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1.  Vascular permeability factor/endothelial growth factor (VPF/VEGF): accumulation and expression in human synovial fluids and rheumatoid synovial tissue 
Vascular permeability factor (VPF, also known as vascular endothelial growth factor or VEGF), is a potent microvascular permeability enhancing cytokine and a selective mitogen for endothelial cells. It has been implicated in tumor angiogenesis and ascites fluid accumulation. Since development of the destructive synovial pannus in rheumatoid arthritis (RA) is associated with changes in vascular permeability (synovial fluid accumulation), synovial cell hyperplasia, and angiogenesis, we examined synovial fluids (SFs) and joint tissue for the expression and local accumulation of VPF/VEGF. VPF/VEGF was detected in all of 21 synovial fluids examined and when measured by an immunofluorimetric assay, ranged from 6.9 to 180.5 pM. These levels are biologically significant, since < 1 pM VPF/VEGF can elicit responses from its target cells, endothelial cells. Levels of VPF/VEGF were highest in rheumatoid arthritis fluids (n = 10), with a mean value (+/- SEM) of 59.1 +/- 18.0 pM, vs. 21.4 +/- 2.3 pM for 11 SFs from patients with other forms of arthritis (p = 0.042). In situ hybridization studies that were performed on joint tissues from patients with active RA revealed that synovial lining macrophages strongly expressed VPF/VEGF mRNA, and that microvascular endothelial cells of nearby blood vessels strongly expressed mRNA for the VPF/VEGF receptors, flt-1 and KDR. Immunohistochemistry performed on inflamed rheumatoid synovial tissue revealed that the VPF/VEGF peptide was localized to macrophages within inflamed synovium, as well as to microvascular endothelium, its putative target in the tissue. Together, these findings indicate that VPF/VEGF may have an important role in the pathogenesis of RA.
PMCID: PMC2191547  PMID: 8006592
2.  Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing 
The Journal of Experimental Medicine  1992;176(5):1375-1379.
Persistent microvascular hyperpermeability to plasma proteins even after the cessation of injury is a characteristic but poorly understood feature of normal wound healing. It results in extravasation of fibrinogen that clots to form fibrin, which serves as a provisional matrix and promotes angiogenesis and scar formation. We present evidence indicating that vascular permeability factor (VPF; also known as vascular endothelial growth factor) may be responsible for the hyperpermeable state, as well as the angiogenesis, that are characteristic of healing wounds. Hyperpermeable blood vessels were identified in healing split-thickness guinea pig and rat punch biopsy skin wounds by their capacity to extravasate circulating macromolecular tracers (colloidal carbon, fluoresceinated dextran). Vascular permeability was maximal at 2-3 d, but persisted as late as 7 d after wounding. Leaky vessels were found initially at the wound edges and later in the subepidermal granulation tissue as keratinocytes migrated to cover the denuded wound surface. Angiogenesis was also prominent within this 7-d interval. In situ hybridization revealed that greatly increased amounts of VPF mRNA were expressed by keratinocytes, initially those at the wound edge, and, at later intervals, keratinocytes that migrated to cover the wound surface; occasional mononuclear cells also expressed VPF mRNA. Secreted VPF was detected by immunofluoroassay of medium from cultured human keratinocytes. These data identify keratinocytes as an important source of VPF gene transcript and protein, correlate VPF expression with persistent vascular hyperpermeability and angiogenesis, and suggest that VPF is an important cytokine in wound healing.
PMCID: PMC2119412  PMID: 1402682
3.  Hypoxia-induced paracrine regulation of vascular endothelial growth factor receptor expression. 
Journal of Clinical Investigation  1996;97(2):469-476.
Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF), an endothelial cell (EC)-specific mitogen, stimulates angiogenesis in vivo, particularly in ischemic regions. VEGF/VPF expression by cells of hypoxic tissues coincides with expression of its two receptors, KDR and flt-1, by ECs in the same tissues. We investigated whether hypoxia or hypoxia-dependent conditions operate in coordinating this phenomenon. Human umbilical vein and microvascular ECs were exposed to direct hypoxia or to medium conditioned (CM) by myoblasts maintained in hypoxia for 4 d. Control ECs were maintained in normoxia or normoxia-CM. Binding of 125I-VEGF to ECs was then evaluated. Hypoxic treatment of ECs had no effect on 125I-VEGF binding. However, treatment of ECs with hypoxia-CM produced a threefold increase in 125I-VEGF binding, with peak at 24 h (P < 0.001, ANOVA). Scatchard analysis disclosed that increased binding was due to a 13-fold increase in KDR receptors/cell, with no change in KDR affinity (Kd = 260 +/- 51 pM, normoxia-CM versus Kd = 281 +/- 94 pM, hypoxia-CM) and no change in EC number (35.6 +/- 5.9 x 10(3) ECs/cm2, normoxia-CM versus 33.5 +/- 5.5 x 10(3) ECs/cm2, hypoxia-CM). Similar results were obtained using CM from hypoxic smooth muscle cells. KDR upregulation was not prevented by addition to the hypoxia-CM of neutralizing antibodies against VEGF, tumor necrosis factor-alpha, transforming growth factor beta 1 or basic fibroblast growth factor. Similarly, addition of VEGF or lactic acid to the normoxia-CM had no effect on VEGF binding. We conclude that mechanism(s) initiated by hypoxia can induce KDR receptor upregulation in ECs. Hypoxic cells, normal or neoplastic, not only can produce VEGF/VPF, but can also modulate its effects via paracrine induction of VEGF/VPF receptors in ECs.
