Effects Of Acidic Conditions On Biomechanical Properties,structure And Interaction With Receptors Of Low Density Lipoprotein

Atherosclerosis(AS)is one of the major causes of cardiovascular disease.At the initial stage of AS,due to excessive phagocytosis of native low-density lipoprotein(LDL)and oxidized low-density lipoprotein(oxLDL),macrophages transform into foam cells and deposit in the intima,、、rigerring the formation of AS.Currently,more and more evidences show that the acidic extracellular microenvironment in the intima of arteries may directly influence the formation of atherosclerosis,but the underlying mechanism remains unclear.Therefore,atomic force microscopy(AFM),transmission electron microscopy(TEM),Fourier infrared spectroscopy(FT-IR),agarose gel electrophoresis,Swiss homology modeling,single molecular force spectroscopy and cell experiments were used to study the mechanical properties,three-dimensional structure,and receptor recognition of LDL/oxLDL under acidic conditions.Thesis paper mainly includes the following aspects:1.The biomechanical properties of single LDL/oxLDL particles were detected dynamically in situ by AFM under different acidic conditions(p H 7.4 ～ p H 4.4).The statistical results showed that oxidation significantly reduced the Young’s modulus and increased the adhesion of LDL and that under slightly acidic condition(p H 6.4)it can significantly reduced the Young’s modulus of LDL and oxLDL but had no obvious effect on the adhesion force.The experimental results showed that oxLDL particles were softer and more viscous than LDL and that the acidified extracellular microenvironment could make LDL/oxLDL softer.2.We verified the feasibility of using NP-40 to remove LDL lipids for the exposure of apoB-100 in physiological state.By integrating existing models and relevant experimental data(including AFM,TEM,FT-IR,Swiss homology modeling,hydrophobicity analysis,etc.),the simulated structure of apoB-100 was modified,and it was concluded that the “α1-β1-α2-β2-α3” structure of apoB-100 in liquid physiological state is "17+22+19+35+13 nm".3.The effects of the extracellular acidic microenvironment on the particle size and structure of LDL/oxLDL were evaluated.The experimental results showed that:(1)In the process of gradual acidification(p H 7.4 ～ p H 4.4),the overall structure of LDL or oxLDL did not collapse and the protein components were not released However,during this process,the height of LDL/oxLDL was significantly reduced and the particle size increased first(p H 6.4)and then decreased(p H 4.4),accompanied by the release of cholesterol;(2)oxidation caused the breakdown of the peptide chain of LDL protein(apoB-100)and the two break sites are likely to be located in the α3 domain of its C-terminal;(3)In the process of gradual acidification,the secondary structure of LDL/oxLDL changed significantly,including the increase of α helix content and the decrease of β folding,and the dramatic change occurred at p H 6.4;(4)acidification and oxidation leaded to condensation of apoB-100 peptide,collapse of its 3-D structure,and decrease of its lipid-binding ability.4.Four methods(single molecular force spectroscopy,immunoelectron microscopy,AFM-based molecular immunity,and cellular immunity)were used to detect the effects of acidic and oxidative conditions on physiological activity of LDL by measuring the interactions between LDL and LDLR/CD36 and between LDL/oxLDL and endothelial cells at different p H values.The results showed that the interactions between LDL and LDLR and between LDL and endothelial cells decreased significantly under acidic condition(p H 6.4)and that oxidation induced excessive recognition and phagocytosis of oxLDL by endothelial cells whereas the interactions between oxLDL and CD36 and between oxLDL and endothelial cells were not influenced by acidification.In summary,this thesis systematically evaluated the changes of biomechanical properties,structure,and receptor recognition of LDL under acidic and oxidized conditions,revised the THREE-DIMENSIONAL structural model of LDL,and preliminarily demonstrated the pathological basis of atherosclerosis induced by LDL/oxLDL under physiological and pathological conditions.This will provide a basis or new idea for the prevention and treatment of atherosclerosis and drug development.