<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is an apparently <em>endothelial</em> cell-specific mitogen that is structurally related to platelet-derived <em>growth</em> <em>factor</em>. By Northern blot and protein analyses, we show that VEGF is produced by cultured <em>vascular</em> smooth muscle cells. Analysis of VEGF transcripts in these cells by polymerase chain reaction and cDNA cloning revealed three different forms of the VEGF coding region, as had been reported in HL60 cells. The three forms of the human VEGF protein chain predicted from these coding regions are 189, 165, and <em>121</em> amino acids in length. Comparison of cDNA nucleotide sequences with sequences derived from human VEGF genomic clones indicates that the VEGF gene is split among eight exons and that the various VEGF coding region forms arise from this gene by alternative splicing: the 165-amino-acid form of the protein is missing the residues encoded by exon 6, whereas the <em>121</em>-amino-acid form is missing the residues encoded by exons 6 and 7. Analysis of the VEGF gene promoter region revealed a single major transcription start, which lies near a cluster of potential Sp1 <em>factor</em> binding sites. The promoter region also contains several potential binding sites for the transcription <em>factors</em> AP-1 and AP-2; consistent with the presence of these sites, Northern blot analysis demonstrated that the level of VEGF transcripts is elevated in cultured <em>vascular</em> smooth muscle cells after treatment with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate.

Angiogenesis is a significant prognostic <em>factor</em> in breast cancer, but the <em>factors</em> that control angiogenesis in vivo are not well defined. Multiple angiogenic polypeptides are known, and we have determined the expression of seven of these in primary human breast cancers; the relationship of expression to estrogen receptor and <em>vascular</em> density was also examined. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and its four isoforms (<em>121</em>, 165, 189, and 206 amino acids), transforming <em>growth</em> <em>factor</em> (TGF)-beta1, pleiotrophin, acidic and basic fibroblast <em>growth</em> <em>factor</em> (FGF), placental <em>growth</em> <em>factor</em>, and thymidine phosphorylase (platelet-derived <em>endothelial</em> cell <em>growth</em> <em>factor</em>) were quantitated by RNase protection analysis. beta-FGF was also measured by ELISA. The estrogen receptor (ER), epidermal <em>growth</em> <em>factor</em> receptor, and <em>vascular</em> density were analyzed in 64 primary breast cancers. All tumors expressed at least six different <em>vascular</em> <em>growth</em> <em>factors</em>. VEGF was most abundant, and the transcript for the <em>121</em>-amino acid form predominated. Other angiogenic <em>factors</em> expressed at high levels were thymidine phosphorylase and TGF-beta1. Expression of most of the angiogenic <em>factors</em> did not correlate with that of ER or <em>vascular</em> density. However, thymidine phosphorylase did, with a correlation coefficient of 0.3 (P = 0.03). There were significant associations of pleiotrophin with acidic FGF expression (P = 0.001) and TGF-beta with platelet-derived <em>endothelial</em> cell <em>growth</em> <em>factor</em> expression (P = 0.001). Thus, angiogenesis may involve a coordinate regulation of some <em>vascular</em> <em>growth</em> <em>factors</em>. High VEGF expression correlated with poor prognosis in univariate analysis (P = 0.03), as did ER and epidermal <em>growth</em> <em>factor</em> receptor expression. Basic FGF was also assessed by ELISA and was more highly expressed in tumors than normal breast tissues (median, 346 microg/ml cytosol; range, 54-1323 versus median, 149; range, 32-509; P = 0.01). Implications for therapy are that broad spectrum agents that block features common to these <em>factors</em> may be useful (e.g., antagonism of heparin-binding activity agents), because so many angiogenic <em>factors</em> are expressed. Inhibiting <em>endothelial</em> migration or agents directly toxic to endothelium would be of value in a combined approach to therapy.