PMCID: PMC507039  PMID: 8567969
4.  Distribution of vascular permeability factor (vascular endothelial growth factor) in tumors: concentration in tumor blood vessels 
The Journal of Experimental Medicine  1991;174(5):1275-1278.
Vascular permeability factor (VPF) is a highly conserved 34-42-kD protein secreted by many tumor cells. Among the most potent vascular permeability-enhancing factors known, VPF is also a selective vascular endothelial cell mitogen, and therefore has been called vascular endothelial cell growth factor (VEGF). Our goal was to define the cellular sites of VPF (VEGF) synthesis and accumulation in tumors in vivo. Immunohistochemical studies were performed on solid and ascites guinea pig line 1 and line 10 bile duct carcinomas using antibodies directed against peptides synthesized to represent the NH2-terminal and internal sequences of VPF. These antibodies stained tumor cells and, uniformly and most intensely, the endothelium of immediately adjacent blood vessels, both preexisting and those newly induced by tumor angiogenesis. A similar pattern of VPF staining was observed in autochthonous human lymphoma. In situ hybridization demonstrated VPF mRNA in nearly all line 10 tumor cells but not in tumor blood vessels, indicating that immunohistochemical labeling of tumor vessels with antibodies to VPF peptides reflects uptake of VPF, not endogenous synthesis. VPF protein staining was evident in adjacent preexisting venules and small veins as early as 5 h after tumor transplant and plateaued at maximally intense levels in newly induced tumor vessels by approximately 5 d. VPF-stained vessels were also hyperpermeable to macromolecules as judged by their capacity to accumulate circulating colloidal carbon. In contrast, vessels more than approximately 0.5 mm distant from tumors were not hyperpermeable and did not exhibit immunohistochemical staining for VPF. Vessel staining disappeared within 24-48 h of tumor rejection. These studies indicate that VPF is synthesized by tumor cells in vivo and accumulates in nearby blood vessels, its target of action. Because leaky tumor vessels initiate a cascade of events, which include plasma extravasation and which lead ultimately to angiogenesis and tumor stroma formation, VPF may have a pivotal role in promoting tumor growth. Also, VPF immunostaining provides a new marker for tumor blood vessels that may be exploitable for tumor imaging or therapy.
PMCID: PMC2118980  PMID: 1940805
5.  Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors. 
Molecular Biology of the Cell  1992;3(2):211-220.
Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), increases microvascular permeability and is a specific mitogen for endothelial cells. Expression of VPF/VEGF previously was demonstrated in a variety of tumor cells, in cultures of pituitary-derived cells, and in corpus luteum. Here we present evidence, by Northern analysis and in situ hybridization, that the VPF/VEGF gene is expressed in many adult organs, including lung, kidney, adrenal gland, heart, liver, and stomach mucosa, as well as in elicited peritoneal macrophages. The highest levels of VPF/VEGF transcripts were found in epithelial cells of lung alveoli, renal glomeruli and adrenal cortex, and in cardiac myocytes. The prominence of VPF/VEGF mRNA in these tissues suggests a possible role for VPF/VEGF in regulating baseline microvascular permeability, which is essential for tissue nutrition and waste removal. We also demonstrate particularly high VPF/VEGF mRNA levels in several human tumors, where it may be involved in promoting tumor angiogenesis and stroma generation, both as an endothelial cell mitogen and indirectly by its permeability enhancing effect that leads to the deposition of a provisional fibrin gel matrix.
PMCID: PMC275520  PMID: 1550962
6.  Mechanism of dexamethasone suppression of brain tumor-associated vascular permeability in rats. Involvement of the glucocorticoid receptor and vascular permeability factor. 
Journal of Clinical Investigation  1996;98(6):1400-1408.
Brain tumor-associated cerebral edema arises because tumor capillaries lack normal blood-brain barrier function; vascular permeability factor (VPF, also known as vascular endothelial growth factor, VEGF) is a likely mediator of this phenomenon. Clinically, dexamethasone reduces brain tumor-associated vascular permeability through poorly understood mechanisms. Our goals were to determine if suppression of permeability by dexamethasone might involve inhibition of VPF action or expression, and if dexamethasone effects in this setting are mediated by the glucocorticoid receptor (GR). In two rat models of permeability (peripheral vascular permeability induced by intradermal injection of 9L glioma cell-conditioned medium or purified VPF, and intracerebral vascular permeability induced by implanted 9L glioma), dexamethasone suppressed permeability in a dose-dependent manner. Since 80% of the permeability-inducing activity in 9L-conditioned medium was removed by anti-VPF antibodies, we examined dexamethasone effects of VPF expression in 9L cells. Dexamethasone inhibited FCS- and PDGF-dependent induction of VPF expression. At all levels (intradermal, intracranial, and cell culture), dexamethasone effects were reversed by the GR antagonist mifepristone (RU486). Dexamethasone may decrease brain tumor-associated vascular permeability by two GR-dependent mechanisms: reduction of the response of the vasculature to tumor-derived permeability factors (including VPF), and reduction of VPF expression by tumor cells.
