The expression of lysyl-oxidase gene family members in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are malignant disorders originating from clonal expansion of a single neoplastic stem cell and characteristically show an increase in bone marrow reticulin fibers. Lysyl oxidases (LOXs) are copper-dependent amine oxidases that play a critical role in the biogenesis of connective tissue by crosslinking extracellular matrix proteins, collagen and elastin. Expression of LOX gene family members is increased in disorders associated with increased fibrosis. To evaluate involvement of LOX gene family in various MPNs. In-situ hybridization was used to detect Lysyl-Oxidase family members in bone marrow biopsies from patients with different MPNs. We compared normal bone marrows and those from patients with polycythemia vera, essential thrombocythemia, chronic myeloid leukemia, and primary myelofibrosis (PMF). Serum levels of lysyl-oxidase from patients with PMF and healthy controls were also examined. LOX gene family was not detected in normal bone marrows. All members of the LOX gene family were over expressed in PMF. In other MPNs a differential pattern of expression was observed. Differences in gene expression were statistically significant (P < 0.010). The medianserum LOX levels in normal controls was 28.4 ± 2.5 ng\ml and 44.6 ± 9.44 ng\ml in PMF (P = 0.02). The varying pattern of expression of LOX genes may reflect differences in the pathophysiology of bone marrow fibrosis in these MPNs. These observations could be used as the basis for future targeted therapy directed against bone marrow fibrosis.

Modeling invasive breast cancer: growth factors propel progression of HER2-positive premalignant lesions.

The HER2/neu oncogene encodes a receptor-like tyrosine kinase whose overexpression in breast cancer predicts poor prognosis and resistance to conventional therapies. However, the mechanisms underlying aggressiveness of HER2 (human epidermal growth factor receptor 2)-overexpressing tumors remain incompletely understood. Because it assists epidermal growth factor (EGF) and neuregulin receptors, we overexpressed HER2 in MCF10A mammary cells and applied growth factors. HER2-overexpressing cells grown in extracellular matrix formed filled spheroids, which protruded outgrowths upon growth factor stimulation. Our transcriptome analyses imply a two-hit model for invasive growth: HER2-induced proliferation and evasion from anoikis generate filled structures, which are morphologically and transcriptionally analogous to preinvasive patients' lesions. In the second hit, EGF escalates signaling and transcriptional responses leading to invasive growth. Consistent with clinical relevance, a gene expression signature based on the HER2/EGF-activated transcriptional program can predict poorer prognosis of a subgroup of HER2-overexpressing patients. In conclusion, the integration of a three-dimensional cellular model and clinical data attributes progression of HER2-overexpressing lesions to EGF-like growth factors acting in the context of the tumor's microenvironment.

Differential expression of neuropilin-1 and neuropilin-2 in arteries and veins.

Neuropilin-1 (np1) and neuropilin-2 (np2) are receptors for class-3 semaphorins and for several isoforms of VEGF. We have cloned and characterized two chick isoforms of np2 cDNA. Expression patterns of np1, np2, and ephrin-B2 were compared in the developing vascular system of 24-72 h old chick embryos. We show for the first time that np2 is expressed in blood vessels in vivo from the earliest stages of their formation. In contrast to ephrin-B2, both np1 and np2 are expressed in blood islands of 24 h old chick embryos. At 48-72 h, np1 expression is localized preferentially in arteries with an expression pattern that resembles that of ephrin-B2. In contrast, np2 is expressed preferentially in veins. Thus, neuropilins may play a role in determining the arterial or venous identity of blood vessels.

Neuroendocrine cells along the digestive tract express neuropilin-2.

