Functions of vascular endothelial cells Cardiovascular disease is initially induced by changes and damage of vascular endothelial cell function. During the detachment of endothelial cells, plasma component (lipid) infiltration, macrophage infiltration and intimal smooth muscle cell proliferation, damage and dysfunction of vascular endothelial cells occur earliest, and play a key role in the occurrence, formation and repair of cardiovascular disease.   Vascular endothelial cells are a cell population as a layer covering the systemic vascular intima. Since this cell population has been successfully cultured in vitro, we have a very in-depth understanding of them. It is now thought that the endothelial layer is not only a barrier to blood and tissue, but also has a variety of other functions, such as reducing vascular permeability, regulating material exchange between tissues and blood, preventing disorderly invasion of plasma components and blood cells, resisting thrombosis, balancing anti-blood coagulation- fibrinolysis system and anti-platelet function, maintaining blood fluidity, adjusting vascular smooth muscle function, synthesizing and secreting factors related to vascular smooth muscle relaxation and contraction, and suppressing migration and proliferation of vascular wall cells[12-27].   Vascular permeability In vivo studies have observed that plasma component infiltration to the endothelium appears during endothelial cell division. To understand how lipid traverses endothelial cells, endothelial cells are co-cultured with low density lipoprotein. Results show that ApoB and ApoE receptors of endothelial cells are not associated with transendothelial transport of low density lipoprotein. In contrast, under histamine and thrombin stimulation, the cell permeability is evidently improved, and abundant low density lipoprotein is detected. It is thus clear that some biological factors (e.g., histamine), shear stress and newborn endothelial cells are associated with endothelial cell permeability.   Antithrombotic effects In the 19th century, pathologist Virchow proposed three main factors of thrombosis including vascular wall damage, blood stagnation and blood anticoagulant system abnormalities. Under normal circumstances, endothelial cells exert antithrombotic effect and maintain blood fluidity. Nevertheless, with blood vessel wall impairment and the presence of cytokines, tissue factor is activated, antithrombotic ability of endothelial cells weakened, and thrombus easily formed within the blood vessel wall. The mechanisms underlying anti-thrombosis of vascular endothelial cells are as follows: (1) synthesis and secretion of anticoagulant factors, such as thrombomodulin, heparin-like substances and tissue factor pathway inhibitor; (2) synthesis and secretion of pro-fibrinolytic factors, such as tissue plasminogen activator; (3) synthesis and secretion of factors that inhibit angiogenesis and platelet aggregation, such as prostacycline and endothelial-derived relaxing factor. These factors keep the blood circulation and prevent thrombosis.   Proliferation and migration of vascular endothelial cells Vascular endothelial cells can be induced to exert inhibitory effects on the proliferation of vascular smooth muscle. This mechanism can be affected by various factors secreted from above endothelial cells. Vascular endothelial cells also can proliferate and migrate under the stimulation of platelet, leukocyte-secreted migration factor and flow shear stress. This function is probably associated with arterial repair and neovascularization.   Tissue-engineered endothelial cells on the surface of grafts Implantation of exogenous endothelial cells Herring (1978) proposed that endothelial cells implanted on the surface of inhibitors could increase the survival of grafts. Theoretically, the presence of a layer of confluent endothelial cells can elevate the antithrombotic ability of grafts, and prevent the occurrence of pseudointimal hyperplasia. The surface of the intact endothelial cells is negatively charged, which can reject platelet adhesion and exhibit obvious anticoagulant activity. Further studies have discovered that the patency rate of the grafts implanted with endothelial cells and antiplatelet is higher than that of grafts without endothelial cells. Moreover, the grafts implanted with endothelial cells show a strong antibacterial ability. However, these grafts show insignificant differences in lessening pseudointimal hyperplasia in the anastomotic area. In fact, high levels of platelet-derived growth factor (PDGF) can be detected in grafts implanted with endothelial cells, which is associated with the strong effect of PDGF on stimulating the migration and proliferation of smooth muscle cells. Thus, pseudointimal hyperplasia appears.   One of the difficulties in endothelial cell culture is the relatively low density of cells used for transplantation, because of which cells cannot fully attach to grafts. In the process of trying to solve these problems, the researchers attempt to cultivate endothelial cells in two stages. The obtained endothelial cells are amplified in vitro until 100% confluence, and then implanted into the human body. Magometschnigg reported that vascular endothelial cells were implanted on fibrin-coated ePTFE grafts. The early patency rate increased by 20%, and the amputation rate decreased by 20% when above grafts were used in femoral-tibial bypass bridging. The disadvantage of this technique is the possibility of potential infections. Cell culture technology is more difficult and requires a second surgery. Another method to achieve the best cell density is to use microvascular endothelial cells. After small retinal vascular cells obtained by enzyme digestion are implanted on small-diameter Dacron grafts, a confluent endothelial cell layer will form with no thrombosis. As previously reported, a dog model exhibited an elevated 1-year patency rate. The current focus is to implant tissue-engineered endothelial cells on the synthesized grafts. Dichek has successfully transferred plasminogen activator genes into endothelial cells. Thus, modified cells could overexpress this fibrinolytic agent and enhance antithrombotic ability of grafts. In order to stimulate the proliferation and migration of endothelial cells and prevent excessive proliferation of smooth muscle cells, it is possible to make excessive secretion of growth factors and growth inhibitory factors from endothelial cells by transgene[28-34].   