The Study Of Multiple Angiogenesis Regulators Associated With Gliomas

Parts1expression of MVD,TSP-1,TGF-β1,PPAR-γ,MMP-9,VEGFand CD36in gliomasBackgrounds:Gliomas is derived from neuroectodermal cells, thus it is also called Neuroectodermal neoplasms or Neuroepithelial neoplasms. It is the most common intracranial tumor, accounting for about forty-five percent of all intracranial tumors in adult. It is reported that in China the incidence of disease is about3-6persons/100,000persons, and almost30,000patients died of gliomas each year. The survival rate for5years of patients with gliomas of high grade is less than3%, and the median live time is less than one year. The current treatment for gliomas is removal of tumor surgically, assisted by radiotherapy and chemotherapy. Although there is rapid progress made in the surgery technique and adjuvant radiotherapy and chemotherapy, it is still difficult to bring gliomas under permanent control. The tumor location and progression speed is the main prognostic factors. Recently progress were made in adjuvant chemotherapy, chemotherapeutics such as Temozolomide were put into clinical use. However, chemotherapeutic drugs being used now are designed for killing the residual tumor cells so as to prevent a quick recurrence of gliomas. It is more effective in some patients with gliomas of specific hereditary features, such as positive for O6-methylguanine-DNA methyl-transferase (MGMT), or loss of heterozygosity (LOH). It now demonstrated that the growth of tumor entity is angiogenesis-dependent. Angiogenesis plays a crucial role in tumor proliferation, invasion and even metastasis. The angiogenesis process is regulated by pro-angiogenesis factors and anti-angiogenesis factors in the microenvironment. In the present study, we investigate the expression of pro-angiogenesis factors and anti-angiogenesis factors and its association with clinicopathologic factors in gliomas, so that we might find new targets for gliomas treatment.Thrombospondin-1(TSP-1) was the first identified natural occurring protein for angiogenesis inhibition. Subsequent studies indicated that TSP-1regulated proliferation, migration, and apoptosis in various physiological and pathological events. It mainly play an important role in the regulation of angiogenesis. The mechanisms may include binding with CD36, in turn affects the proliferation, adhesion and motility of endothelial cells, and regulates angiogenesis eventually. There were findings that indicate TSP-1can induce cell apoptosis directly. Thus, the effect of TSP-1on tumor growth may vary from different tumor types.Transforming growth factor-β1(TGF-β1) is a member of transforming growth factor superfamily. It can bind to the TGF-β receptors in cell membrane and activate the receptors. The signaling pathway includes the SMAD pathway and DAXX pathway; therefore induce cell apoptosis including endothelial cells. However, TGF-β1can also increase the production of VEGF and plasminogen activator inhibitor, which promote the remodeling of tumor vessels, resulting in angiogenesis. So the major effects of TGF-β1on tumors depend on which mechanism is dominant in the microenvironment.Peroxisome Proliferator-activated receptor gamma (PPAR-γ) is a member of type Ⅱ nuclear receptor superfamily, functioned as regulatory factor in nuclear transcription, including α, β, γ sub-types. PPAR have complicated biological function, taking part in the process of lipid metabolism, glycometabolism, adipocyte differentiation, energy balance, inflammatory reactions, arthrosclerosis, tumor cell differentiation and necrosis. PPAR-γ is studied intensively. The main function of which is to promote the synthesis of lipid. Many evidence indicated that the PPAR-γ activator has a distinguished function against tumor. The mechanisms include inhibiting cell proliferation, inducing cell apoptosis and differentiation, inhibiting angiogenesis and reduce the invasive ability of tumor.Matrix metalloproteinases9(MMP-9) is a zinc-dependent proteolytic enzyme that can lyse the extracellular matrix, including collagenase and elastase. It plays an important role in wound healing, neovascularization, tumor remodeling, invasion and metastasis. MMP-9can facilitate the tumor cells to penetrate the basal membrane