Vascular Epidermal Growth Factor Receptor Research Articles

VEGF and Pleiotrophin Modulate the Immune Profile of Breast Cancer

Angiogenesis, the sprouting of the existing vascular network to form new vessels, is required for the growth of solid tumors. For this reason, the primary stimulant of angiogenesis, vascular endothelial growth factor-A (VEGF), is an attractive target for tumor therapy. In fact, there are currently numerous anti-VEGF therapies in clinical development for the treatment of various cancers, including breast cancer. VEGF signals through two primary VEGF receptors, VEGFR1 and VEGFR2. VEGFR2 is the primary angiogenic receptor, and VEGFR1 has been implicated in macrophage chemotaxis and tumor cell survival and invasion. It has only been appreciated recently that the VEGFRs are expressed not only on endothelial cells and tumor cells but also on many host immune cells. Therefore, to better understand the effects of anti-VEGF therapy it is important to consider the effects of VEGF on all cells in the tumor microenvironment, including immune cells. Bevacizumab (Avastin®, Genetech), which binds VEGF and inhibits interaction with VEGFR1 and VEGFR2, was approved for the treatment of metastatic HER2/NEU-negative breast cancer in 2008, however, the majority of human mammary tumors are either innately resistant or will acquire resistance to anti-VEGF therapy. This suggests that these tumors activate alternate angiogenesis pathways. Pleiotrophin (PTN) is an important angiogenic cytokine in breast cancer and is expressed at high levels in approximately 60% of human breast tumors. PTN functions as an angiogenic factor and promotes remodeling of the tumor microenvironment as well as epithelial-mesenchymal transition (EMT). In addition, PTN can have profound effects on macrophage phenotype. The present review focuses on the functions of VEGF and PTN on immune cell infiltration and function in breast cancer. Furthermore, we will discuss how anti-VEGF therapy modulates the immune cell profile.

TRANSLATIONAL IMPACT

von Hippel-Lindau disease (VHL) is a rare, genetic multisystem disorder characterized by the abnormal growth of blood vessels and tumors. Between 60% and 80% of all patients have benign hemangioblastomas of the retina and central nervous system (CNS) that frequently cause neurological morbidity and mortality. VHL patients can also develop retinal neovascularization (also termed angiogenesis; the formation of new blood vessels), similar to diabetic retinopathy and age-related macular degeneration. Angiogenic retinopathies exhibit vascular leakage, macular edema (fluid accumulation) and detachment of the retina, and are the biggest cause of blindness in the Western world, with very limited treatment options. Unfortunately, the current models for angiogenesis do not fully recapitulate the complexity of angiogenic-related disorders. For instance, hemangioblastomas of the retina and CNS have not been found in any of the VHL mouse models.The VHL tumor suppressor gene, VHL, is crucial for oxygen homeostasis. Its loss causes upregulation of HIF (hypoxia-inducible factor), a transcription factor with a central role in oxygen-regulated gene expression, which in turn increases the expression of hypoxia-inducible mRNAs, including those encoding vascular endothelial growth factor A (VEGFA), VEGFA receptors, and proteins involved in the HIF signaling pathwayIn this study, the authors show that zebrafish vhl mutants provide an in vivo model for pathological angiogenesis and vascular retinopathies. Using blood vessel-specific transgenes, they show that vhl mutants develop severe neovascularization in the brain, eye and trunk. The retinal vasculature of vhl−/− zebrafish is leaky, with severe macular edema and retinal detachment, mimicking human angiogenic retinopathies. As in humans, the zebrafish VEGFA orthologs, Vegfaa/Vegfab and Vegfb, are upregulated by loss of vhl, and are key effectors of the vhl−/− angiogenic phenotype; treatment with VEGF receptor inhibitors can block pathological angiogenesis in mutant fish.These zebrafish vhl mutants can be used to understand the mechanisms underlying the development of vascular lesions in VHL disease, and are a unique and clinically relevant angiogenesis model, as pathological angiogenesis and vascular retinopathies can be studied non-invasively under normoxic conditions. Zebrafish vhl mutant embryos with fluorescently-labeled blood vessels are also a cost-effective and efficient model for whole organism screening of anti-angiogenic pharmacological agents and combinatorial therapies.

