The Role And Mechanism Of CGAS-STING Pathway In Hypoxia-induced Proliferation Of Rat Pulmonary Artery Smooth Muscle Cells

BackgroundChronic hypoxia is an important cause of hypoxic pulmonary hypertension（HPH）,and its main pathological features are abnormal pulmonary vasoconstriction and progressive pulmonary vascular remodeling.Hypoxia can cause pulmonary vasoconstriction at the early stage,however,continuous hypoxia can cause nonreversible lung vascular remodeling,Which is the key pathological basis for the development of HPH.A variety of cells are involved in this disease process,including pulmonary artery smooth muscle cells（PASMCs）,lung fibroblasts,pulmonary vascular endothelial cells.The hyperproliferation of PASMCs is considered to be the primary cause of pulmonary vascular remodeling.Therefore,studying the mechanism of hyperproliferation of PASMCs under hypoxic conditions is of great significance for the prevention and treatment of HPH.As the powerhouse of cells,mitochondria not only produce ATP through the tricarboxylic acid cycle and electron transport chain of cellular respiratory but also participate in various cell activities such as oxygen sensing,inflammatory response,cell signal transduction,autophagy regulation,proliferation and apoptosis.Studies have shown that abnormal mitochondrial dynamics,mitochondrial metabolic remodeling and mitochondria-derived damage associated molecular patterns（MTDs）are involved in the occurrence and development of PH.Mitochondria are the evolutionary endosymbionts derived from bacteria,thus,they may carry bacterial molecular motifs.Multiple mitochondrial components can be regarded as MTDs,including:formylated peptides,mitochondrial DNA（mt DNA）,cardiolipin,succinic acid,cytochrome C,reactive oxygen species（ROS）,etc.Normally,mt DNA is located in the mitochondrial stroma,which is released into the cytoplasm or bloodstream in response to cellular stress such as hypoxia,sepsis,trauma,cytotoxicity,or violent fluctuation of mitochondrial membrane potential.Mt DNA,as an important MTD,plays a significant role in the progression of acute kidney injury,cardiovascular disease,arthritis and other diseases.Cyclic GMP-AMP synthase（c GAS）is activated by binding cytoplasmic DNA and catalyzing the production of c GMP.As a second messenger,c GMP activates the stimulator of interferon genes（STING）,participating in autophagy,inflammation,immunity and other pathological processes.Studies have shown that mt DNA,abnormally released from mitochondria,activates the c GAS-STING pathway,and participates in the regulation of endothelial cell proliferation.It was found that mt DNA levels were elevated in plasma and bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis,while mt DNA levels in plasma were correlated with disease severity in patients with COPD.Hypoxia can drive a series of pathological changes such as mitochondrial stress,damaged mitochondrial function,and disturbance of the ROS level.Whether hypoxia can induce mt DNA release or mt DNA is related to cell proliferation are still obscure.We hypothesized that hypoxia can induce PASMCs to release mt DNA and activate the downstream c GAS-STING pathway,mediating hypoxia-induced cell proliferation.Hence,this research devotes to elucidating the role and mechanism of mt DNA and its downstream c GAS-STING pathway in the hypoxia-induced proliferation of PASMCs through small interfering RNA,drug intervention and other technologies,yearning to find a new solution for the prevention and cure of HPH.Methods1.Rat pulmonary artery smooth muscle cells（RPASMCs）were applied to build a cell proliferation model.The experiment is divided into three groups:normoxia group（21%O₂）,hypoxia 24 h group（1%O₂）,hypoxia 48 h group（1%O₂）,to find out the optimal proliferation time as the subsequent experimental conditions.2.Mitochondrial permeability transition pore（MPTP）inhibitor,CSA,was used to inhibit the release of mt DNA,small interfering RNA sequences（si RNA）was used to knock down STING expression.3.The results of cell proliferation activity and PCNA expression together reflected cell proliferation.Western blot was used to evaluate the expression of c GAS and SING,q PCR was used to quantify cytosolic mt DNA release.4.Proteomics was used to detect protein changes in cells which knocked down the STING under hypoxia 48 h（1%O₂）.Results1.In Comparison to the normoxia group,24 h and 48 h exposure to hypoxia（1%O₂）can significantly enhance the proliferation of RPASMCs（P<0.05）,and significantly up-regulate the expression of PCNA（P<0.01）2.After hypoxia exposure,the release of mt DNA from RPASMCs was increased（P<0.01）,and the m RNA and protein expressions of c GAS and STING were significantly up-regulated（P<0.05）.3.Inhibition of MPTP blocked hypoxia-induced mt DNA release（P<0.05）,down-regulated the expressions of c GAS,STING and PCNA（P<0.05）,and significantly inhibited hypoxia-induced RPASMCs proliferation（P<0.05）.4.Interfering STING expression with si RNA significantly down-regulated the expression of PCNA（P<0.05）,and abrogated the hypoxia-induced proliferation of the RPASMCs（P<0.01）.5.Proteomics results demonstrated that compared with the NC group,the regulation of smooth muscle cell proliferation signaling pathway was significantly changed after STING knockdown,and the level of PDCD4 protein was prominently up-regulated,which was further verified by Western blot results（P<0.01）.Conclusion1.Hypoxia induced the release of mt DNA from RPASMCs,promoted the cell proliferation and up-regulated c GAS,STING m RNA and protein expressions.2.Mt DNA regulated the proliferation of RPASMCs by activating the c GAS-STING pathway under hypoxia.3.STING regulated the proliferation of RPASMCs via suppressing the PDCD4 signaling pathway under hypoxia.