We have shown that coculture of bone marrow micro<em>vascular</em> <em>endothelial</em> cells with hematopoietic progenitor cells results in proliferation and differentiation of megakaryocytes. In these long-term cultures, bone marrow micro<em>vascular</em> <em>endothelial</em> cell monolayers maintain their cellular integrity in the absence of exogenous <em>endothelial</em> <em>growth</em> <em>factors</em>. Because this interaction may involve paracrine secretion of cytokines, we evaluated megakaryocytic cells for secretion of cytokines, we evaluated megakaryocytic cells for secretion of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF). Megakaryocytes (CD41a+) were generated by ex vivo expansion of hematopoietic progenitor cells with kit-ligand and thrombopoietin for 10 days and further purified with immunomagnetic microbeads. Using reverse transcription-PCR, we showed that megakaryocytic cell lines (Dami, HEL) and purified megakaryocytes expressed mRNA of the three VEGF isoforms (<em>121</em>, 165, and 189 amino acids). Large quantities of VEGF >> 1 ng/10(6) cells/3 days) were detected in the supernatant of Dami cells, ex vivo-generated megakaryocytes, and CD41a+ cells isolated from bone marrow. The constitutive secretion of VEGF by CD41a+ cells was stimulated by <em>growth</em> <em>factors</em> of the megakaryocytic lineage (interleukin 3, thrombopoietin). Western blotting of heparin-Sepharose-enriched supernatant mainly detected the isoform VEGF165. In addition, immunohistochemistry showed intracytoplasmic VEGF in polyploid megakaryocytes. Thrombin stimulation of megakaryocytes and platelets resulted in rapid release of VEGF within 30 min. We conclude that human megakaryocytes produce and secrete VEGF in an inducible manner. Within the bone marrow microenvironment, VEGF secreted by megakaryocytes may contribute to the proliferation of <em>endothelial</em> cells. VEGF delivered to sites of <em>vascular</em> injury by activated platelets may initiate angiogenesis.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a potent and specific mitogen for <em>endothelial</em> cells. VEGF is synthesized and secreted by many differentiated cells in response to a variety of stimuli including hypoxia. VEGF is expressed in a variety of tissues as multiple homodimeric forms (<em>121</em>, 165, 189, and 206 amino acids/monomer) resulting from alternative RNA splicing. VEGF<em>121</em> is a soluble mitogen that does not bind heparin; the longer forms of VEGF bind heparin with progressively higher affinity. The higher molecular weight forms of VEGF can be cleaved by plasmin to release a diffusible form(s) of VEGF. We characterized the proteolysis of VEGF by plasmin and other proteases. Thrombin, elastase, and collagenase did not cleave VEGF, whereas trypsin generated a series of smaller fragments. The isolated plasmin fragments of VEGF were compared with respect to heparin binding, interaction with soluble VEGF receptors, and ability to promote <em>endothelial</em> cell mitogenesis. Plasmin yields two fragments of VEGF as indicated by reverse phase high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis: an amino-terminal homodimeric protein containing receptor binding determinants and a carboxyl-terminal polypeptide which bound heparin. Amino-terminal sequencing of the carboxyl-terminal peptide identified the plasmin cleavage site as Arg110-Ala111. A heterodimeric form of VEGF165/110, was isolated from partial plasmin digests of VEGF165. The carboxyl-terminal polypeptide (111-165) displayed no affinity for soluble kinase domain region (KDR) or Fms-like tyrosine kinase (FLT-1) receptors. The various isoforms of VEGF (165, 165/110, and <em>121</em>) bound soluble kinase domain region receptor with similar affinity (approximately 30 pM). In contrast, soluble FLT-1 receptor differentiated VEGF isoforms (165, 165/110, 110, and <em>121</em>) with apparent affinities of 10, 30, 120, and 200 pM, respectively. <em>Endothelial</em> cell mitogenic potencies of VEGF110 and VEGF<em>121</em> were decreased more than 100-fold compared to that of VEGF165. The present findings indicate that removal of the carboxyl-terminal domain, whether it is due to alternative splicing of mRNA or to proteolysis, is associated with a significant loss in bioactivity.

For solid tumors and metastatic lesions, tumor <em>vascular</em>ity is a critical <em>factor</em> in assessing response to therapy. Here we report the first example, to our knowledge, of (64)Cu-labeled <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF(<em>121</em>)) for PET of VEGF receptor (VEGFR) expression in vivo.

METHODS

VEGF(<em>121</em>) was conjugated with 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA) and then labeled with (64)Cu for small-animal PET of mice bearing different sized U87MG human glioblastoma xenografts. Blocking experiments and ex vivo histopathology were performed to confirm the in vivo results.

RESULTS

There were 4.3 +/- 0.2 DOTA molecules per VEGF(<em>121</em>), and the VEGFR2 binding affinity of DOTA-VEGF(<em>121</em>) was comparable to VEGF(<em>121</em>). (64)Cu labeling of DOTA-VEGF(<em>121</em>) was achieved in 90 +/- 10 min and the radiolabeling yield was 87.4% +/- 3.2%. The specific activity of (64)Cu-DOTA-VEGF(<em>121</em>) was 3.2 +/- 0.1 GBq/mg with a radiochemical purity of >98%. Small-animal PET revealed rapid, specific, and prominent uptake of (64)Cu-DOTA-VEGF(<em>121</em>) in small U87MG tumors (high VEGFR2 expression) but significantly lower and sporadic uptake in large U87MG tumors (low VEGFR2 expression). No appreciable renal clearance of (64)Cu-DOTA-VEGF(<em>121</em>) was observed, although the kidney uptake was relatively high likely due to VEGFR1 expression. Blocking experiments, immunofluorescence staining, and western blot confirmed the VEGFR specificity of (64)Cu-DOTA-VEGF(<em>121</em>).

CONCLUSIONS

Successful demonstration of the ability of (64)Cu-DOTA-VEGF(<em>121</em>) to visualize VEGFR expression in vivo may allow for clinical translation of this radiopharmaceutical for imaging tumor angiogenesis and guiding antiangiogenic treatment, especially patient selection and treatment monitoring of VEGFR-targeted cancer therapy.