Stimulating re-externalization and angiogenesis of endogenous endothelial cells A mitogen and chemoattractant for endothelial cells can theoretically stimulate trans-interstitial capillary ingrowth, and enhance endothelialization of synthetic vascular grafts, mainly using fibroblast growth factor (FGF)-1(acidic FGF, aFGF) and FGF-2 (basic FGF, bFGF). Blood vessels can be formed in situ through the re-externalization of endogenous endothelial cells. This process mainly contains migration, proliferation and differentiation of vascular cells. Small blood vessels have been prepared successfully in the body. One of the specific models is: capillary endothelial cells induced by basic fibroblast growth factor project into collagen or fibrin matrix and form blood vessels. Angiogenesis induced by fibroblast growth factor is coupled with proteolytic enzymes produced by endothelial cells. The production of protease is a key step in angiogenesis, and provides a tool for endothelial cells to change extracellular matrix and to enter perivascular spaces. The structure of another vascular model in vivo is: cultured endothelial cells, smooth muscle cells and adventitial fibroblasts are paved in remodeled collagen and Dacron culture systems. Two weeks later, a confluent layer is formed to serve as an endothelial barrier. Different from the human body’s blood vessels, the tissue-engineered blood vessels requires a prosthesis to support and to maintain its structure, and do not contain elastin[11, 35-36].   Fixation of cell growth-promoting factor on the surface of polymeric biomaterials To realize the endothelial cellularization on the surface of polymeric biomaterials, bioactive factors should be introduced into the surface of materials. The bioactive factors mainly are proteins. The fixation methods of proteins on the surface of polymers are physical adsorption and chemical fixation. In addition, there are the newly developed ion implantation and self-assembled monolayer methods. Of them, the physical adsorption is relatively convenient, and contains two types: (1) electrostatic adsorption, for example, the heparin containing a negative charge is fixed in a positively charged site of the materials; (2) adsorption by intermolecular force, for example, adsorption between proteins and polymer molecules. In contrast, chemical bonding is more stable and overcomes the shortcoming in physical adsorption, while bioactive molecules cannot exert long-term effects on the surface of the material, and are easy to break away. The chemical fixation usually requires that the substrate surface has hydroxyl group, carboxyl group and amino-group. Therefore, the production of these groups on the surface of the materials by modification is the premise of fixation. Simultaneously, the fixation can cause the degeneration of proteins, and an optimal interaction between cell receptors and proteins cannot be established, so a spacer arm should be inducted in advance on the surface of a polymer. The chemical fixation of proteins usually contains two steps: polymer surface activation and reaction between activated surface and protein[37-53].   Protein fixation on the surface containing hydroxyl polymer Sulfonyl chloride method:Hydroxyl group activated by sulfonyl chloride generates reactive sulfonate, and further produces C-N or C-S bond by reaction with protein amine groups or thiol groups so as to fix proteins.   Carbodiimide method: Hydroxyl group activated by imide generates imide-N-methyl ester, which contains leaving group. Thus, urethane bond is produced by reacting with protein aminogen to fix the protein.   Diisocyanate method: Diisocyanate exerts a coupling effect. Hydroxyl group without activation reacts with one end of the coupling agent. One end of the coupling agent reacts to protein and fixes the protein.   Protein fixation on the surface containing arboxyl polymer The activation of carboxyl group can be carried out by N-hydroxysuccinimide and 1-ethyl-3- (dimethylaminopropyl) carbodiimide.   Protein fixation on the surface containing amino polymers Amino-group can be coupled to proteins through coupling agents diisocyanate, glycol aldehyde and epoxy resin.   Irradiation fixation Irradiation fixation refers to fixation of proteins on the surface of polymers by a bifunctional coupling agent under the condition of irradiation. Light activator includes photosensitive azo, benzophenone and acrylate. Endothelialization of the material surface Endothelialization of the material surface is a new trend of anticoagulant study. The endothelialized surface mainly refers to pseudo-intimal surface or hybridized surface of endothelial cells and high polymer[54-56]. Pseudo-intimal surface refers to a layer of stable red thrombosis film forming on their interfaces when the material contacts with blood. The components of the film include plasma protein, platelet, fibrin and leukocytes. Thus, fibroblasts and endothelial cells grow on the film to form an intima similar to blood vessel wall, i.e., the pseudo-intima. At present, artificial blood vessels made of polytetrafluoroethylene with pseudo-intimal surface have been applied in the clinic[57-58]. It is worth noting that if the pseudo-intima of artificial blood vessels is very thick, nutrition will not support the body, cells will be necrotic and fall off, and coagulation will occur at the exposed part. A large number of studies have been carried out to control the thickness of the pseudo-intima. For example, porous polytetrafluoroethylene invades water-soluble polyvinyl alcohol to form a porous hydrophilic membrane on its surface and to reduce the adsorption of plasma proteins. The pseudo-intima formed on the artificial blood vessel wall is not a true vascular intima. The component and thickness of the formed protein layer cannot be controlled well. Although some improvements have been taken, the pseudo-intima on the artificial blood vessel wall does not achieve an effective compatibility to the transplantation site. Figure 1 shows the artificial blood vessel lined with hybridized endothelial cell polymer[59]. Endothelialization of material surface is a new trend of anticoagulant research. The blood vessel wall made of human endothelial cells is the only known blood-compatible material. Thus, we make hybridization of endothelial cells and polymers by imitating human blood vessels. That is to culture endothelial cells on the surface of material, finally forming a layer of endothelium. This would be a very ideal way for the improvement in blood compatibility of the material, especially the improvement in blood compatibility of small-diameter artificial blood "