Neuropilin-2 (np-2) is a receptor for semaphorin-3F (sema-3F) and semaphorin-3C (sema-3C). These semaphorins repel tips of growing axons that express np-2. In addition, np-2 functions as a receptor for heparin binding forms of the angiogenic factor vascular endothelial growth factor (VEGF) such as VEGF145 and VEGF165. We report that np-2 is strongly expressed in neuroendocrine cells located all along the human digestive tract. Confocal fluorescent microscopy revealed that np-2 is concentrated in vesicle-like structures located near the nucleus at the basolateral side of these cells. In the colon, the np-2-expressing subpopulation of neuroendocrine cell is almost identical with the serotonin-producing subpopulation of neuroendocrine cells. Gastrointestinal carcinoid tumors are digestive tract tumors that develop from neuroendocrine cells. Interestingly, most of the carcinoid tumors derived from the colon and the appendix did not contain np-2-producing cells. However, some carcinoid tumors derived from the small intestine and stomach did express low levels of np-2 in isolated foci of cells. By contrast, strong serotonin and chromogranin-A expression was observed in all of the carcinoid tumors that were examined. These results suggest that loss of np-2 expression may accompany tumor progression in carcinoid tumors.

The contribution of proangiogenic factors to the progression of malignant disease: role of vascular endothelial growth factor and its receptors.

Vascular endothelial growth factor is a major inducer of angiogenesis and a vascular permeability inducing factor. Its expression is upregulated in many types of tumors and it is thought to be a major inducer of tumor angiogenesis. This article focuses on the role of vascular endothelial growth factor in tumor progression and on current efforts aimed at the inhibition of tumor progression through the inhibition of vascular endothelial growth factor activity.

Increased vascular endothelial growth factor 165 binding to kinase insert domain-containing receptor after infection of human endothelial cells by recombinant adenovirus encoding the Vegf(165) gene.

BACKGROUND: The angiogenic effect of vascular endothelial growth factor (VEGF(165)) is mediated mainly through the high-affinity tyrosine kinase receptor VEGF-R2 (KDR/flk-1). This study examined the effects of VEGF overexpression by primary human endothelial cells (ECs), which do not express VEGF under physiological conditions, on cell proliferation, VEGF binding to the kinase insert domain-containing receptor (KDR), and KDR expression. METHODS AND RESULTS: Human primary ECs and SMCs were infected by recombinant adenoviral vector encoding VEGF(165) (rAdVEGF). Proliferation rate, bromodeoxyuridine incorporation, (125)I-labeled VEGF(165) binding to the KDR receptor, and KDR expression were tested in the infected cells and in cells supplemented with VEGF protein. Enhanced proliferation and a significant increase in (125)I-VEGF(165) binding to the KDR receptor were induced by rAdVEGF infection of ECs (autocrine effect) as well as by addition of recombinant VEGF(165) to noninfected cells. Infection of ECs by rAdVEGF led to posttranscriptional upregulation of the KDR receptor, whereas KDR mRNA expression levels remained unchanged. Similar effects were observed with supplemented recombinant VEGF(165) to noninfected ECs; nevertheless, this phenomenon occurred only with high VEGF(165) concentrations (10 ng/mL). CONCLUSIONS: The effect of VEGF(165) on proliferation and upregulation of KDR receptor expression demonstrated an autocrine phenomenon of EC sensitization. The fact that high concentrations of VEGF may be achieved in vivo by local continuous overexpression of VEGF(165) by gene transfer emphasizes the potential advantage of gene transfer over protein supplementation for therapeutic angiogenesis.

Vascular endothelial growth factor receptor-1 and neuropilin-2 form complexes.