Endostatin, a fragment of collagen XVIII, is a potent anti-angiogenic protein, but the molecular mechanism of its action is not yet clear. We examined the effects of endostatin on the biological and biochemical activities of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF). Endostatin blocked VEGF-induced tyrosine phosphorylation of KDR/Flk-1 and activation of ERK, p38 MAPK, and p125(FAK) in human umbilical vein <em>endothelial</em> cells. Endostatin also inhibited the binding of VEGF(165) to both <em>endothelial</em> cells and purified extracellular domain of KDR/Flk-1. Moreover, the binding of VEGF(<em>121</em>) to KDR/Flk-1 and VEGF(<em>121</em>)-stimulated ERK activation were blocked by endostatin. The direct interaction between endostatin and KDR/Flk-1 was confirmed by affinity chromatography. However, endostatin did not bind to VEGF. Our findings suggest that a direct interaction of endostatin with KDR/Flk-1 may be involved in the inhibitory function of endostatin toward VEGF actions and responsible for its potent anti-angiogenic and anti-tumor activities in vivo.

BACKGROUND

"Therapeutic angiogenesis" seeks to improve perfusion by the growth of new blood vessels. The Regional Angiogenesis with Vascular Endothelial growth factor (RAVE) trial is the first major randomized study of adenoviral vascular endothelial growth factor (VEGF) gene transfer for the treatment of peripheral artery disease (PAD).

RESULTS

This phase 2, double-blind, placebo-controlled study was designed to test the efficacy and safety of intramuscular delivery of AdVEGF121, a replication-deficient adenovirus encoding the 121-amino-acid isoform of vascular endothelial growth factor, to the lower extremities of subjects with unilateral PAD. In all, 105 subjects with unilateral exercise-limiting intermittent claudication during 2 qualifying treadmill tests, with peak walking time (PWT) between 1 to 10 minutes, were stratified on the basis of diabetic status and randomized to low-dose (4x10(9) PU) AdVEGF121, high-dose (4x10(10) PU) AdVEGF121, or placebo, administered as 20 intramuscular injections to the index leg in a single session. The primary efficacy end point, change in PWT (DeltaPWT) at 12 weeks, did not differ between the placebo (1.8+/-3.2 minutes), low-dose (1.6+/-1.9 minutes), and high-dose (1.5+/-3.1 minutes) groups. Secondary measures, including DeltaPWT, ankle-brachial index, claudication onset time, and quality-of-life measures (SF-36 and Walking Impairment Questionnaire), were also similar among groups at 12 and 26 weeks. AdVEGF121 administration was associated with increased peripheral edema.

CONCLUSIONS

A single unilateral intramuscular administration of AdVEGF121 was not associated with improved exercise performance or quality of life in this study. This study does not support local delivery of single-dose VEGF121 as a treatment strategy in patients with unilateral PAD.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) was identified as a heparin-binding polypeptide mitogen with a target cell specificity restricted to <em>vascular</em> <em>endothelial</em> cells. Molecular cloning reveals the existence of four species of VEGF having <em>121</em>, 165, 189, and 206 amino acids. These have strikingly different secretion patterns, which suggests multiple physiological roles for this family of polypeptides. The two shorter forms are efficiently secreted, while the longer ones are mostly cell-associated. Alternative splicing of mRNA, rather that transcription from different genes, is the mechanism for their generation. In situ hybridization reveals that the VEGF mRNA is widely distributed in most tissues and organs and expressed at particularly high levels in areas of active <em>vascular</em> proliferation, like the ovarian corpus luteum. Ligand autoradiography on rat tissue sections demonstrates that VEGF binding sites are associated with <em>vascular</em> <em>endothelial</em> cells of both fenestrated and non-fenestrated capillaries and with the endothelium of large vessels, while no displaceable binding is evident on non-<em>endothelial</em> cell types. These findings support the hypothesis that VEGF plays a highly specific role in the maintenance and in the induction of <em>growth</em> of <em>vascular</em> <em>endothelial</em> cells.

Expression of the <em>vascular</em> permeability <em>factor</em>/<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGPF) gene was investigated in human central nervous system (CNS) neoplasms and normal brain. Adsorption of capillary permeability activity from human glioblastoma multiforme (GBM) cell conditioned medium and GBM cyst fluids by anti-VEGPF antibodies demonstrated that VEGPF is secreted by GBM cells and is present in sufficient quantities in vivo to induce <em>vascular</em> permeability. Cloning and sequencing of polymerase chain reaction-amplified GBM and normal brain cDNA demonstrated three forms of the VEGPF coding region (567, 495, and 363 nucleotides), corresponding to mature polypeptides of 189, 165, and <em>121</em> amino acids, respectively. VEGPF mRNA levels in CNS tumors vs. normal brain were investigated by the RNase protection assay. Significant elevation of VEGPF gene expression was observed in 81% (22/27) of the highly <em>vascular</em> and edema-associated CNS neoplasms (6/8 GBM, 8/8 capillary hemangioblastomas, 6/7 meningiomas, and 2/4 cerebral metastases). In contrast, only 13% (2/15) of those CNS tumors that are not commonly associated with significant neo<em>vascular</em>ity or cerebral edema (2/10 pituitary adenomas and 0/5 nonastrocytic gliomas) had significantly increased levels of VEGPF mRNA. The relative abundance of the forms of VEGPF mRNA was consistent in tumor and normal brain: VEGPF495>> VEGPF363>> VEGPF567. In situ hybridization confirmed the presence of VEGPF mRNA in tumor cells and its increased abundance in capillary hemangioblastomas. Our results suggest a significant role for VEGPF in the development of CNS tumor neo<em>vascular</em>ity and peritumoral edema.