PMCID: PMC507566  PMID: 8823305
7.  Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. 
Journal of Clinical Investigation  1989;84(5):1470-1478.
Vascular permeability factor (VPF) is an Mr 40-kD protein that has been purified from the conditioned medium of guinea pig line 10 tumor cells grown in vitro, and increases fluid permeability from blood vessels when injected intradermally. Addition of VPF to cultures of vascular endothelial cells in vitro unexpectedly stimulated cellular proliferation. VPF promoted the growth of new blood vessels when administered into healing rabbit bone grafts or rat corneas. The identity of the growth factor activity with VPF was established in four ways: (a) the molecular weight of the activity in preparative SDS-PAGE was the same as VPF (Mr approximately 40 kD); (b) multiple isoforms (pI greater than or equal to 8) for both VPF and the growth-promoting activity were observed; (c) a single, unique NH2-terminal amino acid sequence was obtained; (d) both growth factor and permeability-enhancing activities were immunoadsorbed using antipeptide IgG that recognized the amino terminus of VPF. Furthermore, 125I-VPF was shown to bind specifically and with high affinity to endothelial cells in vitro and could be chemically cross-linked to a high-molecular weight cell surface receptor, thus demonstrating a mechanism whereby VPF can interact directly with endothelial cells. Unlike other endothelial cell growth factors, VPF did not stimulate [3H]thymidine incorporation or promote growth of other cell types including mouse 3T3 fibroblasts or bovine smooth muscle cells. VPF, therefore, appears to be unique in its ability to specifically promote increased vascular permeability, endothelial cell growth, and angio-genesis.
PMCID: PMC304011  PMID: 2478587
8.  Mast Cells Can Secrete Vascular Permeability Factor/ Vascular Endothelial Cell Growth Factor and Exhibit Enhanced Release after Immunoglobulin E–dependent Upregulation of Fcε Receptor I Expression  
The Journal of Experimental Medicine  1998;188(6):1135-1145.
Vascular permeability factor/vascular endothelial cell growth factor (VPF/VEGF) can both potently enhance vascular permeability and induce proliferation of vascular endothelial cells. We report here that mouse or human mast cells can produce and secrete VPF/VEGF. Mouse mast cells release VPF/VEGF upon stimulation through Fcε receptor I (FcεRI) or c-kit, or after challenge with the protein kinase C activator, phorbol myristate acetate, or the calcium ionophore, A23187; such mast cells can rapidly release VPF/VEGF, apparently from a preformed pool, and can then sustain release by secreting newly synthesized protein. Notably, the FcεRI-dependent secretion of VPF/VEGF by either mouse or human mast cells can be significantly increased in cells which have undergone upregulation of FcεRI surface expression by a 4-d preincubation with immunoglobulin E. These findings establish that at least one cell type, the mast cell, can be stimulated to secrete VPF/VEGF upon immunologically specific activation via a member of the multichain immune recognition receptor family. Our observations also identify a new mechanism by which mast cells can contribute to enhanced vascular permeability and/or angiogenesis, in both allergic diseases and other settings.
PMCID: PMC2212544  PMID: 9743532
allergy; angiogenesis; c-kit; stem cell factor; vascular permeability
9.  The neurotransmitter dopamine modulates vascular permeability in the endothelium 
Vascular permeability factor/Vascular endothelial growth factor (VPF/VEGF), a multifunctional cytokine, is a potent inducer of vascular permeability, an important early step in angiogenesis. It is known that the neurotransmitter dopamine can inhibit VPF/VEGF mediated angiogenesis, in particular microvascular permeability, but the effectors of this action remain unclear.
Here, we define the signaling pathway modulated by dopamine that inhibits VPF/VEGF induced vascular permeability in endothelial cells. Signals from VPF/VEGF lead to changes in the phosphorylation of tight junction protein zonula occludens (ZO-1) and adherens junction proteins like VE-cadherin and associated catenins, thus weakening endothelial cell-cell adhesion and increasing vascular permeability. We found VEGF receptor-2 (VEGFR-2) to be part of a multi-protein complex involving ZO-1, VE-cadherin and β-catenin. VPF/VEGF induced phosphorylations of VE-cadherin, β-catenin and ZO-1 were inhibited by dopamine treatment. Association of occludin with ZO-1 and ZO-1 with VE-cadherin were significantly inhibited by dopamine in VEGF treated cells. Furthermore, we identified Src as an important target for dopamine-mediated inhibition of VPF/VEGF induced permeability.
Taken together, our results provide molecular insights of dopamine function in the vascular endothelium and suggest a central role of Src in regulating key molecules that control vascular permeability.