The products of the neuropilin-1 (Np-1) and neuropilin-2 (Np-2) genes are receptors for factors belonging to the class 3 semaphorin family and participate in the guidance of growing axons to their targets. In the presence of heparin-like molecules, both receptors also function as receptors for the heparin-binding 165-amino acid isoform of vascular endothelial growth factor (VEGF(165)). Both receptors are unable to bind to the 121-amino acid isoform of vascular endothelial growth factor (VEGF(121)), which lacks a heparin-binding domain. Interestingly, complexes corresponding in size to (125)I-VEGF(121).neuropilin complexes are formed when (125)I-VEGF(121) is bound and cross-linked to porcine aortic endothelial cells co-expressing VEGFR-1 and either Np-1 or Np-2. These complexes do not seem to represent complexes of (125)I-VEGF(121) with a truncated form of VEGFR-1, presumably formed as a result of the presence of Np-1 or Np-2 in the cells, because such truncated forms could not be detected with anti-VEGFR-1 antibodies. Antibodies directed against VEGFR-1 co-immunoprecipitated the (125)I-VEGF(121).Np-2 sized cross-linked complex along with (125)I-VEGF(121).VEGFR-1 complexes from cells expressing both VEGFR-1 and Np-2 but not from control cells, indicating that VEGFR-1 and Np-2 associate with each other. To perform the reciprocal experiment we have expressed in porcine aortic endothelial cells a Np-2 receptor containing an in-frame myc epitope at the C terminus. Surprisingly, the myc-tagged Np-2 receptor lost most of its VEGF(165) binding capacity but not its semaphorin-3F binding ability. Nevertheless, when Np-2myc was co-expressed in cells with VEGFR-1, it partially regained its VEGF(165) binding ability. Antibodies directed against the myc epitope co-immunoprecipitated (125)I-VEGF(165).Np-2myc and (125)I- VEGF(165).VEGFR-1 complexes from cells co-expressing VEGFR-1 and Np-2myc, indicating again that VEGFR-1 associates with Np-2. Our experiments therefore indicate that Np-2, and possibly also Np-1, associate with VEGFR-1 and that such complexes may be part of a cell membrane-associated signaling complex.

Isolation and characterization of mitochondrial F(1)-ATPase from crayfish (Orconectes virilis) gills.

A soluble F(1)-ATPase was isolated from the mitochondria of crayfish (Orconectes virilis) gill tissue. The maximal mitochondrial disruption rate (95%) was obtained by sonicating for 4 min at pH 8.6. A 15-fold purification was estimated. The properties for both soluble and membrane-bound enzyme were studied. Both enzyme forms were stable at 4 to -70 degrees C when kept in 20% glycerol. Soluble F(1)-ATPase was more stable at room temperature than membrane-bound enzyme. It displayed a narrower pH profile (pK(1) =6.58, pK(2)=7.68) and more acid pH optimum (7.13) than membrane-bound enzyme (pK(1)=6.42, pK(2)=8.55, optimum pH 7.49). The anion-stimulated activities were in the order HCO(3)(-)>SO(4)(2-)>Cl(-). The apparent K(a) values for soluble enzyme were 11.4, 11.2, and 10.9 mM, respectively, but the K(a) of HCO(3)(-) for membrane-bound enzyme (14.9 mM) was higher than for soluble enzyme. Oligomycin and DCCD inhibited membrane-bound F(1)-ATPase with I(50) of 18.6 ng/ml and 2.2 microM, respectively, but were ineffective in inhibiting soluble enzyme. Both enzyme forms shared identical sensitivity to DIDS (I(50)=12.5 microM) and vanadate (I(50)=9.0 mM). Soluble ATPase was significantly more sensitive to pCMB (I(50)=0.15 microM) and NO(3)(-) (I(50)=28.6 mM) than membrane-bound enzyme (I(50)=1.04 microM pCMB and 81.5 mM NO(3)(-)). In addition, soluble F(1)-ATPase was slightly more sensitive to azide (I(50)=91.8 microM) and NBD-Cl (I(50)=9.18 microM) than membrane-bound enzyme (I(50)=111.6 microM azide and 12.88 microM NBD-Cl). These data suggest a conformational change transmission between F(0) and F(1) sectors and slight conformational differences between soluble F(1) and membrane-bound F(1). In addition, an unmodified F(0) stabilizes F(1) and decreases F(1) sensitivities to inhibitors and modulators.