BACKGROUND

Therapeutic angiogenesis, a new experimental strategy for the treatment of <em>vascular</em> insufficiency, uses the administration of mediators known to induce <em>vascular</em> development in embryogenesis to induce neo<em>vascular</em>ization of ischemic adult tissues. This report summarizes a phase I clinical experience with a gene-therapy strategy that used an E1(-)E3(-) adenovirus (Ad) gene-transfer vector expressing human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) <em>121</em> cDNA (Ad(GV)VEGF<em>121</em>.10) to induce therapeutic angiogenesis in the myocardium of individuals with clinically significant coronary artery disease.

RESULTS

Ad(GV)VEGF<em>121</em>.10 was administered to 21 individuals by direct myocardial injection into an area of reversible ischemia either as an adjunct to conventional coronary artery bypass grafting (group A, n=15) or as sole therapy via a minithoracotomy (group B, n=6). There was no evidence of systemic or cardiac-related adverse events related to vector administration. In both groups, coronary angiography and stress sestamibi scan assessment of wall motion 30 days after therapy suggested improvement in the area of vector administration. All patients reported improvement in angina class after therapy. In group B, in which gene transfer was the only therapy, treadmill exercise assessment suggested improvement in most individuals.

CONCLUSIONS

The data are consistent with the concept that direct myocardial administration of Ad(GV)VEGF<em>121</em>.10 to individuals with clinically significant coronary artery disease appears to be well tolerated, and initiation of phase II evaluation of this therapy is warranted.

Almost any <em>growth</em> of tumors is to some extent associated with an inflammatory reaction which may be anti-tumorigenic by acting directly on tumor cells or protumorigenic cells presumably by inducing tumor-associated angiogenesis. In this study, we have analyzed the angiogenesis-inducing capacity of monocyte chemoattractant protein-1 (MCP-1), a key regulatory molecule of monocyte trafficking to sites of inflammation. MCP-1 was found to be potently angiogenic when implanted into rabbit cornea, exerting potency similar to the specific angiogenic <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)-A(<em>121</em>). MCP-1-induced angiogenesis in the cornea is associated with prominent recruitment of macrophages, whereas VEGF-A(<em>121</em>)-induced corneal angiogenesis is devoid of inflammatory cell recruitment. Based on these findings, we studied MCP-1 expression and macrophage recruitment in human invasive ductal mammary carcinomas in comparison with the physiological angiogenic processes in bovine ovarian corpus luteum. Macrophage recruitment was always associated with MCP-1 expression. High macrophage counts in mammary tumors corresponded with poor prognosis. In contrast, physiological ovarian angiogenesis was associated with only minimal inflammatory recruitment of macrophages. Our data show that MCP-1 is an indirect inflammation-associated inducer of angiogenesis and demonstrate distinct qualitative differences between tumor angiogenesis in human mammary tumors and physiological angiogenesis in the ovary.

OBJECTIVE

Proliferative diabetic retinopathy results from excess blood vessel growth into the vitreous fluid of the eye. Retinal angiogenesis is regulated by expression of vascular endothelial growth factor (VEGF), and many studies have shown that VEGF is critically involved in proliferative diabetic retinopathy. VEGF is alternatively spliced to form the angiogenic (VEGF(xxx)) and potentially anti-angiogenic (VEGF(xxx)b) family of isoforms. The VEGF(xxx)b family is found in normal tissues, but down-regulated in renal and prostate cancer. Previous studies on endogenous expression of VEGF in the eye have not distinguished between the two families of isoforms.

METHODS

We measured VEGF(xxx)b isoform expression in normal human eye tissue (lens, sclera, retina and iris) and vitreous fluid using enzyme-linked immunosorbent assay and Western blotting with a VEGF(xxx)b-specific antibody.

RESULTS

VEGF(xxx)b protein was expressed in lens, sclera, retina, iris and vitreous fluid. Multiple isoforms were seen, including VEGF(165)b, VEGF(121)b, VEGF(145)b, VEGF(183)b and VEGF(189)b. In non-diabetic patients, 64+/-7% of the VEGF in the vitreous was VEGF(xxx)b (n=18), whereas in diabetic patients only 12.5+/-3.6% of total VEGF was VEGF(xxx)b.