Abstract A108: A phase I and pharmalogical study of the broad-spectrum tyrosine kinase inhibitor JNJ-26483327, administered to subjects with advanced or refractory solid tumors

Abstract Background: JNJ-26483327 is a potent reversible multi-targeted tyrosine kinase inhibitor. Tyrosine kinases of Epidermal Growth Factor Receptor (EGFR), Her2, Her4, RET receptor, Vascular Epidermal Growth Factor Receptor (VEGFR)-3, and Src family (Lyn, Fyn, Yes) are inhibited at low nanomolar concentrations. Methods: Objectives were to (1) determine safety, maximum tolerated dose (MTD) and dose-limiting toxicity (DLT), (2) characterize the pharmacokinetic (PK) profile, (3) explore preliminary evidence of antitumor activity, and (4) explore pharmacodynamic (PD) effects in skin biopsies and blood. Results: 19 patients, 16 M/3F, median age 61 yrs (47-74) received JNJ-26483327 twice daily (BID) continuously at 100 mg (n=1), 200 mg (n=1), 400 mg (n=1), 800 mg (n=3), 1200 mg (n=3), 1500 mg (n=6) and 2100 mg (n=4). JNJ-26483327 was administered as oral solution (dose ≤ 1200 mg) or capsules (dose ≥ 1500 mg, 300 mg capsule). Skin biopsies were taken at days 0 and 28. At 2100 mg DLT consisted of a combination of grade 3 anorexia and fatigue in one patient, and due to substantial difficulty in intake in most patients, pill load at 2100 mg was dose limiting. JNJ-26483327 was well tolerated up to 1500 mg BID, with most common reported toxicities being nausea, vomiting, diarrhea, fatigue, skin rash. Plasma concentrations of JNJ-26483327 rapidly increased till about 3–4h post dose. PK showed significant inter-individual variability, but no marked deviation in dose-proportionality was observed up to 1500 mg BID. At 1500 mg BID, steady state plasma concentrations were 2–6 µg/l and AUCs 13–53 µg.h/ml and were in the active range as observed in xenograft mouse models. PD evaluation failed to show a substantial effect on expression of Ki-67, p27kip1, pMAPK, pAKT, and EGFR in paired skin biopsies. Serum levels of sVEGFR-2, VEGF-C and VEGF-D remained unchanged. No antitumor activity was observed in this study. Conclusion: The recommended dose of JNJ-26483327 is 1500 mg BID. JNJ-26483327 shows a predictable pharmacokinetic profile. Additional PD research is planned to demonstrate biological activity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A108.

Vascular endothelial growth factor A and its two receptors in human preantral follicles from fetuses, girls, and women

To investigate the expression of vascular endothelial growth factor A (VEGF-A) and that its two receptors (VEGFR1, VEGFR2) in human preantral follicles. Immunohistochemical, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR) study of the expression of the VEGF-A system in human ovaries. Major tertiary-care academic center. Twenty-two patients who underwent pregnancy terminations at 21-35 gestational weeks and 29 girls/women aged 5-39 years who underwent ovarian laparoscopies. None. Laboratory analysis of human ovarian specimens. Immunhistochemistry and in situ hybridization revealed the expression of the proteins and mRNA transcripts for VEGFR1 and VEGFR2 in oocytes, granulosa cells, and stroma cells from fetuses and girls/women. The protein for VEGF-A was detected immunohistochemically in oocytes, granulosa cells, and stroma cells from fetuses and girls. VEGF-A and VEGFR1 proteins were expressed more strongly than VEGFR2. VEGF-A(121), VEGF-A(165), and VEGF-A(189) isoforms were identified by RT-PCR in the ovarian samples from fetuses and women. The presence of the VEGF-A receptors, particularly in the granulosa cells, suggests that VEGF-A might be involved in proliferation initiation of primordial follicles or play an as yet unknown role in human preantral follicles.

