The Role Of Integrin ?v?3 In Basilar And Femoral Arterial Remodeling In Simulated Microgravity Rats And Influence Of Intermittent Artificial Gravity

Post-flight orthostatic intolerance poses great threat to the health of astronauts and undermines their readaptability to the ground.Upon exposure to microgravity,transmural pressure across arteries could be changed by cephalic shifting of body fluid evoked by the disappearance of hydrostatic pressure.Head-ward fluid shifts are associated with elevated intravascular pressure in the upper body and reduced intravascular pressure in the legs.While arteries especially the resistance arteries are capable of compensating these elevated or reduced intravascular pressure by changing their contraction in the early stage,the structural and functional remodelling may occur to maintain relatively normal stress and tissue perfusion when the pressure changes sustain.Previous human and animal studies in real and simulated microgravity have revealed that arteries in the upper body undergo hypertrophy and exhibit thickened vascular wall and tunica media,increased smooth muscle layers,enhanced vasoconstriction and myogenic tone,while the vessels in the lower body undergo atrophy and exhibit atrophic vascular wall and tunica media,decreased vasoconstriction and myogenic tone.The insufficiency of peripheral resistance and excessive contraction of the cerebral arteries caused by the regional specific arterial remodelling may result in the lower-body blood deposition and impaired autoregulation of the cerebral vasculature,which may act synergistically in the occurrence of post-flight orthostatic intolerance.At present,due to the fact that exercise-based countermeasures are still insufficient to prevent post-flight orthostatic intolerance,it is imperative that more effective countermeasures be developed to meet the demand of future long-term space flight.Our previous animal studies have found that daily 1-h restoration of orthostatic posture by normal standing can completely prevent the region-specific structural and functional arterial remodelling of some arteries induced by simulated microgravity.Therefore,intermittent artificial gravity(IAG)may be an ideal countermeasure against long-term space flight in the future.However,the underlying mechanisms of the simulated microgravity-induced arterial remodelling and the counter effect of IAG are still unknown.Integrins are transmembrane receptors that heterodimerize by ? and ? subunits in a noncovalent fashion.The extracellular domain can recognize a wide variety of extracellular matrix(ECM)including collagen,fibronectin and laminin and their intracellular domain can connect with cytoskeleton and signal protein,thereby forming the extracellular matrixintegrin-cytoskeleton axis,which participates in the mechanical and biological signal transduction of cells and has many important biological functions.Integrins mainly function through various intracellular kinases,among which the integrin-focal adhesion kinase(FAK)signalling pathway is the key pathway.Both vascular smooth muscle cells(VSMCs)and endothelial cells(ECs)are regulated by integrin-FAK signalling pathway,and integrin-FAK signalling pathway is found to be up-regulated in various cardiovascular pathophysiological processes such as hypertension,pulmonary hypertension,atherosclerosis,and pressuremediated arterial myogenic tone and cardiomyocyte remodelling and is involved in diverse cardiovascular functional and structural remodelling such as endothelial dysfunction,proliferation and migration of VSMCs and cardiomyocytes through different downstream mechanisms.Besides,focal adhesion(FA)is a dynamic protein complex that exists in the intracellular domain of integrin and is the convergence point of mechanical signal transduction in integrin signal transduction.However,studies about whether the integrin-FAK signalling pathway participates in the simulated microgravity-induced arterial remodelling and counter effect of IAG are still rare.Thus,in this study,4-week hindlimb unweighted(HU)rats were used to simulate the physiological effects of microgravity,and daily 1-h normal standing was used to simulate the effects of IAG.The structural remodelling,extracellular matrix alteration and changes of integrin signalling pathway in the basilar and femoral arteries of HU and IAG rats were investigated by the Hematoxylin-Eosin staining,Masson staining,Western Blot,Immunohistochemistry and Immunofluorescence,etc.The major findings of the present study are as follows:(1)The basilar and femoral arteries underwent structural remodelling,and some proteins in extracellular-matrix altered after exposure to simulated microgravity.Compared with the CON rats,the wall thickness(Tw)and intima-media area and the content of collagen and laminin of the basilar artery were significantly increased in HU rats;the wall thickness(Tw)and intima-media area and the content of collagen and laminin of femoral artery significantly decreased.(2)The integrin ?v?3 signal pathway was significantly upregulated in basilar artery and was significantly downregulated in femoral arteryWestern blot,immunohistochemistry and immunofluorescence staining were used to detect the expression and localization of integrin ?v?3,focal adhesion kinase(FAK),Src,and extracellular signal-regulated kinase 1/2(ERK1/2)and the formation of focal adhesions(FAs)in VSMCs of basilar and femoral arteries induced by 4-week simulated microgravity.Compared with CON group,the expression of integrin ?v,integrin ?3,p-Src Y418,p-FAK Y397 and p-ERK 1/2 and the number of FAs in the basilar artery of HU rats were significantly increased;while the expression of integrin ?v,integrin ?3,p-Src Y418,p-FAK Y397 and p-ERK 1/2 and the number of FAs in the femoral artery were significantly decreased.(3)Daily 1-h IAG prevented the structural and extracellular-matrix remodelling induced by simulated microgravity.Compared with HU rats,the wall thickness(Tw),the intima-media area and the content of collagen and laminin of the basilar artery in the IAG rats were significantly increased;while the wall thickness(Tw),the intima-media area and the content of collagen and laminin of the femoral artery in the IAG rats were significantly decreased;no significant changes were found between CON and IAG rats.(4)Daily 1-h IAG prevented the upregulated and downregulated integrin ?v?3 signal pathway and the altered number of FAs in basilar and femoral arteries induced by simulated microgravityCompared with HU rats,the expression of integrin ?v,integrin ?3,p-Src Y418,p-FAK Y397 and p-ERK 1/2 and the number of focal adhesions in the basilar artery of IAG rats were significantly decreased;while the expression of integrin ?v,integrin ?3,p-Src Y418,p-FAK Y397,and p-ERK 1/2 and the number of focal adhesions in the femoral artery of IAG rats were significantly increased;except the number of focal adhesions in basilar artery of IAG rats was still more that that of CON rats,no significant difference was found between CON and IAG rats.In summary,after 4-week simulated microgravity,the basilar artery underwent hypertrophy with increased extracellular matrix and upregulated integrin ?v?3 signal pathway,while the femoral artery underwent atrophy with decreased extracellular matrix and downregulated integrin ?v?3 signal pathway.Besides,daily 1-h IAG can prevent the above changes induced by simulated microgravity.Combined previous research results,we speculated that the increased or decreased transmural pressure across the vasculature may be transmitted through the extracellular matrix-integrin-cytoskeletal axis,establishing a signal transduction pathway which involving integrin aggregation,formation of focal adhesion and combination of cytoskeletal and signal proteins and mediating the stressinduced vascular remodelling,while short-period restoration of the stress may block this process.The mechanism by which IAG prevents vascular remodelling may be due to the transient block of the signal transduction.