The Effects Of Substrate Stiffness On HUVEC Adhesion With THP-1 Cells And Molecules Associated With Adhesion

Cardiovascular Disease(CVD) has become the primary threat to human health. According to "Chinese cardiovascular disease report 2013". Almost 4.0 million people in our country die of cardiovascular disease each year, which account for 40% of the total cause of death. The increased vascular stiffness is included in the common features of cardiovascular disease. Bad habits such as excessive smoking, picky eating with no or less exercise even with the growing age promotes the reduction of elastin in vascular wall. On the other hand collagen is accumulated, as well as cross-linkage between collagen is increased, eventually vascular stiffness occurs. Vascular stiffness make the condition favorable for various cardiovascular disease. Apart from collagen and elastin, adhesion between endothelial cells and immune cells mediated by molecules associated with adhesion and Chemokines is also one of the key step in cardiovascular disease. Studies shows that molecules associated with adhesion even in response of mechanical force such as shear stress produced by blood and the improperly expression of gene, but, if substrate stiffness as a feature of cardiovascular disease can influences molecules associated with adhesion even unknown. Finding the relationship between vascular stiffness and endothelial cell adhesion may provide a new target for the treatment of cardiovascular diseases.In this topic, we are adjusting the composition ratio of acrylamide to get polyacrylamide gels(PA gel) with various rigidity, to simulate the stiffness of normal and abnormal vessel, then, assess the influence of substrate stiffness on molecules associated with adhesion in endothelial cell. The main contents and conclusions are as follows:(1)Arcylamide and bis-arcylamide were reacted with different proportion for different polyacrylamide gel. Sulfo-SANPAH and collagen(Col-I) were used for surface modification of gel. The Young's modulus of gel were 11.15±0.1kPa, 32.51 ±1.91 kPa and 80.64±2.11 kPa. This meets the requirements of simulating normal and abnormal vessel. To investigate the effects of substrate stiffness on the ability of HUVECs adhesion THP 1 cells, we co-cultured HUVECs and THP-1 cells.(2)In order to exclude the effect of Col-I on gel, we analyzed Col-I on different stiffness by immunofluorescence. The binding capacity of Col-I with the change of stiffness was not significantly changed. Human umbilical vein endothelial cells(HUVECs) cultured on different gels, when cells more than 80%, to extracted total RNA and protein. Using immunofluorescence, western blotting and fluorescence quantitative PCR technology analysis the influence of stiffness on molecules associated with adhesion. From experimental results, the m RNA and protein expression levels of molecules associated with adhesion HUVECs on normal physiological stiffness(30kPa) were lower than the pathological stiffness(80kPa and 10kPA). Interestingly, we found that both mRNA and protein levels of inhibitors of differentiation-1(Id1) in HUVECs cultured on gel with different stiffness have same trend as molecules associated with adhesion, namely the lowest on 30 kPa.(3)On the basis of Id1 mRNA and protein levels trend are consistent with molecules associated with adhesion. We built Id1 overexpression HUVECs to study whether Id1 has relation with adhesion-related molecules affected by basal stiffness or not. After protein and mRNA were detected determining Id1 overexpression. Using western blotting and fluorescence quantitative PCR technology analysis the influence of Id1 overexpression on molecules associated with adhesion was determined. Both mRNA and protein of molecules associated with adhesion increased in Id1 overexpression HUVECs, which was similar to gels under pathological stiffness. To sum up, through this work we found that change in the stiffness of HUVECs live will influence molecules associated with adhesion, namely molecules associated with adhesion increased in pathological stiffness. Likewise, Id1 is also highly expressed in pathological stiffness. And, molecules associated with adhesion will high expressed in transfected HUVECs, which may provide a new sophisticated way for prevention of cardiovascular disease by controlling vessels stiffness.