PMCID: PMC2516513  PMID: 18662404
10.  Identification of a Human VPF/VEGF 3′ Untranslated Region Mediating Hypoxia-induced mRNA Stability 
Molecular Biology of the Cell  1998;9(2):469-481.
Hypoxia is a prominent feature of malignant tumors that are characterized by angiogenesis and vascular hyperpermeability. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) has been shown to be up-regulated in the vicinity of necrotic tumor areas, and hypoxia potently induces VPF/VEGF expression in several tumor cell lines in vitro. Here we report that hypoxia-induced VPF/VEGF expression is mediated by increased transcription and mRNA stability in human M21 melanoma cells. RNA-binding/electrophoretic mobility shift assays identified a single 125-bp AU-rich element in the 3′ untranslated region that formed hypoxia-inducible RNA-protein complexes. Hypoxia-induced expression of chimeric luciferase reporter constructs containing this 125-bp AU-rich hypoxia stability region were significantly higher than constructs containing an adjacent 3′ untranslated region element without RNA-binding activity. Using UV-cross-linking studies, we have identified a series of hypoxia-induced proteins of 90/88 kDa, 72 kDa, 60 kDa, 56 kDa, and 46 kDa that bound to the hypoxia stability region element. The 90/88-kDa and 60-kDa species were specifically competed by excess hypoxia stability region RNA. Thus, increased VPF/VEGF mRNA stability induced by hypoxia is mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3′ untranslated region and distinct mRNA-binding proteins in human tumor cells.
PMCID: PMC25276  PMID: 9450968
11.  Tissue factor controls the balance of angiogenic and antiangiogenic properties of tumor cells in mice. 
Journal of Clinical Investigation  1994;94(3):1320-1327.
Meth-A sarcoma cells were stable transfected to overexpress (sense construct) or underexpress (antisense construct) tissue factor. In vitro, there was no difference in plating efficiency or growth between these cell lines. In vivo, tumor cells transfected to overexpress tissue factor grew more rapidly, and established larger and more vascularized tumors than control transfectants. Antisense transfectants grew the slowest and were the least vascularized. Anticoagulation of mice with warfarin did not alter the difference between these tumor lines. Tumor cells over-expressing tissue factor released more (compared with control transfectants) mitogenic activity for endothelial cells in parallel with enhanced transcription of vascular permeability factor/vascular endothelial cell growth factor (VEGF/VPF), and diminished transcription of thrombospondin (TSP2), a molecule with anti-angiogenic properties. Antisense tissue factor transfectants, while releasing the lowest amount of mitogenic activity, had increased thrombospondin and decreased VEGF/VPF transcription compared with control transfectants or wild-type cells. Experiments with these sense, antisense, truncated sense, or vector tumor lines gave comparable results in complete medium, serum free medium or in the presence of hirudin, indicating that the activation of the coagulation mechanism was not likely to be responsible for changes in tumor cell properties. These results suggest that tissue factor regulates angiogenic properties of tumor cells by altering the production of growth regulatory molecules of endothelium by a mechanism distinct from tissue factor activation of the coagulation mechanism.
PMCID: PMC295218  PMID: 7521887
12.  Neuropilin-1 Modulates p53/Caspases Axis to Promote Endothelial Cell Survival 
PLoS ONE  2007;2(11):e1161.
Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), one of the crucial pro-angiogenic factors, functions as a potent inhibitor of endothelial cell (EC) apoptosis. Previous progress has been made towards delineating the VPF/VEGF survival signaling downstream of the activation of VEGFR-2. Here, we seek to define the function of NRP-1 in VPF/VEGF-induced survival signaling in EC and to elucidate the concomitant molecular signaling events that are pivotal for our understanding of the signaling of VPF/VEGF. Utilizing two different in vitro cell culture systems and an in vivo zebrafish model, we demonstrate that NRP-1 mediates VPF/VEGF-induced EC survival independent of VEGFR-2. Furthermore, we show here a novel mechanism for NRP-1-specific control of the anti-apoptotic pathway in EC through involvement of the NRP-1-interacting protein (NIP/GIPC) in the activation of PI-3K/Akt and subsequent inactivation of p53 pathways and FoxOs, as well as activation of p21. This study, by elucidating the mechanisms that govern VPF/VEGF-induced EC survival signaling via NRP-1, contributes to a better understanding of molecular mechanisms of cardiovascular development and disease and widens the possibilities for better therapeutic targets.
PMCID: PMC2048754  PMID: 18000534
13.  Src homology 2 (SH2) domain containing protein tyrosine phosphatase-1 (SHP-1) dephosphorylates VEGF Receptor-2 and attenuates endothelial DNA synthesis, but not migration* 
Vascular endothelial growth factor receptor-2 (VEGFR-2, KDR), a receptor tyrosine kinase, regulates mitogenic, chemotactic, hyperpermeability, and survival signals in vascular endothelial cells in response to its ligand vascular permeability factor/ vascular endothelial growth factor (VPF/VEGF). SHP-1 is a protein tyrosine phosphatase known to negatively regulate signaling from receptors such as EGF receptor, IL3 receptor, erythropoietin receptor and also KDR. However, the mechanism by which SHP-1 executes KDR dephosphorylation, the targeted tyrosine residue(s) of KDR and also overall downstream signaling or phenotypic change(s) caused, is not defined.
