Effect Of Tumor Cells On Inducing Human Endothelial Cells To Form New Blood Vessels In Vitro And The Role Of VEGF

IntroductionTumor is characteristic angiogenesis - dependent disease, namely the growth of human tumors and development of metastases depend on the de novo formation of blood vessels. The formation of new blood vessels is tightly regulated by a large number of factors and it is the result of the imbalance between promo tors and inhibitors of tumor angiogenesis. Tumor angiogenesis is defined as a process of micro - vessels growth and blood circulation setting up in tumor. It is a highly complex multistep process that involves the degradation of components of the extracellular matrix and then the proliferation, differentiation, migration of endothelial cells to eventually form tubular structure. New blood vessels increase the blood flow perfusion and promote tumor growth, invasion and metastasis. Inhibition of angiogenesis suppresses tumor growth, which has become a new treatment target in tumor therapy.Vascular endothelial growth factor is a potent direct - acting angiogenic factor secreted by tumor cells, a highly effective specific mitogen for endothelial cells. It plays a key role on tumor angiogenesis. The study on angiogenesis and VEGF has become a hot point in the area of cancer research. Thus the experiment cultured HUVECs in vitro, and set up a stable angiogenesis culture system by Matrigel in order to discuss the influence of tumor cells on inducing human vascular endothelial cells to form new blood vessels and the role of VEGF. Further discuss the relationship between angiogenesis and VEGF and its mechanism.MethodsHUVECs were cultured in 24 - well plates by modified Jaffe method and induced to develop tubular structure by Matrigel. Thus set up a stable angiogenesis system and added the stimulating factors. Oral squamous cell carcinoma cells lines were cultured and passaged, the conditional medium of tumor cells were prepared. Group one, dynamic observing the circumstances of HUVECs induced to form tubular structure by Matrigel . Group two, divided into control group (serum - free DMEM culture medium) and tumor cells conditional medium stimulating groups in varying concentrations ( the ratio of the upper clear liquid of tumor cells to DMEM culture medium is 1- 2, 1-1 and 2- 1,respectively ) then acting 24h. Group three, divided into the conditional medium of tumor cells (the ratio of the upper clear liquid of tumor cells to DMEM culture medium is 1 : 1) stimulating groups in varying time (12hA24h^48h ). The various groups observed and photography by inverted phase contrast microscope , then fixed by 95% ice alcohol. The expression of CD34 and KDR of endothelial cells were detected by immunocytochemical method. The experiment results were tested by calculating the number of tubule's in microscopic obversation and computer image - analysis. Finally the data was analyzed by statistics.ResultsHUVECs can be induced to develop tubular structure by Matrigel in vitro. After treatment with the conditional medium of tumor cells, the number of tubules increased and in certain ranges showing a dose - dependent and time - dependent manner (P <0. 01);with the number of tubules increased, the expression of KDR of vascular endothelial cells increased and in certain ranges showing a dose - dependent and time - dependent manner ( P <0.01).DiscussionMatrigel basement membrane matrix is a solubilized basement membrane preparation extracted from the Engelbreth - Holm - Swarm ( EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins. Its major component is lami-nin, followed by collagen IV , entactin and heparan sulfate proteglycans. It also contains TGF - (3, fibroblast growth factor, tissue plasminogen activator, and other growth factors which occur naturally in the EHS tumor. In room temperature, Matrigel matrix can be polymerized a bioactive matrix material which has adhesive and differentiation effect on vascular endothelial cells resembling the basement membranes of the mammal cells. In this experiment, Matrigel effect on cultured HUVECs was observed and cultured HUVECs was induced to develop tubular structure by Matrigel in vitro successfully.Tumor angiogenesis is controlled by both proangiogenic and antiangiogenic factors. In vivo, tumor angiogenic factors are mainly secreted by tumor cells in which VEGF is the vital positive regulating factor. VEGF is a kind of secretory glucoprotein cytokine. It can be secreted by tumor cells and specially stimulate vascular endothelial cells to proliferate and form new blood vessels. In vitro, VEGF mainly exists in the conditional medium of the tumor cells. In this experiment we observed that after the cultured HUVECs treated by the conditional medium-of the tumor cells, the number of tubules of the cultured system increased and in certain ranges showing a dose - dependent and time - dependent fashion. It can be inferred that tumor cells promoted tumor angiogenesis via secreting VEGF and other growth factors.Vascular endothelial growth factor receptor is specific for VEGF receptor tyrosine kinases( RTKs). These receptors are currently designated VEGFR - 1, VEGFR - 2, and VEGFR - 3 and were originally named Fit ( fms - like tyrosine kinase) , KDR ( kinase insert domain - containing receptor)/Flk - 1 (fetal liver kinase - 1) , and Fit -4, respectively. VEGFR - 1 and VEGFR -2 are expressed mainly in the blood vascular endothelium, whereas VEGFR - 3 is restricted largely to the lymphatic endothelium. It is mainly VEGFR2/KDR thatplays a key role on cells growth and differentiation and involved in endothelial cells proliferation and new blood vessel formation. In this experiment we observed that after the cultured HUVECs treated by the conditional medium of the tumor cells, the expression of KDR of the endothelial cells enhanced with the number of tubules of the cultured system increased. This indicates that tumor cells can upregulate the expression of KDR by releasing VEGF, combining and activation of KDR receptor tyrosine kinase, promote tumor angiogenesis and further facilitate the growth of tumors and development of metastases in a paracrine fashion via VEGF/VEGFR - 2 signal transduction pathways.Conclusions1. HUVECs can be induced to develop tubular structure by Matrigel in vitro.2. Tumor cells can promote in vitro induced human vascular endothelial cells by Matrigel to form new blood vessels and in certain ranges showing a dose- dependent and time - dependent manner.3. Tumor cells can promote in vitro induced human vascular endothelial cells by Matrigel to upregulation KDR receptor expression and in certain ranges showing a dose — dependent and time — dependent manner.