Vascular Endothelial Growth Factor Alpha Research Articles

Evaluation of the effect of autologous mesenchymal stem cell injection in a large‐animal model of bilateral kidney ischaemia reperfusion injury

Adult mesenchymal stem cells (MSC) have been proven to be of benefit to the kidney in different experimental models of renal injuries. All studies have been performed in valuable rodent models, but the relevance of these results to large mammals and ultimately, to humans remains unknown. Therefore, the aim of this study was to investigate the effect of MSC transplantation in an alternative ovine large-animal model of bilateral kidney ischaemia reperfusion injury. Sheep were divided into three groups: one sham-operated group and two groups submitted to renal bilateral ischaemia for 60 min. Animals with ischaemia reperfusion injury were treated with injection of autologous MSCs or with vehicle medium. The model sheep presented with renal histological manefestations that closely resembled lesions seen in patients. Transplanted MSCs were found in glomeruli but not in tubules and did not express glomerular cell markers (podocin, von Willebrand factor), but functional evaluation showed no beneficial effect of MSC infusion. Morphological and molecular analyses corroborated the functional results. MSCs did not repair kidney parenchyma and failed to modulate cell death and proliferation or cytokine release (tumour necrosis factor-alpha, vascular endothelial growth factor alpha (VEGF-alpha), Bcl-2, caspase). In this unique autologous large-animal model, MSCs did not exhibit reparative or paracrine protective properties.

Quantitative PCR validation of hormones and growth and immune factors from gene array of rat cecal cells fed resistant starch and control diets

Previously we reported the gene array results on cecal cells from rats fed three diets: resistant starch (RS, 3.2 kcal/g), standard control (CC, 3.6 kcal/g), and energy balanced control (EC, 3.2 kcal/g). We now can report the validation assay performed by quantitative PCR (n=8) for 15 hormones, and growth and immune factors with the greatest fold changes and levels of significance from the gene array. The 18S ribosomal RNA gene was used as the endogenous control. Three t‐tests, RS‐EC, RS‐CC, and CC‐EC, were performed with results for RS‐EC presented. Fourteen of 15 factors found different by the gene array were validated. Large increases (>2) in fold change and highly significant (p<0.0001) differences for RS were: 1) hormones ‐ adrenomedullin, glucagon, neurotensin and peptide YY; 2) growth factors ‐ amphiregulin, bone morphogenetic protein 2, epiregulin, and vascular endothelial growth factor alpha; and 3) immune factor ‐ interleukin 1 alpha. Results demonstrate dietary RS is associated with increased gene expression of hormones and factors that may positively impact the health of the gut and body of the animals. Funding sources were National Starch and LSU AgCenter.

Cytoprotective effects of a blue light–filtering intraocular lens on human retinal pigment epithelium by reducing phototoxic effects on vascular endothelial growth factor-α, Bax, and Bcl-2 expression

To compare the possible protective effects of the ultraviolet (UV)-filtering and blue light-filtering SN60AT intraocular lens (IOL) and the untinted UV-filtering SA60AT IOL with regard to light-induced stress on human retinal pigment epithelium (RPE). Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. Primary human RPE cells were exposed to white light, and a tinted or untinted IOL was placed in the light beam. After 15 to 60 minutes of irradiation, cell viability was determined by a colorimetric test (tetrazolium dye-reduction assay) and a microscopic live/dead assay. The expression of vascular endothelial growth factor-alpha (VEGF-alpha), Bax, and Bcl-2 and their mRNA was determined by reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting. Without an IOL, white-light exposure decreased cell viability compared with the decrease with the nonirradiated control in a time-dependent manner. Light-induced cell death was significantly reduced by both the tinted IOL and untinted IOL. The combined UV and blue-light filtering attenuated light-induced cell damage significantly more than UV filtering alone. Results of RT-PCR and Western blotting showed a significant time-dependent decrease in Bcl-2 and increase in Bax and VEGF-alpha that were significantly less with the tinted IOL than with the untinted IOL. Both IOLs reduced light-induced RPE damage. The UV- and blue light-filtering IOL reduced damage more than the conventional IOL. This supports the hypothesis that blue light-filtering IOLs may prevent retinal damage in clinical use.