Kinetic studies on mitochondrial F(1)-ATPase from crayfish (Orconectes virilis) gills.

The substrate kinetics and the role of free Mg(2+) and free ATP were studied in membrane-bound F(1)-ATPase from crayfish (Orconectes virilis) gills. It was shown that the MgATP complex was the true substrate for the ATPase activity with a K(m) value of 0.327 mM. In the absence of bicarbonate, the maximum azide-sensitive activities in the presence and absence (<18 microM) of free ATP were 0.878 and 0.520 micromol P(i)/mg protein/min, respectively, while the maximum bicarbonate-stimulated activity in absence of free ATP was 1.486 micromol P(i)/mg protein/min. Free ATP was a competitive inhibitor (K(i)=0.77 mM) and free Mg(2+) was a mixed inhibitor (K(i)=0.81 mM, K(i)'=5.89 mM). However, free ATP also acted as an activator. Lineweaver-Burk plots for MgATP hydrolysis at high free Mg(2+) concentrations exhibited an apparent negative cooperativity, which was not the case for high free ATP levels. These results suggest that, although free ATP inhibited the enzyme by binding to catalytic sites, it stimulated ATPase activity by binding to non-catalytic sites and promoted the dissociation of inhibitory MgADP from the catalytic site.

Sequential V(A)/Q distributions in the normal rabbit by micropore membrane inlet mass spectrometry.

We developed micropore membrane inlet mass spectrometer (MMIMS) probes to rapidly measure inert-gas partial pressures in small blood samples. The mass spectrometer output was linearly related to inert-gas partial pressure (r(2) of 0.996-1.000) and was nearly independent of large variations in inert-gas solubility in liquid samples. We infused six inert gases into five pentobarbital-anesthetized New Zealand rabbits and used the MMIMS system to measure inert-gas partial pressures in systemic and pulmonary arterial blood and in mixed expired gas samples. The retention and excretion data were transformed into distributions of ventilation-to-perfusion ratios (V(A)/Q) with the use of linear regression techniques. Distributions of V(A)/Q were unimodal and broad, consistent with prior reports in the normal rabbit. Total blood sample volume for each VA/Q distribution was 4 ml, and analysis time was 8 min. MMIMS provides a convenient method to perform the multiple inert-gas elimination technique rapidly and with small blood sample volumes.

The VEGF splice variants: properties, receptors, and usage for the treatment of ischemic diseases.

Vascular endothelial growth factor (VEGF) was discovered 10 years ago as a growth factor that can regulate angiogenesis and in addition the permeability of blood vessels. Numerous studies have revealed that it is essential for normal embryonic development and that it plays a major role in physiological and pathological events of angiogenesis in adults. It is unique in that its expression is regulated directly by hypoxia. These properties are now being exploited in attempts aimed at the induction of new blood vessels in pathological situations such as ischemic heart disease. Five VEGF forms of 121 to 206 aminoacids are produced from a single gene by alternative splicing. Cells expressing VEGF usually express several forms simultaneously. VEGF121 does not contain exons 6 and 7 of the gene and consequently lacks a heparin binding ability. However, this form is fully active as an inducer of angiogenesis, and as a blood vessel permeabilizing agent. Exon 6 and 7 contain 2 independent heparin binding domains. The VEGF form containing exon 7 (VEGF165) and the vascular endothelial growth factor form containing exon 6 (VEGF145) display similar biological potencies raising the question of why so many VEGF forms are required. It was found that VEGF121 diffuses better because it does not bind to heparan-sulfate proteoglycans. In contrast, VEGF145 binds to extracellular matrix and is released from it slowly. When the receptor binding properties of VEGF121 and VEGF165 were compared it was found that VEGF165 binds to a class of VEGF receptors that is not recognized by VEGF121. These receptors are encoded by the neuropilin-1 gene, and we have recently found that the related neuropilin-2 gene also encodes a VEGF165 receptor. We have recently found evidence indicating the neuropilins form complexes with another VEGF receptor, VEGFR-1. However, the biological function of this complex remains to be elucidated.

Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms VEGF-145 and VEGF-165 [corrected].

Neuropilin-1 (np-1) and neuropilin-2 (np-2) are receptors for axon guidance factors belonging to the class 3 semaphorins. np-1 also binds to the 165-amino acid heparin-binding form of VEGF (VEGF(165)) but not to the shorter VEGF(121) form, which lacks a heparin binding ability. We report that human umbilical vein-derived endothelial cells express the a17 and a22 splice forms of the np-2 receptor. Both np-2 forms bind VEGF(165) with high affinity in the presence of heparin (K(D) 1.3 x 10(-10) m) but not VEGF(121). np-2 also binds the heparin-binding form of placenta growth factor. These binding characteristics resemble those of np-1. VEGF(145) is a secreted heparin binding VEGF form that contains the peptide encoded by exon 6 of VEGF but not the peptide encoded by exon 7, which is present in VEGF(165). VEGF(145) binds to np-2 with high affinity (K(D) 7 x 10(-10) m). Surprisingly, VEGF(145) did not bind to np-1. Indeed, VEGF(145) does not bind to MDA-MB-231 breast cancer cells, which predominantly express np-1. By contrast, VEGF(145) binds to human umbilical vein-derived endothelial cells, which express both np-1 and np-2. The binding of VEGF(165) to porcine aortic endothelial cells expressing recombinant np-2 did not affect the proliferation or migration of the cells. Nevertheless, it is possible that VEGF-induced np-2-mediated signaling will take place only in the presence of other VEGF receptors such as VEGF receptor-1 or VEGF receptor-2.

Airway cross section strongly influences alveolar plateau slope of capnograms for smaller tidal volumes.

We compared the predictions of the single path convection-diffusion model (SPM) and the well-mixed single acinus model (SAM) with the normalized slopes (NS) of experimentally measured volumetric capnograms in which VT was varied in three healthy spontaneously breathing adults. For values of VT greater than 15 ml/kg, the tidal volume penetrates deep into the acinar airways, mixing by molecular diffusion is rapid and the predictions of the SAM and SPM both agree with the experiment. The SPM however shows much better agreement with the experimental NS data than does the SAM for values of VT less than 10 ml/kg. The explanation for the departure of the SAM from the observed experimental data, at small VT, is that it represents the limiting case (of well-mixed alveolar gas) for the SPM, only at large VT, where the assumption of rapid mixing is most accurate. We conclude that in general, gas phase diffusivity and total acinar airway cross sectional area variation with cumulative volume into the lung are essential to realistically model airway gas exchange between VT and FRC and to obtain agreement with experimental data under the widest range of breathing conditions.

Atypical antipsychotics in older adults.

Psychotic symptoms are common in older adults and reflect a variety of psychiatric and medical conditions. Antipsychotic drugs form the core of the treatment of these symptoms; however, treatment of the elderly is complicated by a high frequency of comorbid medical illnesses, risk of side effects, and age-related changes in pharmacodynamics and pharmacokinetics. The superior safety and efficacy of atypical antipsychotics makes them first-line agents for managing psychotic patients with schizophrenia. Their uses now extend to other conditions such as schizoaffective disorders, delusional disorder, and mood disorders with psychotic features. Although the drugs have been studied extensively in young subjects, well-designed, double-blind, placebo-controlled studies are relatively lacking in the elderly. Our knowledge of their safety, efficacy and dosage in older adults is based on a few studies with small samples or extrapolated from studies of younger patients. Several psychiatric and medical conditions that are associated with psychotic symptoms in older people are reviewed, as well as how these patients may benefit from treatment with these agents.