Use of Proximity Ligation to Screen for Inhibitors of Interactions between Vascular Endothelial Growth Factor A and Its Receptors

Improved methods are required to screen drug candidates for their influences on protein interactions. There is also a compelling need for miniaturization of screening assays, with attendant reductions in reagent consumption and assay costs. We used sensitive, miniaturized proximity ligation assays (PLAs) to monitor binding of vascular endothelial growth factor A (VEGF-A) to 2 of its receptors, VEGFR-1 and VEGFR-2. We measured the effects of proteins and low molecular weight compounds capable of disrupting these interactions and compared the results with those obtained by immunoblot analysis. We analyzed 6 different inhibitors: a DNA aptamer, a mixed DNA/RNA aptamer, a monoclonal VEGF-A neutralizing antibody, a monoclonal antibody directed against VEGFR-2, a recombinant competitive protein, and a low molecular weight synthetic molecule. The PLAs were successful for monitoring the formation and inhibition of VEGF-A-receptor complexes, and the results correlated well with those obtained by measuring receptor phosphorylation. The total PLA time is just 3 hours, with minimal manual work and reagent additions. The method allows evaluation of the apparent affinity [half-maximal inhibitory concentration (IC(50))] from a dose-response curve. The PLA may offer significant advantages over conventional methods for screening the interactions of ligands with their receptors. The assay may prove useful for parallel analyses of large numbers of samples in the screening of inhibitor libraries for promising agents. The technique provides dose-response curves, allowing IC(50) values to be calculated.

Second-Generation Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-small Cell Lung Cancer

Approximately 10 to 15% of patients with non-small cell lung cancer have tumors that depend on activation of the epidermal growth factor receptor (EGFR), as evidenced by mutations in EGFR. In these patients, there is often an initial dramatic response to treatment with the first-generation EGFR tyrosine kinase inhibitors (TKIs) erlotinib or gefitinib. A small number of patients with EGFR mutations have primary resistance to erlotinib and gefitinib, and most patients who initially respond to treatment with erlotinib or gefitinib will develop resistance to first-generation EGFR TKIs. The problems with both primary and acquired resistance to erlotinib and gefitinib support the need for development of additional agents that inhibit EGFR signaling in such patients. This is an overview of three representative second-generation EGFR TKIs. HKI-272, a second-generation irreversible EGFR TKI that also inhibits HER2, has completed accrual of a phase II trial in both untreated patients and patients with acquired resistance to erlotinib or gefitinib. XL647 is a reversible inhibitor of EGFR, HER2, and vascular epidermal growth factor receptor. Preclinical work shows that XL647 can inhibit cell lines bearing mutated forms of EGFR that have been associated with acquired resistance. BIBW2992 is an irreversible EGFR TKI that also inhibits HER2 and vascular epidermal growth factor receptors. In vitro work shows that this compound inhibits wild-type EGFR, EGFR exon 19 deletion, EGFR L858R, and EGFR T790M, the mutation associated with acquired resistance. The preliminary results from phase I and phase II trials for BIBW-2992 and XL647 are discussed.

Cellular distribution of vascular endothelial growth factor A (VEGFA) and B (VEGFB) and VEGF receptors 1 and 2 in focal cortical dysplasia type IIB