that is mainly formed by type Ⅳ collagen, thus promote the neovascularization in the stroma. Vascular endothelial growth factor (VEGF) is an important factor in regulating angiogenesis. VEGF promotes cell division and Chemotaxis, causing the proliferation and migration of endothelial cells. It also increase the permeability of microvessels, leading to leaking of plasma protein to extracellular stroma, facilitating the migration of fibroblasts and endothelial cells. Moreover, VEGF can induce the production of urokinase-type plasminogen activator, tissue plasminogen activator and urokinase-type plasminogen activator receptor, causing coagulation of plasma proteins in the stroma, thus provide braces for neovessels.Objectives:In the present study, we investigated the expression of TSP-1, TGF-β1, PPAR-γ, MMP-9, VEGF and CD36in gliomas. The expression difference was compared between groups to find the potential target for new therapy.Material and methods:Patients and tissues:Gliomas removed surgically in Shandong Provincial Hospital from Jan2009to Aug2011were selected randomly. There were34male, and28female. The median average age is52.3±1.3y, ranging from5y to71y. The pathological diagnosis was confirmed by immunochemical staining.Methods:The expression of TSP-1, TGF-β1, PPAR-γ, MMP-9, VEGF and CD36in gliomas was detected by immunochemical staining, Realtime-PCR and Western Blotting.Results:1.The expression of TSP-1and PPAR-γ was found higher in low-grade gliomas than in high-grade gliomas. Both the expression of TSP-1and PPAR-γ was found reversely correlated to MVD.(P<0.05).2. The expression of TGF-β1, MMP-9, VEGF was found lower in low-grade gliomas than in high-grade gliomas. Both the expression of TSP-1and PPAR-γ was found positively correlated to MVD.(P<0.05).Conclusions:The expression of TSP-1and PPARγ was significantly decreased in high-grade gliomas compared to that in low-grade gliomas. The mechanism may due to their anti-angiogenesis effect. The mechanism underlying the anti-angiogenesis effect of TSP-1may involve the inhibition of MMP-9and VEGF. PPARy can increase the expression of CD36, which may help partly explain anti-angiogenesis effect of PPARy.2. The expression of TGF-β1, MMP-9and VEGF significantly higher in high-grade gliomas compared to that in low-grade gliomas. The mechanism may due to their pro-angiogenic effect. Part2The effect of TSP-1combined with PPAR Y ligands on HUVEC and U251BackgroundAnti-angiogenic therapy have many advantage, including seldom drug resistance, low toxicity, long-term medication, suitable for multiple tumors, co-existing with other chemotherapy drugs. Although Anti-angiogenic drugs made good performance in cell experiment and in vitro, many of them failed to reach the original expectancy. The main reason for this is that the microenvironment is very complicated. There are many cells which can produce pro/anti-angiogenic factor to undermine the effect of drugs.TSP.-1is proved to be function mainly by its binding to CD36. TSP-1can inhibit the adhesion, proliferation, and migration of endothelial cells, which can be agonized by CD36antibody. Transfection of CD36into HUVEC can sensitize ECs to the inhibitory effect of TSP-1on migration.The expression of CD36is heterogonous. It is less expressed in large vessels and some arteries. The inhibitory effects of TSP-1mainly depend on the portion of CD36-positive cells. PPAR can up-regulate the expression of CD36. The mechanism is still unknown.In the lab, HUVEC is often used for the test of ECs. U251is often used for test of gliomas. These two cells were used in our study. Objective:to determine the synergetic effect of TSP-1and PPAR γ in U251and HUVEC.Methods:After applying PPAR Y together with TSP-1, we conduct to determine the expression of CD36in HUVEC and expression of TGF β1,VEGF,MMP-9In U251. MTT and flow cytometry were also used to detect the apoptosis in HUVEC.Results:The CD36expression is elevated by PPAR Y in a time-dependent and dose-dependent way. PPAR Y and TSP-1induce the apoptosis in HUVEC. TSP-1down-regulates the expression of VEGF.Conclusions:PPAR Y and TSP-1have a synergetic effect and can inhibit angiogenesis by inducing CD36expression and ECs apoptosis. TSP-1can also down-regulates the expression of VEGF in vitro.