Effect of vascular endothelial growth factor on hepatic regenerative activity following partial hepatectomy in rats.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) is an angiogenic factor with a growth-promoting effect that is thought to be restricted to vascular endothelial cells. Its essential role during liver regeneration has yet to be determined. The aim of this study was to document the effect of exogenous VEGF administration on liver regeneration in rats undergoing submaximal hepatic resections. METHODS: Adult male Sprague-Dawley rats (n = 4/group) undergoing 30% partial hepatectomy were administered 200 ng VEGF165 intravenously and were sacrificed at 24, 36, and 48 h postoperatively. Liver regeneration was monitored by measuring the restituted liver mass, proliferating cell nuclear antigen (PCNA) immunostaining, and hepatic PCNA protein by Western blot. RESULTS: Changes in restituted liver mass 48 h postsurgery were more prominent, but did not differ statistically between VEGF-treated and control rats (47% vs. 29%; p<0.06). Nevertheless, PCNA immunostaining showed increased labeling index of hepatocytes, apparent at 36 and 48 h after partial hepatectomy (38% vs. 18% [p<0.041 and 42% vs. 11% [p<0.021], respectively). Hepatic PCNA proteins measured by Western blot showed a 3-fold increase in VEGF-treated rats 48 h postsurgery compared with controls (p<0.01). CONCLUSION: Exogenous VEGF administration early after partial hepatectomy stimulates liver regeneration in rats. Whether or not VEGF165 is a direct mitogen for hepatocytes remains to be determined.

Glypican-1 is a VEGF165 binding proteoglycan that acts as an extracellular chaperone for VEGF165.

Glypican-1 is a member of a family of glycosylphosphatidylinositol anchored cell surface heparan sulfate proteoglycans implicated in the control of cellular growth and differentiation. The 165-amino acid form of vascular endothelial growth factor (VEGF165) is a mitogen for endothelial cells and a potent angiogenic factor in vivo. Heparin binds to VEGF165 and enhances its binding to VEGF receptors. However, native HSPGs that bind VEGF165 and modulate its receptor binding have not been identified. Among the glypicans, glypican-1 is the only member that is expressed in the vascular system. We have therefore examined whether glypican-1 can interact with VEGF165. Glypican-1 from rat myoblasts binds specifically to VEGF165 but not to VEGF121. The binding has an apparent dissociation constant of 3 x 10(-10) M. The binding of glypican-1 to VEGF165 is mediated by the heparan sulfate chains of glypican-1, because heparinase treatment abolishes this interaction. Only an excess of heparin or heparan sulfates but not other types of glycosaminoglycans inhibited this interaction. VEGF165 interacts specifically not only with rat myoblast glypican-1 but also with human endothelial cell-derived glypican-1. The binding of 125I-VEGF165 to heparinase-treated human vascular endothelial cells is reduced following heparinase treatment, and addition of glypican-1 restores the binding. Glypican-1 also potentiates the binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1. Furthermore, we show that glypican-1 acts as an extracellular chaperone that can restore the receptor binding ability of VEGF165, which has been damaged by oxidation. Taken together, these results suggest that glypican-1 may play an important role in the control of angiogenesis by regulating the activity of VEGF165, a regulation that may be critical under conditions such as wound repair, in which oxidizing agents that can impair the activity of VEGF are produced, and in situations were the concentrations of active VEGF are limiting.

Vascular endothelial growth factor (VEGF) and its receptors.

