducer of VEGF in vitro. Here we demonstrate that VEGF is induced in vivo by exposing mice to systemic hypoxia. VEGF induction was highest in brain, but also occurred in kidney, testis, lung, heart, and liver. In situ hybridization analysis revealed that a distinct subset of cells within a given organ, such as glial cells and neurons in brain, tubular cells in kidney, and Sertoli cells in testis, responded to the hypoxic stimulus with an increase in VEGF expression. Surprisingly, however, other cells at sites of constitutive VEGF expression in normal adult tissues, such as epithelial cells in the choroid plexus and kidney glomeruli, decreased VEGF expression in response to the hypoxic stimulus. Furthermore, in addition to VEGF itself, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was induced by hypoxia in endothelial cells of lung, heart, brain, kidney, and liver. VEGF itself was never found to be up-regulated in endothelial cells under hypoxic conditions, consistent with its paracrine action during normoxia. Our results show that the response to hypoxia in vivo is differentially regulated at the level of specific cell types or layers in certain organs. In these tissues, up- or down-regulation of VEGF and VEGFR-1 during hypoxia may influence their oxygenation after angiogenesis or modulate vascular permeability.