CONCLUSIONS

Since VEGF(xxx)b inhibits VEGF(xxx)-induced angiogenesis in a one-to-one stoichiometric manner, these results show that in the eye of diabetic patients VEGF splicing was switched from an anti-angiogenic to a pro-angiogenic environment. This occurred through changes to the ratio of VEGF(xxx):VEGF(xxx)b. Alterations to splicing, and through that to the balance of VEGF isoforms, could therefore be a potential therapeutic strategy for diabetic retinopathy.

OBJECTIVE

Activation or overexpression of HER-2/neu is associated with up-regulation of vascular endothelial growth factor (VEGF) in human breast cancer cells in vitro. Preclinical experiments indicate that increased expression of VEGF may in part mediate the biologically aggressive phenotype of HER-2/neu-overexpressing human breast cancer. It was the purpose of this study to: (a). evaluate the association between HER-2/neu and VEGF expression in a large clinical cohort of primary breast cancer patients; (b). compare the prognostic significance of VEGF isoforms; and (c). analyze the combined effects of HER-2/neu and VEGF on clinical outcome.

METHODS

HER-2/neu and VEGF were measured by ELISA in primary breast tumor tissue lysates from 611 unselected patients with a median clinical follow-up of 50 months. At least six VEGF isoforms consisting of 121, 145, 165, 183, 189, or 206 amino acids are generated as a result of alternative splicing. The VEGF(121-206) ELISA uses antibodies that bind to VEGF(121) and, therefore, detects all of the VEGF isoforms with 121 and more amino acids. The VEGF(165-206) ELISA uses antibodies that bind to VEGF(165) and, therefore, detects all of the VEGF isoforms with 165 and more amino acids. VEGF(121-206) and VEGF(165-206) were analyzed both as continuous and categorical variables, using detectable expression as a cutoff for positivity. Cell lines with defined HER-2/neu expression levels were used to establish a cutoff point for HER-2/neu overexpression in breast tumor samples.

RESULTS

Our findings indicate a significant positive association between HER-2/neu and VEGF expression. VEGF(121-206) and VEGF(165-206) expression was detectable in 88 (77.2%) and 100 (87.7%), respectively, of the 114 patients with HER-2/neu-overexpressing tumors, in contrast to 271 (54.5%) and 353 (71.0%), respectively, of the 497 patients with nonoverexpressing tumors (chi(2) test: P < 0.001 for both VEGF(121-206) and VEGF(165-206)). VEGF(121-206) and VEGF(165-206) demonstrate a comparable prognostic significance for survival in unselected primary breast cancer patients (univariate analysis: VEGF(121-206), P = 0.0068; VEGF(165-206), P = 0.0046; multivariate analysis: VEGF(121-206), P = 0.1475; VEGF(165-206), P = 0.1483). When the analyses were performed separately for node-negative and node-positive patients, VEGF(121-206) and VEGF(165-206) were of prognostic significance for survival only in node-positive patients (univariate analysis: VEGF(121-206), P = 0.0003; VEGF(165-206), P = 0.0038; multivariate analysis: VEGF(121-206), P = 0.0103; VEGF(165-206), P = 0.0150). A biological concentration-effect relationship between VEGF expression and survival (VEGF(121-206), P = 0.0280; VEGF(165-206,) P = 0.0097) suggests that VEGF levels, as determined by ELISA, could be of importance as a predictive marker for therapeutic strategies that target VEGF. Combining HER-2/neu and VEGF(121-206)/VEGF(165-206) results in additional prognostic information for survival (VEGF(121-206), P = 0.0133; VEGF(165-206), P = 0.0092).

CONCLUSIONS

The positive association between HER-2/neu and VEGF expression implicates VEGF in the aggressive phenotype exhibited by HER-2/neu overexpression, and supports the use of combination therapies directed against both HER-2/neu and VEGF for treatment of breast cancers that overexpress HER-2/neu.

Acute kidney injury induces the loss of renal microvessels, but the fate of <em>endothelial</em> cells and the mechanism of potential <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)-mediated protection is unknown. Cumulative cell proliferation was analyzed in the kidney of Sprague-Dawley rats following ischemia-reperfusion (I/R) injury by repetitive administration of BrdU (twice daily) and colocalization in <em>endothelial</em> cells with CD31 or cablin. Proliferating <em>endothelial</em> cells were undetectable for up to 2 days following I/R and accounted for only ∼1% of BrdU-positive cells after 7 days. VEGF-<em>121</em> preserved <em>vascular</em> loss following I/R but did not affect proliferation of <em>endothelial</em>, peri<em>vascular</em> cells or tubular cells. <em>Endothelial</em> mesenchymal transition states were identified by localizing <em>endothelial</em> markers (CD31, cablin, or infused tomato lectin) with the fibroblast marker S100A4. Such structures were prominent within 6 h and sustained for at least 7 days following I/R. A Tie-2-cre transgenic crossed with a yellow fluorescent protein (YFP) reporter mouse was used to trace the fate of <em>endothelial</em> cells and demonstrated interstititial expansion of YFP-positive cells colocalizing with S100A4 and smooth muscle actin following I/R. The interstitial expansion of YFP cells was attenuated by VEGF-<em>121</em>. Multiphoton imaging of transgenic mice revealed the alteration of YFP-positive <em>vascular</em> cells associated with blood vessels characterized by limited perfusion in vivo. Taken together, these data indicate that <em>vascular</em> dropout post-AKI results from <em>endothelial</em> phenotypic transition combined with an impaired regenerative capacity, which may contribute to progressive chronic kidney disease.