Thyroxine treatment stimulated ovarian follicular angiogenesis in immature hypothyroid rats.

The development of mature ovarian follicles is greatly dependent on healthy thecal angiogenesis. Recent experimental evidence showed that thyroxine (T4) treatment promoted ovarian follicle development in immature hypothyroid (rdw) rats. However, an involvement of thyroid hormone in ovarian follicular angiogenesis has not yet been demonstrated. By morphological and molecular approaches, the present studies demonstrated that antral follicles in untreated, T4- or equine chorionic gonadotropin (eCG)-treated rdw rats were mainly small and/or atretic, and presented a poorly developed thecal microvasculature with ultrastructural evidence of diffuse quiescent or degenerative thin capillaries. However, T4 together with eCG increased the number of large antral and mature follicles with numerous activated capillaries and ultra-structural evidence of rich and diffuse angiogenesis in the theca layer. While T4 alone significantly increased mRNA expression of vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNFalpha), it decreased that of fetal liver kinase compared with those in the untreated group. Combined treatment of T4 and eCG markedly increased mRNA abundance of not only VEGF and TNFalpha, but also basic fibroblast growth factor. These data suggest that T4 may promote ovarian follicular angiogenesis in rdw rats by up-regulating mRNA expression of major angiogenic factors.

Toll-like receptor 4-mediated growth of endometriosis by human heat-shock protein 70

We investigated the role of human heat-shock protein 70 (Hsp70) in Toll-like receptor 4 (TLR4)-mediated growth of endometriosis. TLR4 expression was examined in macrophages (M) isolated in primary culture from the peritoneal fluid of women with and without endometriosis. The production of a number of macromolecules by non-treated M, Hsp70-treated M and after treatment with anti-TLR4 antibody was examined by enzyme linked immunosorbent assay (ELISA). The single and combined effects of Hsp70 and lipopolysaccharide (LPS) on the growth of endometrial stromal cells were analyzed by 5-bromo-2-deoxyuridine (BrdU) incorporation study. Hsp70 levels in eutopic and ectopic endometria were measured by ELISA. TLR4 was detected in isolated M at protein and gene level. Hsp70 (10 microg/ml) significantly stimulated the production of hepatocyte growth factor, vascular endothelial cell growth factor, interleukin-6 and tumor necrosis factor alpha by M derived from women with endometriosis compared with M derived from women with no endometriosis (P < 0.05 for each). This effect of Hsp70 was abrogated after pretreatment of M with anti-TLR4 antibody. BrdU incorporation indicated that Hsp70 significantly enhanced the growth of endometrial stromal cells ( approximately 50% increase) from women with endometriosis compared to non-treated cells. A synergistic effect on cell proliferation was observed between exogenous Hsp70 and LPS and this was significantly suppressed by pretreatment of cells with anti-TLR4 antibody (P < 0.05). Tissue levels of Hsp70 were significantly higher in the eutopic endometria (P < 0.05) and opaque red lesions (P < 0.01) derived from women with endometriosis than from other peritoneal lesions or from women with no endometriosis. A prominent stress reaction was observed in blood-filled opaque red peritoneal lesions. Human Hsp70 induces pelvic inflammation and may be involved in TLR4-mediated growth of endometrial cells derived from women with endometriosis.

Protective Role for CD1d-Reactive Invariant Natural Killer T Cells in Cauterization-Induced Corneal Inflammation

Corneal inflammation can be induced by various stimuli, such as chemical burns, trauma, and acute bacterial infection, and directly impairs visual acuity. Natural killer T (NKT) cells belong to a specialized population of leukocytes that coexpress the T-cell receptor and NK markers. This study examined the role of CD1d-reactive invariant NKT cells in cauterization-induced acute corneal inflammation. The corneas of CD1d-knockout (KO) mice and Jalpha18-KO mice (both of which are NKT cell deficient) and control mice were cauterized with silver nitrate. Corneal edema and opacity were examined, and the phenotypes of the corneal-infiltrating cells were analyzed histologically at 24 hours and by flow cytometry at 96 hours. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expression of vascular endothelial growth factor (VEGF), interferon (IFN)gamma, and tumor necrosis factor (TNF)alpha in the cauterized corneas. The CD1d-KO and Jalpha18-KO mice had significantly greater levels of corneal edema and opacity than did the control mice. Although the number of infiltrating cells was not significantly different at 96 hours, both groups of NKT cell-deficient mice demonstrated increased early neutrophil accumulation at 24 hours and early expression of VEGF, IFNgamma, and TNFalpha. There was no difference in the level of VEGF-induced corneal neovascularization. NKT cells appear to regulate the early accumulation of neutrophils, protect the cornea from excessive inflammation, and maintain corneal clarity. However, in this study, they did not affect the corneal revascularization process induced by VEGF.