Members of the vascular endothelial growth factor (VEGF) family are key signaling proteins in the induction and regulation of angiogenesis, both during development and in pathological conditions. However, signaling mediated through VEGF family proteins and their receptors has recently been shown to have direct effects on neurons and glial cells. In the present study, we immunocytochemically investigated the expression and cellular distribution of VEGFA, VEGFB, and their associated receptors (VEGFR-1 and VEGFR-2) in focal cortical dysplasia (FCD) type IIB from patients with medically intractable epilepsy. Histologically normal temporal cortex and perilesional regions displayed neuronal immunoreactivity (IR) for VEGFA, VEGFB, and VEGF receptors (VEGFR-1 and VEGFR-2), mainly in pyramidal neurons. Weak IR was observed in blood vessels and there was no notable glial IR within the grey and white matter. In all FCD specimens, VEGFA, VEGFB, and both VEGF receptors were highly expressed in dysplastic neurons. IR in astroglial and balloon cells was observed for VEGFA and its receptors. VEGFR-1 displayed strong endothelial staining in FCD. Double-labeling also showed expression of VEGFA, VEGFB and VEGFR-1 in cells of the microglia/macrophage lineage. The neuronal expression of both VEGFA and VEGFB, together with their specific receptors in FCD, suggests autocrine/paracrine effects on dysplastic neurons. These autocrine/paracrine effects could play a role in the development of FCD, preventing the death of abnormal neuronal cells. In addition, the expression of VEGFA and its receptors in glial cells within the dysplastic cortex indicates that VEGF-mediated signaling could contribute to astroglial activation and associated inflammatory reactions.

Semaphorin3a disrupts podocyte foot processes causing acute proteinuria

Semaphorin3a was discovered as a secreted guidance protein that acts as a chemorepellent to migrating axons and endothelial cells. In the adult mouse kidney, it is expressed in podocytes and collecting tubules. Here, we show that exogenous semaphorin3a caused acute nephrotic range proteinuria associated with podocyte foot process effacement and fusion, endothelial cell damage, decreased vascular endothelial growth factor-A receptor expression, and downregulation of the slit-diaphragm proteins podocin, nephrin, and CD2-associated protein. When vascular endothelial growth factor 165 was administered at the same time as Semaphorin3a, no proteinuria or renal ultrastructural abnormalities occurred, suggesting that semaphorin3a effects may be mediated, in part, by downregulation of vascular endothelial growth factor receptor 2 signaling. Our findings indicate that a balance of semaphorin3a to vascular endothelial growth factor-A may be important for glomerular filtration barrier homeostasis.

Regulation of embryonic lung vascular development by vascular endothelial growth factor receptors, Flk‐1 and Flt‐1

The biological effects of vascular endothelial growth factor A (VEGF-A) are mediated by fetal liver kinase-1 (Flk-1) and fms-like tyrosine kinase-1 (Flt-1). In lung tissue, VEGF-A is diffusely expressed throughout the embryonic stages, whereas the development of vascular endothelial cells is not uniform. Noting the signaling properties of the two receptors, we hypothesized that Flk-1 and Flt-1 regulate the embryonic development of lung vasculature. We herein show the spatiotemporal expression and experimental inhibition of Flk-1 and Flt-1 of embryonic mouse lung tissue. When Flk-1 was predominantly expressed (embryonic day [E] 9.5-E13.5), then vascular endothelial cells actively proliferated. When Flt-1 was enhanced (E14.5-E16.5), these cells less actively proliferated, thereby constituting organized networks. The treatment of cultured lung buds (E11.5) with antisense oligonucleotides complementary to Flk-1 inhibited branching of capillaries and proliferation of endothelial cells. In contrast, the inhibition of Flt-1 promoted the branching of capillaries and enhanced proliferation of endothelial cells. Of interest, inhibition of Flt-1 promoted Flk-1 expression. These results suggest that the two VEGF-A receptors regulate pulmonary vascular development by modulating the VEGF-A signaling.

Integrin α9β1 Directly Binds to Vascular Endothelial Growth Factor (VEGF)-A and Contributes to VEGF-A-induced Angiogenesis

Vascular endothelial growth factor A (VEGF-A) is a potent inducer of angiogenesis. We now show that VEGF-A-induced adhesion and migration of human endothelial cells are dependent on the integrin alpha9beta1 and that VEGF-A is a direct ligand for this integrin. Adhesion and migration of these cells on the 165 and 121 isoforms of VEGF-A depend on cooperative input from alpha9beta1 and the cognate receptor for VEGF-A, VEGF receptor 2 (VEGF-R2). Unlike alpha3beta1or alphavbeta3 integrins, alpha9beta1 was also found to bind the 121 isoform of VEGF-A. This interaction appears to be biologically significant, because alpha9beta1-blocking antibody dramatically and specifically inhibited angiogenesis induced by VEGF-A165 or -121. Together with our previous findings that alpha9beta1 directly binds to VEGF-C and -D and contributes to lymphangiogenesis, these results identify the integrin alpha9beta1 as a potential pharmacotherapeutic target for inhibition of pathogenic angiogenesis and lymphangiogenesis.