Here, we have demonstrated that KDR and SHP-1 are constitutively associated and upon VEGF treatment, the phosphatase activity of SHP-1 is stimulated in a c-Src kinase dependent manner. Knockdown of SHP-1 by siRNA or inhibition of c-Src by an inhibitor, results in augmented DNA synthesis perhaps due to increased phosphorylation of at least three tyrosine residues of KDR 996, 1059 and 1175. On the other hand, neither tyrosine residue 951 of KDR nor VEGF-mediated migration is affected by modulation of SHP-1 function.
Taken together our results define the tyrosine residues of KDR that are regulated by SHP-1 and also elucidates a novel feed back loop where SHP-1 is activated upon VEGF treatment through c-Src and controls KDR induced DNA synthesis, eventually leading to controlled angiogenesis.
PMCID: PMC2292718  PMID: 18377662
14.  Vascular Hyperpermeability, Angiogenesis, and Stroma Generation 
It has been known for more than half a century that the tumor microvasculature is hyperpermeable to plasma proteins. However, the identity of the leaky vessels and the consequences of vascular hyperpermeability have received little attention. This article places tumor vascular hyperpermeability in a broader context, relating it to (1) the low-level “basal” permeability of the normal vasculature; (2) the “acute,” short-term hyperpermeability induced by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A) and other vascular permeabilizing agents; and (3) the “chronic” hyperpermeability associated with longer-term exposure to agents such as VPF/VEGF-A that accompanies many types of pathological angiogenesis. Leakage of plasma protein-rich fluids is important because it activates the clotting system, depositing an extravascular fibrin gel provisional matrix that serves as the first step in stroma generation.
Tumor microvasculature is hyperpermeable to plasma proteins. This is often associated with acute release or chronic synthesis and release of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A).
PMCID: PMC3281587  PMID: 22355795
15.  Vesiculo-vacuolar organelles and the regulation of venule permeability to macromolecules by vascular permeability factor, histamine, and serotonin 
The Journal of Experimental Medicine  1996;183(5):1981-1986.
In contrast to normal microvessels, those that supply tumors are strikingly hyperpermeable to circulating macromolecules such as plasma proteins. This leakiness is largely attributable to a tumor-secreted cytokine, vascular permeability factor (VPF). Tracer studies have shown that macromolecules cross tumor vascular endothelium by way of a recently described cytoplasmic organelle, the vesiculo-vacuolar organelle or VVO (VVOs are grapelike clusters of interconnected, uncoated vesicles and vacuoles). However, equivalent VVOs are also present in the cytoplasm of normal venules that do not leak substantial amounts of plasma protein. To explain these findings, we hypothesized that VPF increased the permeability of tumor blood vessels by increasing VVO function and that the VVOs of normal venules were relatively impermeable in the absence of VPF stimulation. To test this hypothesis, VPF was injected intradermally in normal animals after intravenous injection of a soluble macromolecular tracer, ferritin, whose extravasation could be followed by electron microscopy. VPF caused normal venules to leak ferritin, and, as predicted by our hypothesis, ferritin extravasated by way of VVOs, just as in hyperpermeable tumor microvessels. Ultrathin (14-nm) serial electron microscopic sections and computer-aided three-dimensional reconstructions better defined VVO structure. VVOs occupied 16-18% of endothelial cytoplasm in normal venules. Individual VVOs were clusters of numerous (median, 124) interconnected vesicles and vacuoles that formed complex pathways across venular endothelium with multiple openings to both luminal and abluminal surfaces. Like VPF, histamine and serotonin also stimulated ferritin extravasation across venules by way of VVOs. Together, these data establish VVOs as the major pathway by which soluble plasma proteins exit venules in response to several mediators that increase venular hyperpermeability. These same mediators also increased the extravasation of colloidal carbon, but this large particulate nonphysiological tracer exited venules primarily through endothelial gaps.
PMCID: PMC2192559  PMID: 8642308
16.  Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration 
The Journal of Experimental Medicine  1990;172(6):1535-1545.
Systemic infusion of low concentrations of tumor necrosis factor/cachectin (TNF) into mice that bear TNF-sensitive tumors leads to activation of coagulation, fibrin formation, and occlusive thrombosis exclusively within the tumor vascular bed. To identify mechanisms underlying the localization of this vascular procoagulant response, a tumor-derived polypeptide has been purified to homogeneity from supernatants of murine methylcholanthrene A-induced fibrosarcomas that induces endothelial tissue factor synthesis and expression (half- maximal response at approximately 300 pM), and augments the procoagulant response to TNF in a synergistic fashion. This tumor- derived polypeptide was identified as the murine homologue of vascular permeability factor (VPF) based on similar mobility on SDS-PAGE, an homologous NH2-terminal amino acid sequence, and recognition by a monospecific antibody to guinea pig VPF. In addition, VPF was shown to induce monocyte activation, as evidenced by expression of tissue factor. Finally, VPF was shown to induce monocyte chemotaxis across collagen membranes and endothelial cell monolayers. Taken together, these results indicate that VPF can modulate the coagulant properties of endothelium and monocytes, and can promote monocyte migration into the tumor bed. This suggests one mechanism through which tumor-derived mediators can alter properties of the vessel wall.