Effect of oleic and linoleic acids on the inflammatory phase of wound healing in rats

Inflammation is a crucial step for the wound healing process. The effect of linoleic and oleic acids on the inflammatory response of the skin during the healing process and on the release of pro-inflammatory cytokines by rat neutrophils in vitro was investigated. A wound in the dorsal surface of adult rats was performed and fatty acids were then topically administered. Both oleic and linoleic acids increased the wound healing tissue mass. The total protein and DNA contents of the wounds were increased by the treatment with linoleic acid. The treatments with oleic and linoleic acids did not affect vascular permeability. However, the number of neutrophils in the wounded area and air pouches was increased and the thickness of the necrotic cell layer edge around the wound was decreased. A dose-dependent increase in vascular endothelial growth factor-alpha (VEGF-alpha) and interleukin-1beta (IL-1beta) by neutrophils incubated in the presence of oleic and linoleic acid was observed. Oleic acid was able to stimulate also the production of cytokine-induced neutrophil chemoattractant in inflammation 2 alpha/beta (CINC-2alpha/beta). This pro-inflammatory effect of oleic and linoleic acids may speed up the wound healing process.

The role of immature myeloid suppressor cells (IMSCs) in transgenic mammary tumor chemoprevention and therapy by Celecoxib (49.21)

Abstract Immune manipulation can control tumor initiation and growth while tumor growth may suppress host immunity. In our studies of tumor–host interactions, we observed a tumor associated expansion of IMSCs and suppression of T cell number and tumor infiltration. Further, the cyclooxygenase-2 (COX-2) inhibitor, Celecoxib had chemopreventative and as shown for the first time, therapeutic activity for mammary tumors in HER-2/neu transgenic BALB/c mice. Tumor growth was associated with a significant decrease in T cells and a significant increase in IMSCs (CD11b+Gr1+). In addition type 1 cytokine transcripts were depressed, while transcription levels of COX-2, arginase-1 (ARG1), nitric oxide synthetase-2 (NOS2), granulocyte (G) and G-monocyte colony stimulating factor, and vascular endothelial growth factor-alpha (VEGF-a) were increased in the preneoplastic lesions, tumors, and spleens and to a greater extent tumor infiltrating leukocytes. Celecoxib chemoprevention reduced the changes in IMSC and T cell numbers and cytokine and enzyme transcription levels. We posit that the increase in IMSCs may be due to the increased levels of COX-2, VEGF-a and growth factor(s), while the loss of T cell numbers and function maybe due to the heightened levels of ARG1 and NOS2. Further, as Celecoxib administration does not increase T cell infiltration of tumors or decrease IMSC infiltration, the changes in cytokine and growth factor levels maybe critical to the improved clinical outcomes. Together, these studies suggest an important role for IMSCs and its regulation by Celecoxib in cancer induction, and therapeutic intervention. Supported in part by funding from NIH grant 5 RO1 AT001739, Eppley Cancer Center and Nebraska DHHS LB506.