Molecular Mechanisms of VEGF-A Action during Tissue Repair

Vascular endothelial growth factor-A (VEGF-A) is a crucial mediator of vascular hyperpermeability, angiogenesis, and inflammation, processes intimately involved in tissue repair. Although much emphasis has been placed on understanding the synthesis and stability of VEGF-A mRNA, relatively little attention has been given to the study of the stability and processing of VEGF-A proteins themselves. In recent years, several studies indicated that VEGF-A protein activity is highly controlled through interaction with angiogenic or non-angiogenic mediators. We analyzed mechanisms that might control extracellular VEGF-A processing during wound repair. First, our studies provide evidence that VEGF-A protein is a target of proteases present in the microenvironment of human chronic non-healing wounds. Serine proteases, in particular plasmin cleave VEGF165 and digested VEGF fragments, showed decreased mitogenic activity. Inactivation of the plasmin cleavage site Arg110/Ala111 preserved the structural integrity and increased the angiogenic potency of VEGF165 when tested in an impaired healing mouse model. Secondly, we identified significantly increased levels of the potent VEGF-A inhibitor, the soluble form of the VEGF receptor VEGFR-1 (sVEGFR-1) in non-healing wounds when compared to healing wounds. Wound closure in healing and non-healing wounds correlated significantly with a decline in sVEGFR-1 levels. These observations support the concept that VEGF-A and VEGF-A receptors are important mediators in tissue repair. Further, our data provide mechanisms how VEGF-A-mediated interactions are disturbed during impaired healing.

Human lactoferin upregulates expression of Vascular Endothelial Growth Factor Receptor 2 and stimulates angiogenesis of endothelial cells

Human lactoferrin (LF) is a heparin- and iron-binding glycoprotein present in epithelial secretion and in the secondary granules of neutrophils. Recent studies have demonstrated that ingested human apolactoferrin (apoLF) significantly enhanced vascular endothelial growth factor A (VEGF-A)-mediated angiogenesis in vivo. In this study, we investigated effects of Lf on expression of VEGFR-2, a receptor for VEGF-A and found that treatment of human umbilical vein endothelial cells with apoLF resulted in a significant increase in the level of VEGFR-2 (KDR/Flk-1), but not VEGFR-1 (Flt-1) mRNAs. Quantitative reverse transcription polymerase chain reaction analysis revealed a time- and dose-dependent up-regulation of mRNA for VEGFR-2. Western blot and cell-based ELISA analysis demonstrated that apoLF stimulated VEGFR-2 protein levels in HUVECs. Human apoLF stimulated VEGF-induced phosporlyation of ERK MAP kinase. Furthermore, we showed that human apoLF promoted endothelial cell migration and proliferation mediated by VEGF-A, while holoLF had no effects. Human apoLF stimulated VEGF-A-mediated angiogenesis in the chick chorioallantoic membrane assay. These findings could explain at least in part the stimulatory effects of apoLf on VEFG-A-mediated angiogenesis. This work was supported by Korea Science and Engineering Foundation and Korean Ministry of Science and Technology.

Changes in Fetal Lung Distension Alter Expression of Vascular Endothelial Growth Factor and Its Isoforms in Developing Rat Lung