Neuropilin-1 (NRP1) is a 130-kDa transmembrane receptor for semaphorins, mediators of neuronal guidance, and for <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> 165 (VEGF(165)), an angiogenesis <em>factor</em>. A 2.2-kb truncated NRP1 cDNA was cloned that encodes a 644-aa soluble NRP1 (sNRP1) isoform containing just the a/CUB and b/coagulation <em>factor</em> homology extracellular domains of NRP1. sNRP1 is secreted by cells as a 90-kDa protein that binds VEGF(165), but not VEGF(<em>121</em>). It inhibits (125)I-VEGF(165) binding to <em>endothelial</em> and tumor cells and VEGF(165)-induced tyrosine phosphorylation of KDR in <em>endothelial</em> cells. The 3' end of sNRP1 cDNA contains a unique, 28-bp intron-derived sequence that is absent in full-length NRP1 cDNA. Using a probe corresponding to this unique sequence, sNRP1 mRNA could be detected by in situ hybridization differentially from full-length NRP1 mRNA, for example, in cells of liver, kidney, skin, and breast. Analysis of blood vessels in situ showed that NRP1, but not sNRP1, was expressed. sNRP1 was functional in vivo. Unlike control tumors, tumors of rat prostate carcinoma cells expressing recombinant sNRP1 were characterized by extensive hemorrhage, damaged vessels, and apoptotic tumor cells. These results demonstrate the existence of a naturally occurring, soluble NRP1 that is expressed differently from intact NRP1 and that appears to be a VEGF(165) antagonist.

Neuropilin-1 (NRP1) was first described as a receptor for the axon guidance molecule, Semaphorin3A, regulating the development of the nervous system. It was later shown that NRP1 is an isoform-specific receptor for <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), specifically VEGF(165). Much interest has been placed on the role of the various VEGF isoforms in <em>vascular</em> biology. Here we report that blocking NRP1 function, using a recently described antibody that inhibits VEGF(165) binding to NRP1, surprisingly reduces VEGF(<em>121</em>)-induced migration and sprout formation of <em>endothelial</em> cells. Intrigued by this observation, direct binding studies of NRP1 to various VEGF isoforms were performed. We show that VEGF(<em>121</em>) binds directly to NRP1; however, unlike VEGF(165), VEGF(<em>121</em>) is not sufficient to bridge the NRP1.VEGFR2 complex. Additionally, we show that VEGFR2 enhances VEGF(165), but not VEGF(<em>121</em>) binding to NRP1. We propose a new model for NRP1 interactions with various VEGF isoforms.

Here, we report an intrinsic property of gold nanoparticles (nanogold): they can interact selectively with heparin-binding glycoproteins and inhibit their activity. Gold nanoparticles specifically bound <em>vascular</em> permeability <em>factor</em>/<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VPF/VEGF)-165 and basic fibroblast <em>growth</em> <em>factor</em>, two <em>endothelial</em> cell mitogens and mediators of angiogenesis resulting in inhibition of <em>endothelial</em>/fibroblast cell proliferation in vitro and VEGF-induced permeability as well as angiogenesis in vivo. In contrast, nanogold did not inhibit VEGF-<em>121</em> or epidermal <em>growth</em> <em>factor</em>, two non-heparin-binding <em>growth</em> <em>factors</em>, mediated cell proliferation. Gold nanoparticles significantly inhibited VEGF receptor-2 phosphorylation, intracellular calcium release, and migration and RhoA activation in vitro. These results report for the first time a novel property of gold nanoparticles to bind heparin-binding proteins and thereby inhibit their subsequent signaling events.

Inhibitors of angiogenic factors are known to be upregulated, and their levels increase in the maternal circulation before the onset of preeclampsia. We reproduced a previously characterized model of preeclampsia by adenoviral overexpression of the soluble vascular endothelial growth factor (VEGF) receptor sFlt-1 (also referred to as sVEGFR-1) in pregnant and nonpregnant Sprague-Dawley rats. Animals were treated with VEGF121 at 0, 100, 200, or 400 microg/kg once or twice daily (n=8 per group; 64 total) and compared with normal control animals (n=4 per group) by examination of systolic blood pressure, urinary albumin and creatinine, renal histopathology, and glomerular gene expression profiling. sFlt-1 expression induced hypertension with proteinuria and glomerular endotheliosis and significant changes in gene expression. VEGF121 treatment alleviated these symptoms and reversed 125 of 268 sFlt-1-induced changes in gene expression. VEGF121 had beneficial effects in this rat model of preeclampsia without apparent harm to the fetus. Further study of VEGF121 as a potential therapeutic agent for preeclampsia is warranted.