Soluble Signalling Factors Derived from Differentiated Cartilage Tissue Affect Chondrogenic Differentiation of Rat Adult Marrow Stromal Cells

Background: Chondral defects show lack of proper regeneration whereas osteochondral lesions display limited regeneration capacity. Latter is probably due to immigration of chondroprogenitor cells from the subchondral bone. Known chondroprogenitor cells for cartilage tissues are multi-potent adult marrow stromal or mesenchymal stem cells (MSCs). In vitro chondrogenic differentiation of these precursor cells usually require cues from growth and signalling factors provided in vivo by surrounding tissues and cells. We hypothesise that signalling factors secreted by differentiated cartilage tissue can initiate and maintain chondrogenic differentiation status of MSCs. Methods: To study such paracrine communication between allogenic rat articular cartilage and rat MSCs embedded in alginate beads a novel coculture system without addition of external growth factors has been established. Results: Impact of cartilage on differentiating MSCs was observed at two different time points. Firstly, sustained expression of Sox9 was observed at an early stage which indicated induction of chondrogenic differentiation. Secondly, late stage repression of collagen X indicated pre-hypertrophic arrest of differentiation. In the culture supernatant we have identified vascular endothelial growth factor alpha (VEGF-164α), matrix metalloproteinase (MMP) -13 and tissue inhibitors of MMPs (TIMP-1 and TIMP-2) which could be traced back either to the cartilage explant or to the MSCs under the influence of cartilage. Conclusion: The identified factors might be involved in regulation of collagen X gene and protein expression and therefore, may have an impact on the control and regulation of MSCs differentiation.

SEX DIMORPHISMS IN ACTIVATED MESENCHYMAL STEM CELL FUNCTION

The plasticity of bone marrow-derived stem cells (BMSCs) has resulted in positive remodeling and the regeneration of viable tissues. However, BMSC release of growth factors, which limit apoptosis and inflammation, may play an important role in conferring organ protection. Recent studies also indicate that those patients with higher circulating BMSC counts may be more resistant to septic and traumatic insults. There are clear sex differences in response to such insults. Within the population of BMSC, mesenchymal stem cells (MSCs) may have clinical advantages. Therefore, we hypothesize that sex differences in the MSC paracrine response to acute injury exist. Mesenchymal stem cells were obtained from male and female mice. One million MSCs per well (triplicate wells per group) were stressed by hypoxia and increasing doses of endotoxin (lipopolysaccharide [LPS]) and hydrogen peroxide. Mesenchymal stem cell activation was determined by measuring vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha production by enzyme-linked immunosorbent assay. Differences were considered significant if P < 0.05. Lipopolysaccharide resulted in significant activation of both male and female MSCs. However, LPS provoked significantly more VEGF production in female MSCs versus male MSCs at all LPS doses. Hypoxia of 1 h and hydrogen pyroxide exposure also caused significantly more VEGF production in female MSCs versus male MSCs. Female MSCs expressed significantly less tumor necrosis factor alpha than male MSCs after acute LPS and hypoxia. This study constitutes the first demonstration that sex differences exist in activated MSC function. Sex differences in progenitor cell function may have important implications in understanding the observed sex differences in the host's response to injury.

Glioma cells under hypoxic conditions block the brain microvascular endothelial cell death induced by serum starvation

Angiogenesis is one of essential components for the growth of neoplasms, including malignant gliomas. However, tumor vascularization is often poorly organized and marginally functional due to tumor structural abnormalities, inducing regional or temporal hypoxic conditions and nutritional shortages in tumor tissues. We investigated how during angiogenesis migrating endothelial cells survive in these hypoxic and reduced nutritional conditions. Human brain microvascular endothelial cells (HBMECs) underwent apoptosis and necrosis after serum withdrawal. This endothelial cell death was blocked by recombinant VEGF protein or the culture medium of U251 glioma cells exposed to hypoxia (H-CM). Hypoxic treatment increased vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNF-alpha) expression in U251 glioma cells. H-CM activated nuclear factor-kappaB (NFkappaB) protein and increased the gene expression of antiapoptotic factors including Bcl-2, Bcl-X(L), survivin and X-chromosome-linked inhibitor of apoptosis protein (XIAP) in endothelial cells. The survival activity of H-CM for endothelial cells was abolished by two kinds of VEGF inhibitors {Cyclopeptidic VEGF inhibitor and a VEGF receptor tyrosine kinase inhibitor (4-[(4'-chloro-2'-fluoro) phenylamino]-6, 7-dimethoxyquinazoline)} or NFkappaB inhibitors (ALLN and BAY 11-7082). These VEGF inhibitors did not block the activation of NFkappaB induced by H-CM in endothelial cells. On the contrary, TNF-alpha antagonist WP9QY enhanced the survival activity of H-CM for endothelial cells and blocked NFkappaB activation induced by H-CM under serum-starved conditions. Taken together, our data suggest that both the secretion of VEGF from glioma cells and activation of NFkappaB in endothelial cells induced by TNF-alpha are necessary for endothelial cell survival as they increase the expression of antiapoptotic genes in endothelial cells under conditions of serum starvation. These pathways may be one of the mechanisms by which angiogenesis is maintained in glioma tissues.

