Alteration Of Human Umbilical Vein Vascular Endothelial Cells Exposed To Modeled Microgravity And Repair Effect Of Carthamin To Microgravity-induced Cellular Abnormality

The cardiovascular system undergoes major changes in stress with space flight. Even with very short-duration exposure to microgravity, the cardiovascular system meets the return to gravity with a syndrome of characteristics collectively termed cardiovascular deconditioning. The susceptible subjects mainly show orthostatic intolerance,combined with heart palpitations, dizziness and pre-syncopal feelings. While the underlying mechanism remains unclear. The endothelium is located in a strategic anatomical position within the blood vessel wall and thereby plays a crucial role in maintaining the integrity of the vasculature. The endothelial cells form a smooth layer which prevents blood cell interaction with the vessel wall. Due to its unique position in the vessel wall, endothelial cells get the ability of selective permeation, anti-thrombus, and may respond to changes in local conditions such as blood pressure, oxygen tension and blood flow by secreting substances, which have powerful effects on the tone of vascular smooth muscle, causing either contraction or relaxation. Under normal conditions, the endothelium induces vasodilation, limits vascular inflammation and decreases platelets adhesion. So the abnormality of endothelial cells may cause kinds of cardiovascular deconditioning.Recently some stimulated weightlessness studies have found endothelial dysfunction might participate in cardiovascular deconditioning. There is an increased circulating apoptotic endothelial cells in -6°chronic bed rest subjects; and induced nitric oxide synthesis (iNOS) was strongly enhanced in the Hind-limb unloading (HLU) rodents model.The machines mimic microgravity carried out in this study is based on the hypothesis that sensing no weight would have similar effects as being weightless, two clinostat modes around one horizontal axis or around the other horizontal axis. After modeled microgravity (MMG), the function and morphology of Human Umbilical Vein Endothelial Cells (HUVECs) were envalued to proved endothelial cells dysfunction could be a possible mechanism of CD. More and more researches report that"Huo-Xue-Hua-Yu"herbs can protect endothelial cells through secreting different kinds of vasoactive substance. And Carthamin could improve endothelial cells function by blocking the cell damage. Based on this evidence the aim of this study is that: 1. to assay whether endothelial cells are damage in MMG and to what extent then we can identify abnormal endothelial cells could be a mechanism of CD; 2. to prove the reverse and protect effect of Carthamin to HUVECs induced by MMG.PartⅠEffect of MMG on cytoskeleton architecture and function of HUVECsObjective: The objective of this study was to investigate the cytoskeleton, proliferation and migration function of HUVECs under MMG.Method: Isolation and culture of HUVECs by enzyme digestion, and cells were identified by flow cytometry (FCM). The 3～4 passage cells were used in this study. The stimulated weightlessness was mimicked by 2D- clinostat. HUVECs were exposed to clinorotation for 48 h at 30 rpm. Cell counting and FCM was used to analyze cell proliferation, apoptosis and cell cycle. Transwell was used to detected cell migration. Confocal microscopy and image analysis were used to analyze modifications of the cytokeratin network, and chromatin structure.Results:①HUVECs'culture and indentify: The expression of CD31 and Factor-Ⅷwere positive in our primary cultured HUVECs;②Cytoskeleton: F-actin in NG controls display their typical radial array with long strong fibers bundles. Disruption of stress fibers and cortical actin ring formation was not evident in MMG samples. In contrast to the preferential orientation toward the cell periphery and parallel to the coverslip, obvious in the NG controls, F-actin in MMG groups with no preferential orientation, some seemed to be perpendicular to the substratum. Instead of long, strongly labeled microtubules radiating throughout the cytoplasm, only a few filaments could be distinguished against the strong background. This more or less diffuse labeling could correspond to either labeled free tubulin subunits or numerous but very short microtubules;③Proliferation: HUVECs proliferation was greatly decreased in MMG while no apoptosis was detected and cell cycling was mainly blocked in G2/M phase (MMG, 27.6%; NG, 18.1%; P<0.05).