BACKGROUND

Vascular endothelial growth factor (VEGF) is a protein with antiapoptotic, mitogenic, and permeability-increasing activities specific for vascular endothelium. VEGF mRNA, which has five isoforms, is produced by nonmalignant cells in response to hypoxia and inflammation and by tumor cells in constitutively high concentrations. Because VEGF plays a crucial role in physiological and pathophysiological angiogenesis, measurements of circulating VEGF are of diagnostic and prognostic value, e.g., in cardiovascular failures, inflammatory diseases, and malignancies. However, there are major quantitative differences in the published results. This review attempts to identify reasons for these disparities.

METHODS

The literature was reviewed through a Medline search covering 1995 to 2000. A selection of exemplary references had to be made for this perspective overview.

BACKGROUND

Data are included from studies on healthy humans, gynecological patients, and persons suffering from inflammatory or malignant diseases. The results indicate that competitive immunoassays detect the total amount of circulating VEGF, which enables observations regarding the increase in VEGF in pregnancy and preeclampsia to be made. In these cases, capture immunoassays utilizing neutralizing antibodies are insufficient because of an accompanying increase in VEGF-binding soluble receptors (sFlt-1). Measurements of circulating free VEGF are useful for study of malignant diseases, which are associated with both genetically and hypoxia-induced overproduction of VEGF. The VEGF isoform specificity of the antibodies is also critical because both VEGF(121) and VEGF(165) are secreted. It is important to consider that platelets and leukocytes release VEGF during blood clotting.

CONCLUSIONS

Future efforts should concentrate on the balance between free VEGF, total VEGF, and sFlt-1. Plasma, rather than serum, should be used for analysis.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a key <em>factor</em> in <em>endothelial</em> cell biology and blood vessel formation and a candidate therapeutic for the stimulation of angiogenesis-dependent tissue regeneration. The objective of this study was to confer the angiogenic activity of VEGF(<em>121</em>) upon the biomaterial fibrin, a natural substrate for <em>endothelial</em> cell <em>growth</em> and clinically accepted as 'fibrin glue'. To achieve this, we engineered fibrin-based hydrogels that were covalently modified with VEGF(<em>121</em>). Our laboratory has recently developed novel methodology that allows the covalent incorporation of exogenous bioactive peptides by the transglutaminase activity of <em>factor</em> XIIIa into fibrin during coagulation. Here, this ability of <em>factor</em> XIIIa to crosslink additional proteins within fibrin was employed to covalently incorporate VEGF(<em>121</em>). By recombinant DNA methodology, a mutant VEGF(<em>121</em>) variant, alpha(2)-PI(1--8)-VEGF(<em>121</em>), which contains an additional <em>factor</em> XIIIa substrate sequence NQEQVSPL at the aminoterminus, was expressed in E. coli. In soluble form, the mutant protein fully retained its mitogenic activity for <em>endothelial</em> cells. Using (125)I-labeled alpha(2)-PI(1--8)-VEGF(<em>121</em>), its covalent incorporation and the efficiency of incorporation into fibrin was demonstrated and characterized. The immobilized, fibrin-conjugated VEGF(<em>121</em>) protein remained an active and very efficient mitogen for human <em>endothelial</em> cells grown on two-dimensional VEGF(<em>121</em>)-modified fibrin surfaces, and the incorporation of increasing amounts of alpha(2)-PI(1--8)-VEGF(<em>121</em>) resulted in dose-dependent enhancement of <em>endothelial</em> cell <em>growth</em>. The VEGF-modified fibrin matrices can be formed as injectable gels in a single-step reaction under physiological conditions in vivo. When used as a in<em>growth</em> matrix, such VEGF incorporating materials could be useful in a variety of clinical situations that require an angiogenic response into an ischemic region or inplant.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a well known <em>factor</em> that induces angiogenesis. Four isoforms, i.e. VEGF206, 189, 165, and <em>121</em>, have been identified. We examined the isoform patterns of VEGF mRNA using reverse transcription polymerase chain reaction (RT-PCR) analysis in 61 colon cancers. All the colon cancers examined expressed VEGF<em>121</em>. The isoform patterns were classified into three groups: type 1, VEGF<em>121</em>; type 2, VEGF<em>121</em> + VEGF165; type 3, VEGF<em>121</em> + VEGF165 + VEGF189. Three of the 61 colon cancers examined showed type 1 expression, 26 showed type 2 expression and 32 showed the type 3 pattern. The patients with liver metastases showed the type 3 isoform expression pattern at a significantly higher incidence (12 of 16, 75%) than those without liver metastasis (20 of 45, 44%) (P=0.036). The type 3 isoform pattern was significantly associated with M1 stage (P=0.019). The patients with colon cancer and the type 3 isoform pattern showed significantly poor prognosis (P < 0.01, Cox-Mantel). The colon cancers with the type 3 pattern showed a significantly higher involvement of veins (P=0.006). These observations suggest that the aberrant type 3 expression pattern of VEGF189 mRNA isoforms is correlated with liver metastasis, M stage, and poor prognosis in colon cancer.