Proteins with an endostatin-like domain in a mouse model of oxygen-induced retinopathy

Neovascularization in the retinopathy of prematurity (ROP) mouse eye is a self-limiting phenomenon. Free endostatin is known to be anti-angiogenic. In this study, we identified the localization of endostatin-like protein (ELP) sequences and investigated their possible role in this process. ROP was induced in C57Bl/6 mice and the eyes observed 1–11 days after termination of high oxygen supply (P13–P21). Sagittal sections and retinal flatmounts were double-stained with antibodies against a protein-sequence of endostatin, vascular endothelial growth factor (VEGF), lectin, and smooth-muscle alpha actin. The fluorescence was visualized by traditional and confocal microscopy. Intense staining for VEGF in the inner retina was limited to the early stages of neovascularization and diminished at P19–P21. In contrast, staining for ELPs appeared at P15 around the newly formed vessels and remained even after degeneration of their endothelial cells. Staining of the inner retinal vasculature for ELPs was restricted to P17–P19, the known maximum of the neovascular response. Outer retinal vessels did not show presence of ELPs at any time. Our study demonstrates that ELPs, absent at the beginning of neovascular sprouting, increases with the amount of neovascularization and thus, varies reciprocally to VEGF in the time period investigated. ELPs remain during the regression of the vessels and might therefore play an important role in the self-limiting process of ROP neovascularization.

N-acetylglucosaminyltransferase I Activity of Bovine Oviduct Epithelial Cells: Stimulation by Luteinizing Hormone, Vascular Endothelial Growth Factor and Tumor Necrosis Factor .ALPHA.

N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101), which catalyzes the first step in the conversion of oligomannose to complex or hybrid N-glycans of glycoproteins, was found in media cultured with bovine oviduct epithelial cells (BOEC) obtained from non-pregnant cows during the follicular phase. Combined treatment with specific hormones increased GnT I release from BOEC. Luteinizing hormone (LH; 10 ng/ml) alone slightly, but together with 17beta-estradiol (E2; 1 ng/ml), synergistically increased GnT I activity. Vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF) alpha, which have been shown to have their highest activities in the bovine oviduct during the periovulatory period, also increased in GnT I activity. This study provides the first evidence of an increase of GnT I release from BOEC in vitro, and shows that endocrine as well as local factors such as LH, VEGF and TNFalpha increase this activity. The results suggest that GnT I activity in the bovine oviduct may contribute to the induction of glycosylation and thereby contributing to the provision of the optimal microenvironment for fertilization and early development of the embryos.

Hypoxia-inducible factor and vascular endothelial growth factor are expressed more frequently in embolized than in nonembolized cerebral arteriovenous malformations.

In previous studies, we documented a marked neoangiogenesis and endothelial proliferation in cerebral arteriovenous malformations (AVMs) that were embolized before surgery compared with those that were not embolized. We hypothesized that embolization caused a local hypoxia that promotes neoangiogenesis as a possible pathomechanism. To support this hypothesis, we now examined the angiogenesis-related proteins in a larger cohort of patients. In addition, we investigated hypoxia-inducible factor-1 alpha as a possible protein operative during neoangiogenesis of cerebral AVMs. Paraffin-embedded specimens of 56 AVMs obtained from surgical resection and 14 brain tissue controls were immunohistochemically stained with antibodies to proliferating cell nuclear antigen, MIB-1, vascular endothelial growth factor, Flk1, and hypoxia-inducible factor-1 alpha by standard protocols. In AVMs treated with embolization before surgery (n = 35, 63%), the expression of hypoxia-inducible factor-1 alpha (P = 0.0101) and vascular endothelial growth factor (P = 0.0007) was significantly higher (Fisher's exact test) than in patients who did not have previous endovascular treatment. Differences in the expression of Flk-1 (P = 0.0798) and proliferating cell nuclear antigen (P = 0.0423) were in the same direction but were not significant when corrected for multiple testing. Our results provide circumstantial evidence that a partial occlusion of cerebral AVMs might induce local hypoxia-related neoangiogenesis. To support these data, future animal studies should be performed.