④Migration: Compared to NG controls, the number of cells migrated through reconstituted basal membrane was significantly decreased in MMG groups (-60%, P<0.05).Conclusion: MMG could affect cellular morphology, migration and proliferation. And the decreased cell proliferation is greatly associated with architecture of microtubules. PartⅡEffect of MMG on FAs architecture, relocation and function of HUVECsObjective: The objective of this study was to investigate this part was①to detect the parameters of FAs (mean vinculin spot number per cell, VN; mean vinculin spot area, VA; the area percentage of overlap of FAs, OSP; average distance of vinculin spots from the cell edge, DS);②to analyze the activity of tyrosine of FAs;③to evaluate the expression of kinases and integrins of FAs.Method: Semiquantitative calculating FAs parameters by confocal and high-pass filtered image. Immunofluorescence staining to detected the PTyr signaling. Total and phosphorylation expression of FAK, pyk2 and ILK were got by Western blot and the expressions of integrins were detected by RT-PCR and Western blot.Result:①FAs alteration: After 48 h of microgravity exposure, the HUVECs presented a major reduction in VN (-50%) (P<0.05) and VA(-60%) (P<0.05), and the DS were different from NG controls(-28%) (P<0.05);②The PTyr signaling was decreased in MMG condition;③FAs kinases: There was no significant change in FAK, ILK and PYK2 protein expression between cells cultured in NG or in MMG condition. The phosphorylation levels of FAK, ILK and PYK2 were decreased compared with NG control cells;④Integrins: MMG inhibited the expression of integrinβ1 andβ4 both in protein and mRNA level. Conclusion: MMG could affect FAs formation and location, moreover it could down-regulate the PTyr signaling, key kinases and integrins expression.PartⅢEffect of Carthamin on altered proliferation, migration and cytoskeleton of HUVECs induced by MMG.Objective: The objective of this study was to investigate this part was to observe the interfering effect of Carthamin on proliferation, migration and cytoskeleton of HUVECs under MMG.Method: Get the proper Carthamin concentration by MTT. HUVECs were exposed to clinorotation for 48 h at 30 rpm. All the samples were divided into 3 groups: NG controls, MMG controls and Carthamin+ MMG. Cell counting and FCM was used to analyze cell proliferation. Transwell was used to detected cell migration. Confocal microscopy and image analysis were used to analyze modifications of the cytokeratin network, and chromatin structure.Results:①Drug dosage: the proper constration of Carthamin is 10μg/ml②Proliferation: HUVECs proliferation was inhibited in MMG. And it shows no signs of recovery when treated with Carthamin;③Migration: The migration was improved when cells were incubated with Carthamin. Compared with MMG controls, the migrate cell number in MMG +Carthamin were increased 38% (P<0.05), while it still less than the NG controls;④Cytoskeleton: After incubated with Carthamin, the amellipodia observed in MMG +Carthamin cells were more plentiful than in MMG controls although the f-actin still loose their typical radial array. Carthamin did not refine microtubules architecture the microtubules in MMG +Carthamin were still show diffuse pattern.Conclusion: Carthamin could repair the blocked HUVECs migration caused by MMG and it is greatly associative with refinement of f-actin structure.PartⅣFAs and RhoA mediate refined migration of Carthamin to HUVECs induced by MMG.Objective: The objective of this study was to investigate this part was to clear whether Carthamin can alter FAs architecture, and discussed the RhoA- mediated effect of Carthamin'refinement of migration arresting induced by MMG.Method: HUVECs were exposed to 2D- clinostat to mimic weightlessness. Investigating the alteration of FAs parameters (VA, VN and DS) after cultured with Carthamin. Evaluated cellular migration, the activity of PTyr, and phosphorylation of its down-stream protein ILK and Cofilin after the RhoA activity was blocked by Exoenzyme C3 Transferase.Results:①FAs architecture: the abnormal FAs parameters induced by MMG can by refined by 10μg/ml Carthamin. The number and area of FAs in MMG+ Carthamin groups were increased compared with MMG controls, VN increased30.3% (P<0.05),VA increased 25.8% (P<0.05). And the DS was also increased 36.4% (P<0.05).②Migration alteration: compared with MMG+ Carthamin groups, the migration cells were less than in the Exoenzyme C3 blocking groups33.3% (P<0.05), it shows in signs of different compared with MMG controls groups.③The activity of PTyr and ILK,Cofilin: the PTyr activity in was down-regulated when the RhoA was blocked by Exoenzyme C3 (-57.1%). The phosphorylation of ILK and Cofilin were higher in MMG+ Carthamin groups than the MMG controls groups, while this refinement were disappear when the RhoA activity were inhibited by Exoenzyme C3 and the ph-ILK and ph-Cofilin were decreased 41.6% and 64.3%(P<0.05)respectively.Conclusion: The repair effect of Carthamin to migration arresting of HUVECs induced by MMG was FAs-- and RhoA-- mediated.