Remodeling And Local RAS Activity In Elastic Arteries Of Simulated Microgravity Rats And Its Prevention By-G_x Gravitation

Terrestrial animals and humans have adapted to a constant force of 1 G. Exposed to microgravity, undoubtedly all gravitational blood pressure gradients from head to feet that are associated with upright posture on Earth disappear, and a redistribution of transmural pressure and blood flows across and within the arterial vasculature is induced by the removal of hydrostatic pressure gradients. Therefore, in humans, vessels in brain and upper body regions are chronically exposed to higher than normal upright 1-G blood pressure, whereas vessels in dependent body regions are exposed to lower than normal 1-G blood pressure. Our previous ground-based animal studies have shown for the first time that simulated microgravity may induce upward and downward regulations in function, structure, and innervation state of the medium- and small-sized muscular arteries from fore (cerebral) and hind body parts of the same animal subjected to tail-suspended head-down tilt (SUS). On the basis of these findings and the relevant ground-based and spaceflight studies reported recently, we have raised the"peripheral effector mechanism hypothesis". We suggested that, in addition to hypovolemia, the microgravity-induced adaptation changes in function and structure of cardiac muscle and vascular smooth muscle might be another important factor responsible for postflight cardiovascular dysfunction. We further showed that the ion channel remodeling mechanism of vascular smooth muscle cells (VSMCs) and vascular local renin-angiotensin system (L-RAS) might be involved in vascular region-specific adaptational changes to microgravity. In the study of countermeasure, we have shown for the first time that daily 1-h standing (STD), which mimics the effect of intermittent artificial gravity (IAG), can surprisingly prevent the adaptational changes in cardiac muscle and muscular arteries due to microgravity alone.However, there remain several problems to be further elucidated. First, our previous data have been mainly from the studies with medium- and small-sized muscular arteries of rats and little information is available regarding the elastic large arteries. In order to obtain a perfect understanding of vascular adaptation to microgravity, these data are indispensable. For example, numerous studies have demonstrated a direct relationship between the circumferential stress and the structure of the vessel wall itself. Furthermore, at higher pressure loadings, the circumferential stress is highest at the inner wall, and declines across the wall. If redistribution of vascular transmural pressure is the primary stimulus initiating regional adaptation in arteries during microgravity, then the remodeling changes of innermost SM layers would be most significant. Second, recently more attention has been focused on the problem of whether microgravity can induce a decrease in aortic compliance. Therefore, the changes in extracellular matrix in the media of elastic large arteries should be further studied. Thirdly, the role of L-RAS in mediating vascular adaptation to microgravity still need be further clarified by pharmacological blockade and relevant methods. Furthermore, many sophisticated experimental methods can be used with specimens from large arteries. Finally, though we have shown that the gravity-based countermeasure might have a surprising counteracting effect on the adaptation remodeling of medium-sized muscular arteries, it still need be further examined with large elastic arteries.In order to elucidate the aforementioned problems, we used SUS rat model to simulate the microgravity-induced effects on arterial vasculature and daily 1-h standing to simulate the countermeasure effect of IAG. Moreover, we measured the thickness of each smooth muscle layer and elastic lamina, and the cross-sectional area of the wall and luminal diameter of large arteries and etc. by histomorphometry. we measured the expression of the key elements of L-RAS, angiotensinogen (A_O) and angiotensinâ…¡receptor type 1 (AT1R) in the wall of large arteries using immunohistochemistry, Western blot analysis, in situ hybridization, and real-time PCR analysis. Finally, using the established ex vivo perfusion artery organ culture system, we made a preliminary observation on the effect of daily short-duration restoration to low pressure in preventing vascular remodeling due to a sustained high pressure.The main findings of the present work are as follows:(1) A 28-d simulated microgravity may induce region-specific adaptation changes in medium-sized muscular arteries from fore and hind body in rats.The results observed with electron microscope in our previous work were again validated with optical microscope by histomorphometry. The results showed that compared with control group (CON), the media thickness (T), media cross-sectional area (CSA), and number of smooth cell layers (N_CL) of basilar artery from tail-suspension group (SUS) increased by 25.9%, 17.6%, and 20.5% (P<0.01), respectively, after a 28-d simulated microgravity. Both the luminal diameter (D) and mean CSA of smooth muscle cell (AC) showed no significant differences between CON and SUS. Whereas, compared with CON, the T, CSA, and NCL of the anterior tibial artery from SUS decreased by 15.2%, 17.3%, and 13.6% (P<0.01), respectively, and both D and AC showed no significant differences between the two groups.(2) A 28-d simulated microgravity may induce region-specific adaptation changes in large elastic arteries from fore and hind body in rats.The results observed with optical microscope showed that the average thickness of the wall (T_W) of the common carotid artery in SUS was significantly greater than CON group by 25.6% (P<0.01), and there were no significant differences in the parameters, such as the D, mean number of smooth muscle layers (N_M), and mean number of elastic luminae (N_EL) in the two kinds of arteries and in the TW of the abdominal aorta between CON and SUS groups. In the wall of common carotid artery, there are four smooth muscle layers and four elastic laminae. In SUS rats, the thickness of each smooth muscle layer from M1 to M4 increased by 44.0%, 42.4%, 22.2%, and 39.8% (P<0.01), respectively, as compared with that of CON rats. In the wall of abdominal aorta, there are nine muscle layers and nine elastic laminae. In SUS rats, the thickness of each muscle layer from M1 to M4 decreased by 17.0%, 11.7%, 11.4%, (P<0.01) and 10.1% (P<0.05), respectively, but from M5 to M9 it did not show significant changes. Furthermore, In the common carotid artery of SUS group, the CSA of each muscle layer increased significantly, with the trend that the maximum enlargement was in M₁ and M₂. While in the abdominal aorta of the SUS rats, the maximum decrease was in M₁, M₂, and M₃ layer. On the contrary with changes of smooth muscle layer, the thickness and CSA of each elastic lamina showed a trend of decrease and increase in the common carotid artery and abdominal aorta, respectively, after SUS, and some of these differences were statistically significant.