Effect And Mechanism Of Vascular Endothelial Growth Factor On The Permeability Of Blood-tumor Barrier

PrefaceThere are two pathways of drugs being delivered into the tumor cells of blood-brain barrier(BBB):paracellular pathway and transcellular pathway.The pathway chosen depends primarily on the physiochemical properties of each pharmaceutical agent.Hence lipophilic drugs traverse the biological membrane by the transcellular pathway,while hydrophilic ones cross the membrane through the paracellular route.The paracellular route is usually the main route of absorption for hydrophilic drugs,however most drugs are transported transcellularly depending on their physiochemical properties.The transcellular pathway transport plasma proteins similar with or larger than albumin.In normal BBB and blood-tumor barrier(BTB), brain microcapillary endothelium cells(BMECs)have poor endocytosis and pinocytosis. How to enhance transcellular transport is the key that improve anti-cancer drugs entering tumor tissue and theraputic effect of chemotherapy.VEGF is a secreted mitogen associated with angiogenesis and is also a potent vascular permeability factor,belonging to growth factors family.As the strongest vascular permeability factor,the role of increasing vascular permeability was 50,000 times stronger than histamine.Studies have indicated that administration of rhVEGF165 after focal cerebral ischemia might exacerbate the permeability of BBB. Analysis of ultrastructure of endothelial cells showed VEGF played its role by caveolae. Caveolae are specialized plasma membrane structure,located in or near the cell membrane.They are flask-shaped or like Greek letterΩ.Caveolins are major structural proteins of caveolae.VEGF could bind with caveolae to form many pores on endothelial cell membrane which allowed large molecule substances pass through,and promoted transmembrane transportation of biologic molecule to increase the permeability of blood vessels.However,whether administration of VEGF to the rat C6 brain tumor model has the potential to increase permeability of BTB has not been tested.And its mechanism is also not clear.The current study first established rat C6 brain tumor model and administered VEGF.We then measured the BTB permeability through evans blue staining,and observed the number of transendothelial pinocytopic vesicles by electron microscope, examined the experession of caveolin-1 and caveolin-2 to evaluate the relationship between the BTB permeability and content of caveolin-1 and caveolin-2,which would provide theoretic evidence to the mechanism underlying how caveolin-1 and caveolin-2 participate in the modulating of BTB permeability,and eventually provided new target and method to the clinical treatment of brain glioma.Methods1.Establishment of rat C6 brain tumor model.2.In C6 brain glioma rats,the BTB permeability was determined by Evans blue assay before and after VEGF administered.3.In C6 brain glioma rats,the quantity of pinocytotic vesicles were determined by transmission electron microscopy(TEM)before and after VEGF administered.4.Before and after VEGF administering,immunohistochemistry were used to determine the distribution and expression of caveolin-1 and caveolin-2 in C6 brain glioma rats.5.Before and after VEGF administering,western blot assessment was used to detect the proteins expression levels of caveolin-1 and caveolin-2 in C6 brain glioma rats.Results1.C6 glioma rat models were established successfully.2.Administration of 0.01 ng/g,0.05 ng/g,0.1 ng/g,0.2 ng/g and 0.4 ng/g VEGF respectively,When the dosage of VEGF was 0.05 ng/g,the permeability of BTB was increased significantly.3.In C6 glioma rats,the permeability of BTB was increased significantly at 45 min after VEGF administering.After that,they recovered gradually and restored to the level of VEGF 0 min group.4.In C6 glioma rats,the quantity of pinocytotic vesicles were increased obviously about 45 min after administrstion of VEGF,finally returned to the level of VEGF 0 min group.5.In C6 glioma rats,the protein expression level of caveolin-1 and caveolin-2 was increased after VEGF administration.It formed a peak at 45 min,then decreased and finally restored to the level of VEGF 0 min group.DiscussionOur results revealed that VEGF could increase the permeability of BTB significantly,and increase the quantity of pinocytotic vesicles.The expression level of caveolae structural protein caveolin-1 and caveolin-2 is significantly increased compared with the control group after applying VEGF to BTB model in vivo.The peak is formed at 45 min after VEGF administering.Those results showed that VEGF could increase the permeability of BTB by transcellular pathway of increasing the quantity of pinocytotic vesicles.Vascular endothelial growth factor was first described as a tumor-derived factor with potent ability to induce endothelial cell permeability,proliferation and angiogenesis.Many studies indicated proper dosage of VEGF could increase the permeability of BBB and BTB.Our assay of EB extravasation proved that the permeability of BTB was increased obviously in experimental glioma rats after administration of 0.05 ng/g VEGF,and the permeability of EB reached to the peak at 45 min then recovered gradually,finally returned to the level of VEGF 0 min group. The results of other researchers were correspond with us.But whether VEGF increase the permeability of BBB by transcellular pathway or paracellular pathway is not very clear.Many researchers considered VEGF could selectively act on typeⅢtyrosine kinase receptor of endothelial cell membrane to modulate the permeability of endothelial cells.Large molecule substances could be transported through caveolae induced by VEGF.To summarize our TEM studies of microvessels,we observed that the quantity of endothelial pinocytotic vesicles increased apparently about 45 min after administration of VEGF,finally returned to the level of VEGF 0 min group.While extensive research has shown that VEGF was related with the formation of pinocytotic vesicles and fenestrations.But its identified mechanism is unclear. Classical transcellular pathway involving clathrin-coated pits,other ligand induced internalization pathways have been described that involves caveolae and other clathrin/caveolae independent mechanisms.Caveolae are surface invaginations playing key roles in vesicular transport and signal transduction.The structural protein of these plasmalemmal microdomains,caveolin,acts as a scaffold for many caveolar residents. The caveolin-1 isoform is particularly abundant in endothelial cells where it regulates various functions including transcytosis,permeability,vascular tone,and angiogenesis. Caveolin-1 is essential for the formation of caveolae and is a marker for caveolae. Many investigations reveal the change of the caveolin-1 expression level is associated with the increasing of the permeability of BBB and caveolin-2 collaborative caveolin-1 completed endocytosis function.We used immunohistochemistry and western blot assays to research the distribution and expression of caveolin-1 and caveolin-2 after VEGF administering.The results showed that the distribution was changed and the expression level was increased of caveolin-1 and caveolin-2,and the peak was appeared at 45 min then decreased gradually,finally returned to the level of VEGF 0 min group.So we considered that VEGF could increase the permeability of BTB through caveolin-1 and caveolin-2 mediated transcellular pathway.All the results mentioned above indicate that VEGF could increase the quantity of pinocytotic vesicles by increasing the expression of caveolin-1 and caveolin-2.For the first time we prove VEGF increases the permeability of BTB by transcellular pathway at molecular biology level.Conclusions1.VEGF could increase the permeability of BTB significantly and raise the quantity of pinocytotic vesicles in BMECs2.VEGF could promote the expression of caveolin-1 and caveolin-2 in C6 brain glioma rats.The up-regulating of caveolin-1 and caveolin-2 might be one of the reasons of the increasing of pinocytotic vesicles after VEGF administration.