Human Pulmonary Artery Endothelial Cells Senescence Induced By Cigarette Smoke Extract Was Involved In The Process Of Pulmonary Vascular Remodeling

A variety of reasons could lead to chronic obstructive pulmonary diseases and the most dominated factor was cigarette smoking. Pulmonary hypertension(PH) is the most risky symptom among the complications of COPD and contributed to right ventricular failure. The pathological traits of PH is the pulmonary vascular remodeling(PVR) which origins from proliferation of pulmonary artery smooth muscle cells and muscularization of nonmuscular vessels. The underlying mechanism was still not clear yet. It is widely known that cigarette smoking could induce the reactive oxidant species(ROS) accumulated, we could assume that cigarette smoking is the important environmental factor for PVR. Besides, a large amount of ROS accumulation led to cells senescence, cigarette smoking could accelerate the cells telomere attrition and the unstable chromosomes which aggravated the level of cell senescence. Senescence-associated secretory phenotypes(SASP), which is derived from the senescent cells, contains all kinds of inflammation and cell growth factors and could induce human pulmonary artery smooth muscle cells proliferation and differentiation. Nrf2 is the master protein which is the most crucial to resist ROS and cell senescence to keep cells from exogenous injuries by regulating the expression of the downstream genes. Nevertheless, the Nrf2 expression decreased in the patient suffered from COPD. To our knowledge, there are few studies about the role of Nrf2 in pulmonary vascular remodeling induced by cigarette smoking. In the previously study, we have confirmed that human pulmonary artery endothelial cells were senescent after 72 h cigarette smoke extract(CSE) exposure. At the beginning of exposure to the CSE, Nrf2 increased while it reduced as the CSE-exposure prolonged.Therefore the relations between Nrf2 protein and cell senescence may be critical to the process of PVR.Part 1 CSE could induce human pulmonary artery endothelial cells senescent and lead to DNA injury.Objective:To observe the CSE exposure how to influence on the HPAECs after 72 h. Methods: HPAEC s exposed to CSE for different time periods and cell cycle was detected by flowcytometry analysis. The senescence-associated markers, p53 and p21, were analyzed by Real-time PCR and the senescence-associated ?-galactosidase assay was used to calculate the number of senescent cells. Flow-fish was used to measure the length of cells which were treated by CSE for different times. Results: Compared with the control group, the CSE group cells cycle arrested apparently after 72 h exposure and telomere shortened(P<0.05);The p53 and p21 expression increased gradually and cells became senescent. Conclusion: CS exposure could accelerate HPAECs senescent and DNA damaged.Part 2 Nrf2 played an important role in the process of HPAEC senescence induced by prolonged CSE-exposure.Objective: to detect the relationships between CSE impact on HPAEC and the Nrf2 protein expression. Methods: HPAECs were transfected with Nrf2 si RNA and revealed the cell senescence, Then the cells were transfected with pc DNA3.1-Nrf2 to up regulate Nrf2 expression and identified that the senescence was related to Nrf2. furthermore, tert-Butylhydroquinone(t BHQ), the Nrf2 activator, is used to exert a protective role to show the relation between Nrf2 and cells senescence induced by the long term CSE exposure. Nrf2?DJ-1?HO-1?NQO1?P53or P21, senescence-associated proteins, were measured by western blotting, and the m RNA were detected by Real-time PCR. The senescence-associated ?-galactosidase assay was used to detect the senescent cells. Results:(1) After 12 hours exposed to CSE, cells transfected with si Nrf2 would be premature senescent. The senescence-related protein P53 and P21 increased and the antioxidant function declined.(2) After 72 hours exposed to CSE, cells transfected with pc DNA3.1-Nrf2 which upregulated cytoprotective response were not evidently senescent.(3) The effect was similar with the pc DNA3.1-Nrf2 group and the senescent related genes and proteins were lower than the normal. Conclusion: In the first 12 hours after CSE stimulation, the cytoprotective role of Nrf2 was powerful to resist ROS. As the exposure time prolonged, Nrf2 consumed and CSE induced cell premature senescence.Part 3 SASP, secreted by premature senescent HPAEC, induced human pulmonary artery smooth muscle cells(HPASMC) proliferation.Objective: To observe the HPASMC proliferation induced by the SASP which secreted by HPAEC after CSE exposure. Methods: Real-time PCR was used to detect the expression of SASP related genes including IL-6?IL-8?MCP-1?TNF-? and so on.. The condition medium was prepared to culture HPASMC and the cells proliferation was analyzed by CCK8. Transwell assay was used to detect the migration of HPASMC which was cultured in the condition medium. The flowcytometry and ki-67 immunofluorescence was used to reveal the relation between the proliferation of HPASMC and the Nrf2 expression in HPAEC. Results : b FGF?IL-6?IL-8?MCP-1?TNF-??TGF-? decreased in the first 24 hours and then it increased gradually during CSE exposure. For promoting the cell proliferation, the 72 hours condition medium has the most evident positive effects. The promotion of cells proliferation was evident with the prolonged CSE exposure. The result of transwell assay was consistent with that of the CCK8 assay. Compared with the t BHQ and pc DNA3.1-Nrf2 groups, the CSE group cells showed significant proliferation statistically. The ki-67 immunofluorescence showed the similar trends as the cell cycle analysis did. Conclusion: The senescent HPAECs could secreted SASP which induced HPASMCs proliferated.