Hypoxia up-regulates the effects of prostaglandin E 2 on tumor angiogenesis in ovarian cancer cells

Objective. Emerging evidence supports a role for prostaglandins (PG) and their synthesizing enzyme, cyclooxygenase (COX), in tumor angiogenesis. The objective of this study was to investigate the effects of prostaglandin E 2 (PGE 2) on the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible transcription factor-1 alpha (HIF-1α) genes in epithelial ovarian cancer (EOC) cells. Methods. Two human EOC cell lines, MDAH-2774 and SKOV-3, were treated with exogenous dimethyl prostaglandin E 2 (dmPGE 2) at two doses of 10 and 50 μg/ml and cultured for 24 h under both normoxic and hypoxic conditions. Total RNA was extracted from EOC cells with the use of a monophasic solution of phenol and GITC/Trizol method. The levels of COX-2, VEGF, and HIF-1α mRNA were measured by quantitative reverse transcription PCR (RT-PCR). Results. Under normoxic conditions, treatment of both ovarian cancer cell lines with dmPGE 2 resulted in a significant increase in VEGF expression but had no effect on HIF-1α. Culturing the cells under hypoxic conditions resulted in an increase in HIF-1α and VEGF mRNAs. The combination of hypoxia and dmPGE 2 treatment resulted in the highest levels of VEGF and HIF-1α when compared to either individual treatment. Conclusion. PGE 2 is a potent stimulator of VEGF expression in ovarian cancer cells. This effect of PG is further potentiated under hypoxic conditions where it is also associated with a significant increase in HIF-1α expression.

Omega-3 fatty acid supplementation reduces tumor growth and vascular endothelial growth factor expression in a model of progressive non-metastasizing malignancy.

Omega-3 fatty acids, the principal component of fish oil, have been demonstrated to have antiinflammatory properties. The role of eicosapentaenoic acid (EPA) supplementation for cancer patients is currently under investigation; however, the mechanisms of EPA activity have not been defined. The purpose of this study was to characterize tumor-specific and treatment-specific effects of supplemental dietary EPA in an animal model of progressive malignancy. Fischer 344 rats (200-250 g) underwent flank implantation of the methycholanthrene (MCA)-induced fibrosarcoma on day 0. Rats were randomly divided into 3 treatment groups on day 13: EPA (1 mL, 5.0 g/kg per day) + 10 IU vitamin E; corn oil (1 mL) + 10 IU vitamin E, and saline (1 mL) + 10 IU vitamin E (vitamin E was used to prevent fatty acid oxidation). On day 14, gavage feeding was started and was continued through day 28. The animals were killed on day 29, and the tumors were removed. The tumors were weighed and divided by the tumor-free carcass weight to obtain percentage of tumor volume, and the livers were flash frozen. Vascular endothelial growth factor-alpha (VEGF-alpha) and cyclo-oxygenase 2 (COX-2) mRNA were measured by reverse transcription-polymerase chain reaction (RT-PCR). EPA rats had significant reductions in tumor volume compared with isocaloric corn oil and control saline animals (25%, p < .01 and 33%, p < .01, respectively). Rats receiving EPA demonstrated decreased VEGF-alpha mRNA levels (0.023 +/- 0 0.001) compared with those receiving corn oil (0.129 +/- 0.047) or saline (0.150 +/- 0.026; p < .05). These data demonstrate that EPA supplementation inhibits tumor growth, potentially through alterations in the expression of the pro-angiogenic VEGF-alpha. The mechanism(s) of EPA as an inhibitor of tumor-related angiogenic growth factors may be associated with COX-2 enzyme fatty acid metabolism and merits further study.