Vascular endothelial growth factor (VEGF) is a highly specific mitogen for vascular endothelial cells. Five VEGF isoforms are generated as a result of alternative splicing from a single VEGF gene. These isoforms differ in their molecular mass and in biological properties such as their ability to bind to cell-surface heparan-sulfate proteoglycans. The expression of VEGF is potentiated in response to hypoxia, by activated oncogenes, and by a variety of cytokines. VEGF induces endothelial cell proliferation, promotes cell migration, and inhibits apoptosis. In vivo VEGF induces angiogenesis as well as permeabilization of blood vessels, and plays a central role in the regulation of vasculogenesis. Deregulated VEGF expression contributes to the development of solid tumors by promoting tumor angiogenesis and to the etiology of several additional diseases that are characterized by abnormal angiogenesis. Consequently, inhibition of VEGF signaling abrogates the development of a wide variety of tumors. The various VEGF forms bind to two tyrosine-kinase receptors, VEGFR-1 (flt-1) and VEGFR-2 (KDR/flk-1), which are expressed almost exclusively in endothelial cells. Endothelial cells express in addition the neuropilin-1 and neuropilin-2 coreceptors, which bind selectively to the 165 amino acid form of VEGF (VEGF165). This review focuses on recent developments that have widened considerably our understanding of the mechanisms that control VEGF production and VEGF signal transduction and on recent studies that have shed light on the mechanisms by which VEGF regulates angiogenesis.

Neuropilin-1 is a placenta growth factor-2 receptor.

Placenta growth factor (PlGF) belongs to the family of vascular endothelial growth factors (VEGFs). It binds to the flt-1 VEGF receptor but not to the KDR/flk-1 receptor which is thought to mediate most of the angiogenic and proliferative effects of VEGF. Three PlGF isoforms are produced by alternative splicing. PlGF-1 and PlGF-3 differ from PlGF-2 since they lack the exon 6 encoded peptide which bestows upon PlGF-2 its heparin binding properties. Cross-linking experiments revealed that 125I-PlGF-2 binds to two endothelial cell surface receptors in a heparin dependent fashion. The binding of 125I-PlGF-2 to these receptors was inhibited by an excess of PlGF-2 and by the 165-amino acid form of VEGF (VEGF165), but not at all by VEGF121 and very marginally if at all by PlGF-1. The apparent molecular weight and the binding characteristics of these receptors correspond to those of the recently identified VEGF165 specific receptor neuropilin-1, and we therefore conclude that neuropilin-1 is a receptor for PlGF-2. The binding of 125I-PlGF-2 as well as the binding of 125I-VEGF165 to these receptors was inhibited by a synthetic peptide derived from exon 6 of PlGF. Furthermore, the binding of 125I-PlGF-2, but not that of 125I-VEGF165, was also inhibited by a synthetic peptide derived from exon 7 of PlGF. These observations indicate that the peptides encoded by these exons probably participate in the formation of the domain which mediates the binding of PlGF-2 to these receptors. We have also determined, using chemically modified heparin species, that the presence of sulfate moieties on the glucosamine-O-6 and on the iduronic acid-O-2 groups of heparin was required for the potentiation of 125I-PlGF-2 binding to these receptors. To determine if PlGF-2 is able to induce biological responses that are not induced by PlGF-1, we compared the effects of PlGF-1 and PlGF-2 on the migration and proliferation of endothelial cells. Both PlGF forms induced migration of endothelial cells. However, there was no quantitative difference between the response to PlGF-2 and the response to PlGF-1. Furthermore, neither PlGF-1 nor PlGF-2 had any effect upon the proliferation of the endothelial cells.

Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF), a major regulator of angiogenesis, binds to two receptor tyrosine kinases, KDR/Flk-1 and Flt-1. We now describe the purification and the expression cloning from tumor cells of a third VEGF receptor, one that binds VEGF165 but not VEGF121. This isoform-specific VEGF receptor (VEGF165R) is identical to human neuropilin-1, a receptor for the collapsin/semaphorin family that mediates neuronal cell guidance. When coexpressed in cells with KDR, neuropilin-1 enhances the binding of VEGF165 to KDR and VEGF165-mediated chemotaxis. Conversely, inhibition of VEGF165 binding to neuropilin-1 inhibits its binding to KDR and its mitogenic activity for endothelial cells. We propose that neuropilin-1 is a novel VEGF receptor that modulates VEGF binding to KDR and subsequent bioactivity and therefore may regulate VEGF-induced angiogenesis.