(3) A 28-d simulated microgravity may induce up- and down- regulation of gene and protein expression of A_O and AT1R in the wall tissue of carotid artery and abdominal aorta in rats.The immunohistochemical results showed that stained A_O and AT1R are in brown color and mainly located in adventitia and perivascular tissue, but less in media. In the wall of the common carotid artery from a SUS rat, more intensive A_O and AT1R immunoreactivity were detected in the media and adventitia as compared with that of a CON rat. On the contrary, in the wall of the abdominal aorta from a SUS rat, these immunoreactivities were scarcely detected as compared with that of a CON rat. The results further indicated by Western blot analysis that in the common carotid artery from SUS rats, the A_O and AT1R protein expression increased by 130.0% and 50.0% (P<0.05), respectively, as compared with CON rats. Whereas in the abdominal aorta from SUS rats, the A_O and AT1R protein expression decreased by 48.9% and 36.8% (P<0.05), respectively, as compared with CON rats. The results by in situ hybridization showed that the specific signals of the A_O and AT1R mRNA were located in the media and adventitia of the two kinds of vessels. In the wall of the common carotid artery from a SUS rat, more intensive A_O and AT1R mRNA signals were detected in the media and adventitia as compared with that of a CON rat. On the contrary, in the wall of the abdominal aorta from a SUS rat, these mRNA signals were scarcely detected as compared with that of a CON rat. The results were further indicated by real-time PCR. Compared with CON rats, in the common carotid artery from SUS rats, A_O and AT1R mRNA expression increased by 164.7% (P<0.01) and 70.2% (P<0.05), respectively. Whereas in the abdominal aorta from SUS rats, A_O and AT1R mRNA expression decreased by 32.6% (P<0.05)and 55.1% (P<0.01), respectively, as compared with that of CON rats.(4) Chronic blockade of AT1R cannot prevent completely hypertrophic changes in basilar and carotid arteries due to simulated microgravity in rats.After four weeks of treatment by losartan, all the morphometric parameters of basilar artery, anterior tibial artery, common carotid artery, and abdominal aorta from the two treated group [treated control group (C+L) and treated tail suspension group (S+L)] significantly decreased as compared with untreated groups. For example, compared with control group (C), the T, CSA, Ac, and NCL decreased by 15.5%, 17.6%, 11.3%, and 10.3% (P<0.05 or <0.01) in the basilar artery from C+L, respectively, and these parameters decreased by 30.2%, 21.1%, 16.1 %, and 23.7% (P<0.05 or <0.01) in the anterior tibial artery from C+L, respectively. Compared with C, the T decreased by 29.9% and 8.2% (P<0.01) of common carotid artery and abdominal aorta from C+L, respectively. However, compared with C+L, the T, CSA, and NCL increased by 12.2%, 14.3%, and 5.7% (P<0.05 or <0.01) in basilar artery from S+L, respectively, and these parameters of anterior tibial artery from S+L showed no significant changes. In common carotid artery from S+L the T increased by 23.0% (P<0.05) as compared with C+L. Compared with C, A_O and AT1R expressions of common carotid artery from C+L showed no significant changes, and both expressions of abdominal aorta from C+L significantly decreased. The A_O and AT1R expressions of common carotid artery from S+L showed no significant changes, and the AT1R expression of abdominal aorta from S+L obviously decreased (P<0.01) as compared with C+L.(5) Daily 1-h STD over 28 days fully prevented remodeling and L-RAS changes in large elastic arteries that might occur due to simulated microgravity alone in rats.The surprising effectiveness demonstrated in medium-sized muscular arteries was further confirmed in the present study with large elastic arteries. Compared with CON, the T_W, CSA and thickness of each smooth muscle layer of common carotid artery from suspension for 23 h/day and STD for 1 h/day group (SUS+STD1) showed no significant changes, but these parameters showed significant differences (P<0.01) as compared with SUS. Moreover, compared with CON, the thickness of each smooth muscle layer from M₁ to M₄ and CSA from M₁ to M₄ in SUS+STD1 group showed no significant changes, but these parameters showed significant differences (P<0.05 or <0.01) as compared with SUS. Furthermore, Daily 1-h STD fully prevented changes of protein and gene expression of AO and AT1R in the two kinds of elastic large arteries as well.(6) The hypothesis that pressure is the primary stimulus is supported by the preliminary result from the ex vivo experiment.Segments of rat common carotid artery can be maintained under different perfusion pressure schedules in organoid culture for three days. At 150 mmHg, not 0 or 80 mmHg, a marked expression of c-fibronectin (c-FN) was observed in the innermost smooth muscle layers of the media. However, daily 4-h restoration to 0 mmHg during the sustained high pressure perfusion under 150 mmHg prevented the enhanced c-FN expression.In conclusion, these findings have provided data to further support our hypothesis that pressure itself is the primary stimulus that initiates regional adaptation of arteries during microgravity and vascular L-RAS plays an important role in its regulation. Our work has also provided physiological evidence in support of the effectiveness of gravity-based countermeasure, like the IAG. The artery organoid culture system might provide an ideal ex vivo experimental model for further mechanistic study of mechanism.