Research On The Expression Of INOS And Its Transcriptional Regulation Mechanism In Human Umbilical Vein Endothelial Cell Induced By Clinorotation

Individuals exposed to extended periods of microgravity during spaceflight or prolonged 6°head-down tilt often suffer from symptoms of cardiovascular deconditioning when returning to Earth or upright posture, respectively. The cardiovascular dysfunctions include reduced exercise capacity, increased incidence of orthostatic intolerance, and frank syncope. These alterations in orthostatic stability named postflight orthostatic intolerance(POI)can adversely affect the performance of physical work. The underlying mechanisms are multifactorial and recent works show that this is mostly due to reduce plasma volume, attenuated baroreflex sensitivity, inadequate peripheral resistance response, increased venous compliance, and impaired vasoconstrictive response in such individuals, but the exact etiology remains elusive. Evidences from human and animal studies indicate that vascular contractile hyporesponsiveness is a valuable contributor in the occurrence of POI. Yet, the cellular and molecular mechanism underlying vascular contractile hyporesponsiveness remain to be fully elucidated.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(ECs)are in close contact and form a smooth layer that prevents blood cell interaction with the vessel wall. Due to its unique position in the vessel wall, ECs 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. Nitric oxide (NO), an endogenous modulator which is produced by various cell types in different tissues, has been shown to regulate numerous biological processes. This potent endogenous vasodilator plays an important role in the regulation of cardiovascular function. NO is synthesized by the enzyme nitric oxide synthase(NOS)from an L-arginine substrate. Three isoforms of NOS enzymes have been characterized, two of which are constitutively expressed and a third which is inducible. In contrast to the two constitutive enzymes, iNOS produces large, sustained amounts of NO. It has recently become evident that ECs are highly sensitive to microgravity, including the alterations in morphology and gene expression. The studies in the Hind-limb unloading(HLU)rodents model, which has been extensively used as a model to simulate cardiovascular consequences of microgravity in humans, have clearly shown that NO synthesis was strongly enhanced and NO released from the endothelium is an important factor inducing vasodilation. It has been reported that both activity and expression of iNOS are increased in many vasculatures such as the femoral artery from HLU rodents. However, to our knowledge, the alterations of the iNOS and the possible mechanisms underlying the regulation of iNOS in ECs cultured in real or simulated microgravity remain to be determined. In this study, we determined the changes of expression and promoter activity of iNOS in HUVEC-C after 24 h simulated microgravity exposure and further explored the mechanism of its transcriptional regulation. The main results and findings of this work are as follows:1. Effects of simulated microgravity on the expression and promoter activity of iNOS in HUVEC-C We investigated the effects of simulated microgravity on the expression and promoter activity of iNOS in HUVEC-C cultured for 24 h. Results were as follows: The level of iNOS mRNA and protein was significantly increased in clinorotation group. And the level of iNOS mRNA and protein maintained very low level in stationary and rotational controls. Reporter gene assays showed 2.29-fold induction of luciferase activity in clinorotation HUVECs transfected with phiNOS(7.2)Luc compared with that in controls. These data indicated that iNOS was activated at the transcriptional level by 24 h simulated microgravity in HUVEC-C.2. Effects of simulated microgravity on the transactivations of NF-κB and AP-1 in HUVEC-C We investigated the effects of simulated microgravity on the transactivations of NF-κB and AP-1 in HUVEC-C cultured for 24 h by transfecting with cis-reporter plasmid. Results were as follows: The relative luciferase activity in the clinorotation cells cotransfected with pNF-κB-Luc and pRL-TK was markedly decreased about 48% in comparison to that of stationary or horizontal rotation controls. And the relative luciferase activity in the clinorotation cells cotransfected with pAP-1-Luc and pRL-TK was markedly decreased approximately 38% in comparison to that of stationary or horizontal rotation controls. These data indicated that the transactivation of NF-κB and AP-1 can be downregulated by 24 h simulated microgravity.3. The mechanisms underlying regulation of iNOS induced by simulated microgravity in HUVEC-C We investigated the alterations of expression and promoter activity of iNOS by changing the activities of NF-κB and AP-1 after 24 h simulated microgravity to explore the mechanisms of iNOS regulation in transcriptional level. Results were as follows: The expression of iNOS mRNA and protein were not altered by PDTC. And there was no significant change in iNOS promoter-driven luciferase activity with treatment of PDTC. The data shown here indicated that NF-κB was not involved in the regulation of iNOS by clinorotation. The expression of iNOS mRNA and protein were markedly enhanced in 24 h clinorotation with treatment of 20μM SP600125, an inhibitor of AP-1. Consistent with the result of iNOS expression, relative luciferase activity of iNOS was significantly increased in clinorotation with treatment of SP600125 compared with that in no treatment cells. The expression of iNOS mRNA and protein were evidently attenuated in 24h clinorotation by cotransfection of c-Jun/c-Fos. Overexpression of AP-1 significantly reduced the luciferase activity in phiNOS(7.2)Luc transfected cells. These data indicated that a decrease in AP-1 activity, but not NF-κB, contributes to increased expression of iNOS by clinorotation.4. The binding regions of transcriptional factors related to the iNOS expression induced by 24 h clinorotation To delineate the binding regions of transcriptional factors on the human NOS2 promoter, we stimulated HUVEC-C cells with 24 h clinorotation following transfection of iNOS luciferase constructs contain different length promoter regions. Results were as follows: Construction of phiNOS(0.6)Luc and phiNOS(1.0)Luc, a plasmid contains 0.6 kb and 1.0 kb of upstream 5'flanking DNA linked to the luciferase reporter gene, has been studied respectively. The PGL3-basic served as negative control. Transient transfections of the iNOS promoter-driven luciferase constructs into HUVECs and stimulation with clinorotation revealed no significant induction of luciferase activity in phiNOS(0.6)Luc and phiNOS(1.0)Luc in comparison with that of non-clinorotation. These data indicated that clinorotation did not increase promoter activity of constructs containing≤1.0kb of proximal human iNOS promoter region and there is no DNA-binding region of transcriptional factors related to iNOS expression induced by 24 h clinorotation.In conclusion, we examined effects of 24 h clinorotation on the expression of iNOS and explored the mechanism in transcriptional level. Our present results show that the iNOS expression and promoter activity can be induced by simulated microgravity in HUVECs. The transactivation of NF-κB and AP-1 can be downregulated by 24 h simulated microgravity. The expression of iNOS can be inhibited by overexpression of AP-1 and not by inhibition of NF-κB. Our present results indicate that the inhibition of AP-1 rather than the inhibition of NF-κB is involved in the pathways of iNOS activation by clinorotation. Our finding might help to know the possible mechanism of the vasculature dysfunctions induced by microgravity and provide some theoretical basis to the protection of postflight orthostatic intolerance.