The Role And Mechanism Of Megakaryocyte Pyroptosis In Vascular Remodeling Of Pulmonary Hypertension

Background and objectives:Pathological pulmonary vascular remodeling is a common feature of pulmonary arterial hypertension(PAH),which is mainly manifested by the abnormal proliferation and migration of pulmonary arterial smooth muscle cells(PASMC).Abnormal activation of platelets and cytokine secreted disorders participated in pulmonary vascular remodeling.Megakaryocytes play a decisive role in platelet formation and differentiation.In recent years,many authoritative journals such as "Nature" and "J Clin Invest" have found that there are a large number of megakaryocytes in the lungs,and they show unique immune plasticity.The immune plasticity of pulmonary megakaryocytes depends on the tissue and inflammatory environment,and is driven by the environment.An increase in megakaryocyte rupture under inflammatory conditions leads to changes of platelet function and increased release of cellular contents.Whether megakaryocyte function is changed in pulmonary hypertension,resulting in abnormal platelet activation and functional expression has not been studied.Pyroptosis is a novel inflammatory cell death characterized by rapid rupture of cell membrane.It has not been studied whether pyrosis is involved in the regulation of PAH on megakaryocytes.This study is to demonstrate the level of animal and cellular molecules:Gasdermin D mediates megakaryocyte pyroptosis and releases a large number of activated platelets and related cytokines to aggravate pulmonary vascular remodeling.Method:1.Rat models of pulmonary hypertension were induced by monocrotaline(60 mg/kg).The right ventricular systolic pressure(RVSP)was measured by right heart catheterization.The right heart hypertrophy was measured by the ratio of right ventricular weight to right ventricular weight+septum weight.Pulmonary remodeling was observed by H&E staining of lung tissue.2.The expression of GSDMD-N in pulmonary megakaryocytes was detected by immunofluorescence.Flow cytometry was used to detect activation of platelets.Western Blot was used to detect the protein expression of GSDMD and NLRP3 pathway in lung tissues.Serum levels of PDGF,IL-1β and HMGB-1 were determined by Elisa.3.Megakaryocyte were incubated with pulmonary hypertension related factors such as TNF-α to establish a model of megakaryocyte pyroptosis,and Western blot was used to detect the expression of NLRP3、Cleaved-IL-1β、Cleaved-Caspase-1 and GSDMD-N.Flow cytometry was used to detect the activation of platelet-like particles.4.Extract the supernatant as conditioned medium(TNF-a-CM and Control-CM)for culturing PASMC.CCK8 was used to detect cell proliferation and cell migration was detected by scratch test.The content changes of PDGF,IL-1β and HMGB-1 in supernatant were detected by Elisa.5.GSDMD inhibited C57 mice of pulmonary hypertension were induced by monocrotaline(600 mg/kg).RVSP,RVHI and pulmonary artery remodeling were detected respectively.Elisa detection of serum PDGF,IL-1β and HMGB-1 levels.6.Megakaryocytes were treated with disulfiram which is an inhibitor of GSDMD-induced pore formation.Western Blot was used to detect protein expression changes of Cleaved-IL-1β and GSDMD-N.Proliferation and migration of PASMC was detected by CCK8 and scratch test.Results:1.The RVSP and RVHI of the rats with pulmonary arterial hypertension induced by monocrotaline were significantly increased compared to control group(P<0.01).As assessed by H&E staining,the PAH rats showed severe pulmonary vascular remodeling,proliferation of smooth muscle cells in the tunica media,and narrowing of vascular lumen.The proportion of activated platelets in PAH rats was much higher than that in control group.Serum PDGF,IL-1β and HMGB-1 levels were observably increased(P<0.01).2.Immunofluorescence results showed that the number of megakaryocytes increased significantly in rats with pulmonary hypertension induced by monocrotaline,accompanied by pyrophosis of megakaryocytes.GSDMD-N was remarkably expressed in megakaryocytes of lung.The expressions of GSDMD splicing form GSDMD-N in lung tissue were significantly increased(P<0.01).The expressions of NLRP3,Cleaved-Caspase-1 and Cleaved-IL-1β proteins in lung tissue were significantly increased(P<0.05).3.More active platelet-like-particles generated from TNF-α-incubated megakaryocyte.PDGF,IL-1β and HMGB-1 were increased in supernatant(P<0.01).Compare with the control group,the migration and proliferation of PASMC in TNF-a-CM group were increased.By CCK8 experiments,it was proved the cell viability of TNF-α-CM group increased(P<0.01).In addition,the expression of proliferating protein PCNA was significantly increased(P<0.01),and the migration ability of PASMC was enhanced by scratch test(P<0.01).4.Compared with the control group,TNF-α stimulated megakaryocyte had obvious morphological changes of pyroptosis.The cells showed swelling,foaming rupture and finally ruptured.The release of lactate dehydrogenase was significantly increased and GSDMD-N was up-regulated(P<0.01).NLRP3,Cleaved-IL-1β and Cleaved-Caspase-1 proteins of the TNF-α group were significantly up-regulated(P<0.05).5.Silence of GSDMD inhibits monocrotaline induced pulmonary artery remodeling and right ventricular remodeling in C57 mice(P<0.01).The loss of weight was lower than PAH mice too.In addition,the PDGF,IL-1β and HMGB-1 release was significantly reduced.6.Disulfiram inhibits proliferation and migration in TNF-α-CM by blocking cleaved N-terminal GSDMD and megakaryocyte pyroptosis(P<0.01).Through disulfiram ameliorated the morphology and LDH release of pyroptosis(P<0.05).Moreover,the expression of GSDMDN and Cleaved-IL-1β were decreased.Conclusion:1.Megakaryocyte pyroptosis exacerbates pulmonary vascular remodeling in pulmonary hypertension.2.The mechanism is related to GSDMD-mediated pyroptosis of megakaryocyte and the release of a large number of activated platelets,provascular proliferation factors and inflammation factors.