PMCID: PMC2188755  PMID: 2258694
17.  The genesis of peritumoral vasogenic brain edema and tumor cysts: a hypothetical role for tumor-derived vascular permeability factor. 
Cerebral edema and fluid-filled cysts are common accompaniments of brain tumors. They contribute to the mass effect imposed by the primary tumor and are often responsible for a patient's signs and symptoms. Cerebral edema significantly increases the morbidity associated with tumor biopsy, excision, radiation therapy, and chemotherapy. Both edema and cyst formation are thought to result from a deficiency in the blood-brain barrier, with consequent extravasation of water, electrolytes, and plasma proteins from altered tumor microvessels. The resultant expansion of the cerebral interstitial space contributes to the elevated intracranial pressure observed with brain tumors. Departure from the typical blood-brain barrier microvascular architecture may only partially explain the occurrence of edema and tumor cyst formation. Biochemical mediators have also been implicated in vascular extravasation. Vascular permeability factor or vascular endothelial growth factor (VPF/VEGF) is a protein that has recently been isolated from a variety of tumors including human brain tumors. VPFb is an extraordinarily potent inducer of both microvascular extravasation (edemagenesis) and the formation of new blood vessels (angiogenesis). Its role in tumor growth and progression would therefore appear pivotal. Herein, the author presents an updated account of the investigation of VPF. Historical and clinical perspectives of the study and treatment of tumor associated edema are provided. The efficacy of high-dose dexamethasone in the treatment of neoplastic brain edema is discussed. A hypothetical role for VPF in edemagenesis is presented and discussed. It is hoped that an expanded understanding of the mechanisms responsible for the genesis of edema will ultimately facilitate therapeutic intervention.
PMCID: PMC2588896  PMID: 7516104
18.  Synthesis, Storage, and Release of Vascular Endothelial Growth Factor/Vascular Permeability Factor (VEGF/VPF) by Human Mast Cells: Implications for the Biological Significance of VEGF206 
Molecular Biology of the Cell  1998;9(4):875-884.
Mast cells have been implicated in various diseases that are accompanied by neovascularization. The exact mechanisms by which mast cells might mediate an angiogenic response, however, are unclear and therefore, we have investigated the possible expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in the human mast cell line HMC-1 and in human skin mast cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that mast cells constitutively express VEGF121, VEGF165, and VEGF189. After a prolonged stimulation of cells for 24 h with phorbol 12-myristate 13-acetate (PMA) and the ionophore A23187, an additional transcript representing VEGF206 was detectable, as could be verified by sequence analysis. These results were confirmed at the protein level by Western blot analysis. When the amounts of VEGF released under unstimulated and stimulated conditions were compared, a significant increase was detectable after stimulation of cells. Human microvascular endothelial cells (HMVEC) responded to the supernatant of unstimulated HMC-1 cells with a dose-dependent mitogenic effect, neutralizable up to 90% in the presence of a VEGF-specific monoclonal antibody. Flow cytometry and postembedding immunoelectron microscopy were used to detect VEGF in its cell-associated form. VEGF was exclusively detectable in the secretory granules of isolated human skin mast cells. These results show that both normal and leukemic human mast cells constitutively express bioactive VEGF. Furthermore, this study contributes to the understanding of the physiological role of the strongly heparin-binding VEGF isoforms, since these were found for the first time to be expressed in an activation-dependent manner in HMC-1 cells.
PMCID: PMC25314  PMID: 9529385
19.  Vascular Permeability Factor/Vascular Endothelial Growth Factor Induces Lymphangiogenesis as well as Angiogenesis 
The Journal of Experimental Medicine  2002;196(11):1497-1506.
Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A) is a multifunctional cytokine with important roles in pathological angiogenesis. Using an adenoviral vector engineered to express murine VEGF-A164, we previously investigated the steps and mechanisms by which this cytokine induced the formation of new blood vessels in adult immunodeficient mice and demonstrated that the newly formed blood vessels closely resembled those found in VEGF-A–expressing tumors. We now report that, in addition to inducing angiogenesis, VEGF-A164 also induces a strong lymphangiogenic response. This finding was unanticipated because lymphangiogenesis has been thought to be mediated by other members of the VPF/VEGF family, namely, VEGF-C and VEGF-D. The new “giant” lymphatics generated by VEGF-A164 were structurally and functionally abnormal: greatly enlarged with incompetent valves, sluggish flow, and delayed lymph clearance. They closely resembled the large lymphatics found in lymphangiomas/lymphatic malformations, perhaps implicating VEGF-A in the pathogenesis of these lesions. Whereas the angiogenic response was maintained only as long as VEGF-A was expressed, giant lymphatics, once formed, became VEGF-A independent and persisted indefinitely, long after VEGF-A expression ceased. These findings raise the possibility that similar, abnormal lymphatics develop in other pathologies in which VEGF-A is overexpressed, e.g., malignant tumors and chronic inflammation.
