The Alteration In Apoptosis And Proliferation Of Arterial Smooth Muscle Cells In Simulated Microgravty Rats And The Influence Of Intermittent Artificial Gravity On These Changes

The results from both spaceflights and ground-based bed experimentssuggested that humans exposed to microgravity exhibit signs of cardiovasculardeconditioning which was characterized by orthostatic intolerance and reducedexercise capacity. In recent years, many researches about the mechanism oforthostatic intolerance have indicated that the occurance of orthostaticintolerance involves multiple mechanisms. Besides the primaryfactor hypovolemia, diminished compliance of venous systems in legs,changes of vestibular function, altered cardiovascular function and structures arealso involved in it. Findings from human and animal experiments recentlyshowed that the regional special remodeling function and structure in arterialvasculature may play an important role in occrance of postflight cardiovascular dysfunction. Large numbers of relative work at home and abroad showed thatblood vessels of arterial vasculature in upper body regions or forequarters areexposed to higher than normal upright1-G blood pressure, the arteries haveundergone hypertrophic changes, and the vascular wall and media thicken, thenumber of smooth muscle cell layers increases, the myogenic tone andresponsiveness enhance, too. whereas blood vessels of arterial vasculature independent body regions or hindquarters are exposed to lower than normal1-Gblood pressure, such as, the arteries have undergone atrophic changes, and thevascular wall and media thinnen, the number of smooth muscle cell layersdecreases, the myogenic tone and responsiveness attenuate. Our previous studiesalso indicated that ion channel remodeling mechanisms of vascular smoothmuscle cells (VSMCs) and vascular local renin-angiotensin system (L-RAS)might be involved in vascular region-special adaptional changes to microgravity.And at present, exercise-based countermeasures seem insufficient to prevent theoccurrence of orthostatic intolerance in the long-duration spaceflights. When itcomes to countermeasures, our lab has shown that daily1-h standing (STD)first time, which simulates the effects of intermittent artificial gravity (IAG),can obviously prevent the adaptional changes of cardiovascular function andstructures both in short term (3-day) and in medium-term (28-day) simulatedweightlessness. So we think that IAG may be one of the ideal countermeasuresfor the long-duration spaceflight. But up till now, the concrete mechanism of thevascular region-special adaptional changes of structure and function tomicrogravity and IAG is not clear, so it deserves further study. As we know, theproliferation, differentiation and apoptosis are the basic clauses of tissues andorgans keeping cell numbers relatively constant. This study is on the basis of such hypothesis that whether the proliferation and apoptosis of VSMCs inarteries contribute to the vascular region-special adaptional changes of structureand function to microgravity and IAG, and there no related research has beenreported.To verify this hypothesis mentioned above, we used the tail-suspension(SUS) rat model to simulate microgravity and standing countermeasure (STD)rats to simulate the countermeasure effect of IAG, moreover, apoptosis events ofVSMCs were observed by TUNEL and M30staining, and then, the expressionsof apoptosis related proteins were measured,such as Caspase3、Fasl、Bad、Bcl-2and PCNA, in the walls of arteries by immunohistochemistry and Western blotanalysis. Finally, expressions of some growth factors were also measured in thewalls of arteries by Western blot analysis including PDGFA、PDGFRA andVEGF.The main findings of the present work are as follows:Part121days simulation microgravity can lead to the changes ofapoptosis and proliferation of VSMCs from forequarter and hindquarter.inrats.After21days simulated microgravity, the M30CytoDEATH Fluorescein（M30）staining positive cells of common carotid artery and femoral arterysmooth muscle cells decreased and increasd respectively, and the percent ofterminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (Tunel)staining positive cells of thoracic aorta smooth muscle cells decreasedsignificantly (P <0.05. And the results observed with optical microscope byhistomorphometry showed that, as compared with CON, the expressions ofCaspase3and Fasl in thoracic aorta of SUS rats decreasd respectively（P <0.01）, and that of Bcl-2increasd significantly（P <0.05）.Furthermore, the resultsindicated by Western blot analysis that in the thoracic aorta of SUS rats, ascompared with CON rats, the expressions of Caspase3(P <0.01), Bad(P <0.01)and Fasl (P <0.05) decreased, respectively, and the expressions of Bcl-2andPCNA increasd, respectively (P <0.05). whereas in the abdomen aretery fromSUS rats, the expressions of Caspase3(P <0.05), Fasl(P <0.05) and Badincreasd, and the expression of Bcl-2（P <0.05）and PCNA (P <0.01) decreasedsignificantly. These findings suggested that, in SUS rats, the apoptosis ofVSMCs in forequarter decreasd, and increasd in hindquarter, as compared withCON rats. thus, on the basis of these findings, it can be confirmed that theapoptosis and proliferation of VSMCs may play an important role in thevascular region-special adaptional changes of structure and function induced bymicrogravity.Part2The expressions of PDGFA, PDGFRA and VEGF inforequater/hindquater arteries of SUS rats might be involved in vascularregion-special adaptional changes of structure and function toMedium-term (21days) simulation microgravityAfter21days simulated microgravity,the results indicated by Western blotanalysis that in the thoracic aorta of SUS rats, as compared with CON rats, theexpressions of PDGFA (P <0.01), PDGFRA (P <0.01) and VEGF (P <0.05)increasd significantly, whereas in the abdomen aretery, the expressions ofPDGFA、PDGFRA and VEGF decreased significantly(P <0.01). These findingssuggested that, in SUS rats, the expressions of PDGFA, PDGFRA and VEGFin forequater/hindquater arteries might be involved in vascular region-special adaptional changes of structure and function to Medium-term (21days)simulation microgravityPart3Daily1-h standing can aggravate or attenuate the apoptosis ofVSMCs in forequater/hindquater arteries of SUS ratsDaily1-h standing can prevent the hypertrophic or atrophic changes in thewalls of forequater/hindquater areteries to simulated microgravity. As comparedwith either CON rats or SUS rats, M30staining positive VSMCs in commoncarotid areteries increased significantly in STD rats, and in femoral areteries,M30staining positive VSMCs in STD rats increased (vs CON) and decreased(vs SUS)respectively. and the percent of TUNEL staining positive SMCs intharotic aorta of STD rats increased (P <0.05, vs CON) and (P <0.01, vs SUS),respectively. And the results observed with optical microscope byimmunohistochemistry showed that, as compared with CON or SUS rats, theexpressions of Caspase3and Fasl in tharotic aorta of STD rats increasdsignificantly (P <0.01), respectively. and that of Bcl-2decreased with nostatical significance, as compared with SUS rats. and there was only increasingtendency as compared with CON rats. Furthermore, the results indicated byWestern blot analysis that in the tharotic aorta of STD rats, as compared witheither CON rats or SUS rats, the expressions of Caspase3, Bad and Faslincreased, respectively (P <0.01), and that of PCNA increasd (P <0.01)or (P <0.05), respectively, and that of Bcl-2increased or decreasd, respectively, withno statical significance; whereas in the abdominal aretery from STD rats, theexpression of Caspase3, Bad and Fasl increased (vs CON) and decreased (vsSUS) respectively without statical significance, and the expressions of Bcl-2increased (P <0.05, vs CON)or (P <0.01, vs SUS), respectively, as compared with SUS rats, the expressions of PCNA increased significantly (P <0.01). Insum, on the basis of these findings, we can speculate that IAG induced by daily1-h standing can prevent the decreasing apoptosis of VSMCs in the walls offorequater areteries to simulated microgravity, and also can prevent theincreasing apoptosis of VSMCs in the walls of hindquater areteries.Part4Daily1-h standing can incease the expressions of PDGFA、PDGFRA and VEGF in forequater/hindquater arteries of SUS ratsAfter21-day daily1-h standing to simulated microgravity, the resultsindicated by Western blot analysis that in the thoracic aorta of STD rats, ascompared with either CON or SUS rats, the expressions of PDGFA, PDGFRAand VEGF increasd significantly (P <0.01), respectively, and in the abdominalaretery from STD rats, the expressions of PDGFA, PDGFRA and VEGF (P <0.01), as compared with SUS rats. and the expressions of VEGF also increasedsignificantly (P <0.05), as compared with CON rats, but there were onlyincreasing tendencies of PDGFA, PDGFRA. Conclusively, the expressions ofPDGFA, PDGFRA and VEGF in STD rats all increased. These findingssuggested that the increasing expressions of PDGFA, PDGFRA and VEGF inforequater/hindquater arteries might play an important role in the vascularregion-special adaptional changes of structure and function to daily1-h standingto simulated microgravity.In summary, our working hypothesis has been confirmed by these results.Firstly, a decrease or increase of apoptosis of VSMCs was induced by SUSalone rats in thoracic aorta/abdominal arteries, respectively. but daily1-hstanding can prevent these changes. Secondly, the expressions of PDGFA,PDGFRA and VEGF increase or decrease in thoracic aorta/abdominal arteries of SUS rats, respectively. While in STD rats, their expressions are all increased,as compared with both SUS and CON. These findings suggest that apoptosis andproliferation may play an important role in the vascular regional specificremodelings of structure and function to simulated microgravity and IAG.