Vascular endothelial growth factor A (VEGF-A) is essential for normal pulmonary vascular and parenchymal development. Changes in fetal lung distension profoundly affect lung growth and maturation, including vascular development. To define developmental lung expression of VEGF-A and its receptors and investigate effects of changes in fetal lung distension, we studied fetal rats at embryonic day (ED) 16, 19, and 22, postnatal rats at postnatal day (PD) 5, 10, and 21, and adult rats. We used reverse transcriptase PCR to measure mRNA expression for VEGF-A isoforms (VEGF-A(120), (-144), (-164), and (-188)) and VEGF-A receptors, Flt-1 and Flk-1. With advancing development, mRNA content increased only for VEGF-A(188) (p < 0.05) and for Flt-1 (p < 0.02) and Flk-1 (p < 0.005). As a percentage of total VEGF-A mRNA, VEGF-A(188) (15% at ED 16) increased to become the dominant isoform at PD 21 (40%, p < 0.005) and adulthood; in contrast, there were decreases in both VEGF-A(144) (p < 0.05) and (-120) (p < 0.005). VEGF-A protein was expressed in alveolar epithelium (type I and II cells) and interstitium. Increasing fetal lung distension by tracheal occlusion (TO) accelerated the normal maturational pattern of VEGF-A isoforms and increased VEGF-A protein; decreasing fetal lung distension by congenital diaphragmatic hernia (CDH) retarded the normal developmental pattern and decreased VEGF-A protein. Neither TO nor CDH consistently affected Flt-1 or Flk-1 mRNA content. These results show that mechanical factors significantly affect lung VEGF-A expression and suggest that VEGF-A mediates previously described changes in lung vascular and parenchymal development caused by CDH and by TO.

Role of Vascular Endothelial Growth Factor–A in Recurrent Respiratory Papillomatosis

Vascular endothelial growth factor-A (VEGF-A) is known to play an important role in the angiogenic response essential for tumor growth in a variety of human and experimental tumors. This study was designed to investigate whether VEGF-A may play a role in the pathogenesis of recurrent respiratory papillomatosis (RRP). A retrospective study with institutional review board approval was performed at a tertiary care medical center on 12 patients with a history of laryngeal RRP. Their ages at the time of initial diagnosis ranged from 19 to 96 months (mean, 56 months). All patients had involvement of right and left true vocal cords. All patients required multiple endoscopic procedures (range, 4 to 66; mean, 12). Normal pediatric larynx samples from 5 autopsy patients were used as controls. Formalin-fixed, paraffin-embedded sections of laryngeal squamous papillomas from the 12 patients with a diagnosis of RRP and the 5 control patients were examined by in situ hybridization for the presence of messenger RNA (mRNA) for VEGF-A and vascular endothelial growth factor receptor 1 (VEGFR-1) and vascular endothelial growth factor receptor 2 (VEGFR-2). The biopsy specimens were from the true vocal cord (N = 10) or subglottis (N = 2) in the patients with RRP and consisted of large sections of larynx including the true vocal cord in the control patients (N = 5). Strong expression of VEGF-A mRNA was noted in the squamous epithelium of papillomas of all 12 patients. Strong expression of VEGFR-1 and VEGFR-2 was noted in the endothelial cells of the underlying vessels in all 12 patients. Neither strong labeling of VEGF-A mRNA nor labeling of its receptors wasnoted in the control patients. We conclude that the angiogenic growth factor VEGF-A is strongly expressed in the epithelium of squamous papillomas in RRP. Also, VEGFR-1 and VEGFR-2 mRNAs are strongly expressed by underlying vascular endothelial cells, suggesting an important role in the pathogenesis of RRP.

The VEGF receptor tyrosine kinase inhibitor, ZD6474, inhibits angiogenesis and affects microvascular architecture within an orthotopically implanted renal cell carcinoma