PMCID: PMC2194262  PMID: 12461084
20.  Dermal injection of immunocytes induces psoriasis. 
Journal of Clinical Investigation  1996;98(8):1878-1887.
Establishing direct and causal relationships among the confederacy of activated cell types present in psoriasis has been hampered by lack of an animal model. Within psoriatic plaques there are hyperplastic keratinocytes, infiltrating immunocytes, and activated endothelial cells. The purpose of this study was to determine if psoriasis is primarily a disorder of keratinocytes or the immune system. Using a newly developed experimental system in which full-thickness human skin is orthotopically transferred onto severe combined immunodeficient mice, autologous immunocytes were injected into dermis, and the resultant phenotype characterized by clinical, histologic, and immunophenotypic analyses. Engraftment of samples included both uninvolved/ symptomless (PN) skin removed from patients with psoriasis elsewhere, or from healthy individuals with no skin disease (NN skin). In 10 different experiments involving 6 different psoriasis patients, every PN skin was converted to a full-fledged psoriatic plaque skin by injection of autologous blood-derived immunocytes. In all but one psoriatic patient, the immunocytes required preactivation with IL-2 and superantigens to convert PN skin into psoriatic plaque skin. In every case, resultant plaques were characterized by visible presence of flaking and thickened skin, loss of the granular cell layer, prominent elongation of rete pegs with a dermal angiogenic tissue reaction, and infiltration within the epidermis by T cells. Lesional skin displayed 20 different antigenic determinants of the psoriatic phenotype. None of the four NN skin samples injected with autologous immunocytes converted to psoriatic plaques. We conclude that psoriasis is caused primarily by the ability of pathogenetic blood-derived immunocytes to induce secondary activation and disordered growth of endogenous cutaneous cells including keratinocytes and vascular endothelium.
PMCID: PMC507628  PMID: 8878440
21.  Vascular permeability, vascular hyperpermeability and angiogenesis 
Angiogenesis  2008;11(2):109-119.
The vascular system has the critical function of supplying tissues with nutrients and clearing waste products. To accomplish these goals, the vasculature must be sufficiently permeable to allow the free, bidirectional passage of small molecules and gases and, to a lesser extent, of plasma proteins. Physiologists and many vascular biologists differ as to the definition of vascular permeability and the proper methodology for its measurement. We review these conflicting views, finding that both provide useful but complementary information. Vascular permeability by any measure is dramatically increased in acute and chronic inflammation, cancer, and wound healing. This hyperpermeability is mediated by acute or chronic exposure to vascular permeabilizing agents, particularly vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A). We demonstrate that three distinctly different types of vascular permeability can be distinguished, based on the different types of microvessels involved, the composition of the extravasate, and the anatomic pathways by which molecules of different size cross-vascular endothelium. These are the basal vascular permeability (BVP) of normal tissues, the acute vascular hyperpermeability (AVH) that occurs in response to a single, brief exposure to VEGF-A or other vascular permeabilizing agents, and the chronic vascular hyperpermeability (CVH) that characterizes pathological angiogenesis. Finally, we list the numerous (at least 25) gene products that different authors have found to affect vascular permeability in variously engineered mice and classify them with respect to their participation, as far as possible, in BVP, AVH and CVH. Further work will be required to elucidate the signaling pathways by which each of these molecules, and others likely to be discovered, mediate the different types of vascular permeability.
PMCID: PMC2480489  PMID: 18293091
Vascular permeability; Basal vascular permeability; Acute vascular hyperpermeability; Chronic vascular hyperpermeability; VEGF-A; VVO; Angiogenesis
22.  Role of VEGF Receptors in Normal and Psoriatic Human Keratinocytes: Evidence from Irradiation with Different UV Sources 
PLoS ONE  2013;8(1):e55463.
Vascular endothelial growth factor (VEGF) promotes angiogenesis and plays important roles both in physiological and pathological conditions. VEGF receptors (VEGFRs) are high-affinity receptors for VEGF and are originally considered specific to endothelial cells. We previously reported that VEGFRs were also constitutively expressed in normal human keratinocytes and overexpressed in psoriatic epidermis. In addition, UVB can activate VEGFRs in normal keratinocytes, and the activated VEGFR-2 signaling is involved in the pro-survival mechanism. Here, we show that VEGFRs were also upregulated and activated by UVA in normal human keratinocytes via PKC, and interestingly, both the activated VEGFR-1 and VEGFR-2 protected against UVA-induced cell death. As VEGFRs were over-expressed in psoriatic epidermis, we further investigated whether narrowband UVB (NB-UVB) phototherapy or topical halomethasone monohydrate 0.05% cream could affect their expression. Surprisingly, the over-expressed VEGFRs in psoriatic epidermis were significantly attenuated by both treatments. During NB-UVB therapy, VEGFRs declined first in the basal, and then gradually in the upper psoriatic epidermis. VEGFRs were activated in psoriatic epidermis, their activation was enhanced by NB-UVB, but turned undetectable after whole therapy. This process was quite different from that by halomethasone, in which VEGFRs and phospho-VEGFRs decreased in a gradual, homogeneous manner. Our findings further suggest that UV-induced activation of VEGFRs serves as a pro-survival signal for keratinocytes. In addition, VEGFRs may be involved in the pathological process of psoriasis, and UV phototherapy is effective for psoriasis by directly modulating the expression of VEGFRs.