Antagonism of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) signaling by soluble fms-like tyrosine kinase 1 occurs during preeclampsia and is proposed to play an important role in the pathogenesis of preeclampsia. We reported recently that hypertension associated with chronic reductions in uteroplacental perfusion pressure (RUPP) is associated with increased soluble fms-like tyrosine kinase 1 and decreased free VEGF. Whether restoration of circulating VEGF can restore renal function and chronically decrease arterial pressure associated with placental ischemia remains unknown. We hypothesized that chronic infusion of VEGF(<em>121</em>) would attenuate hypertension, increase glomerular filtration rate, and reverse the <em>endothelial</em> dysfunction associated with chronic RUPP. VEGF(<em>121</em>) (at either 90 or 180 microg/kg per day) was administered for 5 days via osmotic minipump placed IP. Mean arterial pressure, renal function, and tissues were obtained on day 19 of pregnancy from RUPP+VEGF, RUPP, and normal pregnant dams. Mean arterial pressure was increased in the RUPP (131+/-3 mm Hg) compared with the normal pregnant (102+/-1 mm Hg) rats, and infusion of VEGF(<em>121</em>) resolved the hypertension (105+/-5 mm Hg). Glomerular filtration rate was decreased in the RUPP dams (1.5+/-0.3 mL/min) and restored to normal pregnant levels (3.1+/-0.5 mL/min) by VEGF(<em>121</em>) treatment (3.1+/-0.4 mL/min). Effective renal plasma flow, decreased by RUPP, was also increased by VEGF(<em>121</em>) infusion. Relaxation to acetylcholine was enhanced by the VEGF treatment (P<0.05). These data demonstrate that chronic infusion of VEGF(<em>121</em>) during late gestation restores glomerular filtration rate and <em>endothelial</em> function and reduces high blood pressure associated with placental ischemia. The present results suggest that VEGF(<em>121</em>) may be a candidate molecule for management of preeclampsia and its related complications.

The two most abundant secreted isoforms of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGF(165) and VEGF(<em>121</em>)) are formed as a result of differential splicing of the VEGF-A gene. VEGF(165) and VEGF(<em>121</em>) share similar affinities at the isolated VEGF receptor (VEGFR)-2 but have been previously demonstrated to have differential ability to activate VEGFR-2-mediated effects on <em>endothelial</em> cells. Herein we investigate whether the recently described VEGF(165) isoform-specific receptor neuropilin-1 (Npn-1) is responsible for the difference in potency observed for these ligands. We demonstrate that although VEGFR-2 and Npn-1 form a complex, this complex does not result in an increase in VEGF(165) binding affinity. Therefore, the differential activity of VEGF(165) and VEGF(<em>121</em>) cannot be explained by a differential binding affinity for the complex. Using an antagonist that competes for VEGF(165) binding at the VEGFR-2.Npn-1 complex, we observe specific antagonism of VEGF(165)-meditated phosphorylation of VEGFR-2 without affecting the VEGF(<em>121</em>) response. These data indicate that the formation of the complex is responsible for the increased potency of VEGF(165) versus VEGF(<em>121</em>). Taken together, these data suggest a receptor-clustering role for Npn-1, as opposed to Npn-1 behaving as an affinity-converting subunit.

Blood vessels form either by the assembly and differentiation of mesodermal precursor cells (vasculogenesis) or by sprouting from preexisting vessels (angiogenesis). <em>Endothelial</em>-specific receptor tyrosine kinases and their ligands are known to be essential for these processes. Targeted disruption of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) or its receptor kdr (flk1, VEGFR2) in mouse embryos results in a severe reduction of all blood vessels, while the complete loss of flt1 (VEGFR1) leads to an increased number of hemangioblasts and a disorganized vasculature. In a large-scale forward genetic screen, we identified two allelic zebrafish mutants in which the sprouting of blood vessels is specifically disrupted without affecting the assembly and differentiation of angioblasts. Molecular cloning revealed nonsense mutations in flk1. Analysis of mRNA expression in flk1 mutant embryos showed that flk1 expression was severely downregulated, while the expression of other genes (scl, gata1, and fli1) involved in vasculogenesis or hematopoiesis was unchanged. Overexpression of vegf(<em>121</em>+165) led to the formation of additional vessels only in sibling larvae, not in flk1 mutants. We demonstrate that flk1 is not required for proper vasculogenesis and hematopoiesis in zebrafish embryos. However, the disruption of flk1 impairs the formation or function of vessels generated by sprouting angiogenesis.