The proangiogenic vascular endothelial growth factor-A (VEGF) is essential for the development of new tumor vessels. ZD6474 is a novel inhibitor of VEGF receptor-2 (VEGFR-2) tyrosine kinase activity, which also has additional activity against epidermal growth factor (EGF) receptor tyrosine kinase. The antitumor activity of different schedules of ZD6474 in a clinically relevant, metastasizing, murine renal cell carcinoma (RENCA) model was evaluated in this study. RENCA cells were inoculated into the left kidney of 24 mice (day 0). Daily ZD6474 (50 mg/kg p.o.) treatment was initiated 1 day or 10 days after tumor cell inoculation and continued until day 21. Following treatment, kidney weight and volume were assessed and blood vessel density determined by CD31 staining. Visible metastases in the lungs, spleen, and lymph nodes were quantified using a dissection microscope. In an additional study, animals were treated according to the same regimen and quantitative three-dimensional microvascular corrosion casting was performed to enable detailed assessment of the tumor vascular architecture. Therapy initiated on day 1 or day 10 resulted in a 79% and 59% reduction in primary tumor volume, a 79% and 60% reduction in the number of lung metastases, and a 58% and 59% reduction in vessel density of primary tumors compared with the control group, respectively. Corrosion casting proved a 5.4- and 3.2-fold lower vascular volume compared with untreated tumors, observations that paralleled with significant architectural alterations. In this RENCA model, ZD6474 was a highly active inhibitor of tumor angiogenesis, primary tumor growth and tumor metastasis.

Polarized Vascular Endothelial Growth Factor Secretion by Human Retinal Pigment Epithelium and Localization of Vascular Endothelial Growth Factor Receptors on the Inner Choriocapillaris: Evidence for a Trophic Paracrine Relation

The retinal pigment epithelium (RPE) maintains the choriocapillaris (CC) in the normal eye and is involved in the pathogenesis of choroidal neovascularization in age-related macular degeneration. Vascular endothelial growth factor-A (VEGF) is produced by differentiated human RPE cells in vitro and in vivo and may be involved in paracrine signaling between the RPE and the CC. We investigated whether there is a polarized secretion of VEGF by RPE cells in vitro. Also, the localization of VEGF receptors in the human retina was investigated. We observed that highly differentiated human RPE cells, cultured on transwell filters in normoxic conditions, produced two- to sevenfold more VEGF toward their basolateral side as compared to the apical side. In hypoxic conditions, VEGF-A secretion increased to the basal side only, resulting in a three- to 10-fold higher basolateral secretion. By immunohistochemistry in 30 human eyes and in two cynomolgus monkey eyes, KDR (VEGFR-2) and flt-4 (VEGFR-3) were preferentially localized at the side of the CC endothelium facing the RPE cell layer, whereas flt-1 (VEGFR-1) was found on the inner CC and on other choroidal vessels. Our results indicate that RPE secretes VEGF toward its basal side where its receptor KDR is located on the adjacent CC endothelium, suggesting a role of VEGF in a paracrine relation, possibly in cooperation with flt-4 and its ligand. This can explain the known trophic function of the RPE in the maintenance of the CC and its fenestrated permeable phenotype and points to a role for VEGF in normal eye functioning. Up-regulated basolateral VEGF secretion by RPE in hypoxia or loss of polarity of VEGF production may play a role in the pathogenesis of choroidal neovascularization.

The angiogenesis induced by HIV–1 Tat protein is mediated by the Flk–1/KDR receptor on vascular endothelial cells

The HIV-1 Tat protein transactivates HIV, viral and some host cell genes. Tat can be released by infected cells and acts extracellularly in the microenvironment, regulating functions of immunocompetent and mesenchymal cells. One of the most striking effects of Tat is the induction of a functional program in vascular cells related to angiogenesis and inflammation (migration, proliferation and expression of plasminogen activator inhibitor-1 and E selectin). Tat induces growth of Kaposi's sarcoma (KS) spindle cells and is angiogenic in vivo and in transgenic mice10-12. We previously reported that Tat is a direct angiogenic factor and noted the Tat arginine- and lysine-rich sequence is similar to that of other potent angiogenic growth factors, such as vascular endothelial growth factor-A (VEGF-A). It is possible that Tat mimics one of these factors by interacting with its growth factor tyrosine kinase receptor. Here we demonstrate that Tat specifically binds and activates the Flk-1/kinase insert domain receptor (Flk-1/KDR), a VEGF-A tyrosine kinase receptor (for review see ref. 13), and that Tat-induced angiogenesis is blocked by agents blocking the Flk-1/KDR receptor. Endothelial cell stimulation by Tat occurs in the absence of activation of FLT-1, another VEGF-A tyrosine kinase receptor.