PMCID: PMC3561271  PMID: 23383198
23.  Transgenic expression of the human amphiregulin gene induces a psoriasis-like phenotype. 
Journal of Clinical Investigation  1997;100(9):2286-2294.
Amphiregulin (AR) is a heparin-binding, heparin-inhibited member of the epidermal growth factor (EGF) family and an autocrine growth factor for human keratinocytes. Previous studies have shown that AR expression is increased in psoriatic epidermis. To test the hypothesis that aberrant AR expression is central to the development of psoriatic lesions, we constructed a transgene (K14-ARGE) encoding a human keratin 14 promoter-driven AR gene. Our results indicate that transgene integration and subsequent expression of AR in basal keratinocytes correlated with a psoriasis-like skin phenotype. Afflicted mice demonstrated shortened life spans, prominent scaling and erythematous skin with alopecia, and occasional papillomatous epidermal growths. Histologic examination revealed extensive areas of marked hyperkeratosis with focal parakeratosis, acanthosis, dermal and epidermal lymphocytic and neutrophilic infiltration, and dilated blood vessels within the papillary dermis. Our results reveal that AR exerts activity in the skin that is distinct from that of transgenic transforming growth factor-alpha or other cytokines, and induces skin pathology with striking similarities to psoriasis. Our observations also link the keratinocyte EGF receptor-ligand system to psoriatic inflammation, and suggest that aberrant expression of AR in the epidermis may represent a critical step in the development or propagation of psoriatic lesions.
PMCID: PMC508424  PMID: 9410906
24.  Vascular density in melanoma xenografts correlates with vascular permeability factor expression but not with metastatic potential. 
British Journal of Cancer  1997;76(5):561-570.
We studied the relation between tumour vascular density and tumour growth rate, metastatic incidence and vascular permeability factor (VPF) mRNA levels in a human xenograft model described previously. Vascular density was determined by automated image analysis. Xenografts derived from cell lines BLM and MV3 showed the highest mean vascular density (MVD), the highest in vivo growth rate, high VPF mRNA levels and rapid development of lung metastases. Xenografts of cell lines M14, Mel57 and MV1 showed a significantly lower degree of vascularization, lower in vivo growth rates and lower levels of VPF mRNA, but formed lung metastases with a similar incidence as those of BLM and MV3. Xenografts from cell line 1F6 did not form lung metastases, whereas tumours derived from a spontaneous mutant of 1F6, designated 1F6m, gave rise to lung metastases to the same extent as Mel57, M14 and MV1 tumours. MVD values in 1F6 and 1F6m xenografts, VPF mRNA levels and in vivo growth rates of 1F6 and 1 F6m xenografts, however, were similar. In conclusion, in the melanoma xenograft model vascular density is correlated with in vivo growth rate and with in vitro VPF mRNA levels, but not with the ability to metastasize.
PMCID: PMC2228011  PMID: 9303353
25.  DEP Induction of ROS in Capillary-like Endothelial Tubes Leads to VEGF-A Expression 
Toxicology  2012;297(1-3):34-46.
Inhalation of diesel exhaust particles (DEPs) is associated with pulmonary and cardiovascular disease. One contributor to pathogenesis is inhaled particles reaching and injuring the lung capillary endothelial cells, and possibly gaining access to the blood stream. Using in vitro capillary tubes as a simplified vascular model system for this process, it was previously shown that DEPs induce the redistribution of vascular endothelial cell-cadherin (VE-Cad) away from the plasma membrane to intracellular locations. This allowed DEPs into the cell cytoplasm and tube lumen, suggesting the tubes may have become permeable (Chao et al., 2011). Here some of the mechanisms responsible for endothelial tube changes after DEP exposure were examined. The results demonstrate that endothelial tube cells mounted an oxidative stress response to DEP exposure. Hydrogen peroxide and oxidized proteins were detected after 24 hr of exposure to DEPs. Particles induced relocalization of Nrf2 from the cytoplasm to the nucleus, upregulating the expression of the enzyme heme oxygenase-1 (HO-1). Surprisingly, vascular endothelial cell growth factor-A (VEGF-A), initially termed “vascular permeability factor” (VPF), was found to be up-regulated in response to the HO-1 expression induced by DEPs. Similar to DEPs, applied VEGF-A induced relocalization of VE-Cadherin from the cell membrane surface to an intracellular location, and relocalization of VE-cadherin was associated with permeability. These data suggest that the DEPs may induce or contribute to the permeability of capillary-like endothelial tube cells via induction of HO-1 and VEGF-A.
PMCID: PMC3387988  PMID: 22507881
diesel exhaust particles; endothelial tubes; toxicity; ROS; VEGF-A; VEGFR2; VE-cadherin; endothelial permeability

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