A Reduction in Serum Cytokine Levels Parallels Healing of Venous Ulcers in Patients Undergoing Compression Therapy

Introduction vascular endothelial growth factor (VEGF) and tumour necrosis factor alpha (TNFα) have been specifically implicated in the tissue damage associated with chronic venous disease (CVD). Furthermore, production of both factors is known to be upregulated in vessel wall cells subject to hypertension. The aim of this study was to determine the local venous levels of VEGF and TNFα in limbs with venous ulcers before and after treatment with graduated compression. Patients and methods eight patients with venous ulcers and 8 patients with varicose veins only were included in the study. For ulcer patients, serum samples were taken from the superficial veins in lower limbs and repeated after 4 weeks of treatment with 4-layered graduated compression. Serum from the arms of the same patients served as controls. Determination of the concentrations of VEGF and TNFα proteins were performed with sandwich enzyme-linked immunosorbent assays. Results both groups of patients had elevated levels of VEGF and TNFα. In patients with venous ulcers there was a reduction in the levels of both cytokines to below control values with treatment. These changes correlated with healing of the ulcers as determined by reduction in ulcer size. Conclusion these data, for the first time, suggest a central role for both TNFα and VEGF in the pathogenesis of venous ulceration which may constitute a causative link between venous hypertension and tissue pathology.

Detection of Vascular Endothelial Growth Factor and Tumor Necrosis Factor Alpha in Epiretinal Membranes of Proliferative Diabetic Retinopathy, Proliferative Vitreoretinopathy and Macular Pucker

Purpose: To evaluate epiretinal membranes in proliferative eye disease for the presence of vascular endothelial growth factor (VEGF) and tumor necrosis factor α (TNF-α). Methods: Membranes were surgically removed from 66 patients with proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR) and macular pucker (MP). Cytokine concentrations were determined by ELISA (VEGF) and bioassay (TNF-α). Results: VEGF was detected in all 66 membranes investigated. The highest VEGF values were found in patients with type I diabetes (mean = 5,994 pg/mg protein). In patients with type II diabetes, the values were at a mean of 1,242 pg/mg protein. When coagulation therapy was performed for longer than 3 months prior to surgery, VEGF was significantly (p < 0.05) reduced. Intermediate levels of VEGF were found in PVR membranes (mean = 1,417 pg/mg protein). The lowest activity was found in MP (mean = 216 pg/mg protein). In contrast, TNF-α was present in 16 PDR membranes, 9 PVR membranes and 8 MP membranes. Conclusion: The presence of VEGF in all membranes investigated indicates that this cytokine plays an important role in angiogenesis in ischemic retinal disease and in membrane growth in proliferative disorders.

Cytokine signaling through the novel tyrosine kinase RAFTK in Kaposi's sarcoma cells.

A number of cytokines, including basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), oncostatin M (OSM), IL-6, and tumor necrosis factor alpha (TNF-alpha), have been postulated to have a role in the pathogenesis of Kaposi's sarcoma (KS). The proliferative effects of bFGF and OSM may be via their reported activation of the c-Jun NH2-terminal kinase (JNK) signaling pathway in KS cells. We now report that KS cells express a recently identified focal adhesion kinase termed RAFTK which appears in other cell systems to coordinate surface signals between cytokine and integrin receptors and the cytoskeleton as well as act downstream to modulate JNK activation. We also report that the tyrosine kinase receptor FLT-4, present on normal lymphatic endothelium, is robustly expressed in KS cells. Treatment of KS cells with VEGF-related protein (VRP), the ligand for the FLT-4 receptor, as well as with the cytokines bFGF, OSM, IL-6, VEGF, or TNF-alpha resulted in phosphorylation and activation of RAFTK. Following its activation, there was an enhanced association of RAFTK with the cytoskeletal protein paxillin. This association was mediated by the hydrophobic COOH-terminal domain of the kinase. Furthermore, JNK activity was increased in KS cells after VEGF or VRP stimulation. We postulate that in these tumor cells RAFTK may be activated by a diverse group of stimulatory cytokines and facilitate signal transduction to the cytoskeleton and downstream to the growth promoting JNK pathway.