The Role Of MAMs In Hypoxia-induced Smooth Muscle Cell Proliferation And Endothelial Cell Injury

Background:Hypoxic pulmonary hypertension is characterized by persistent pulmonary vasoconstriction and obvious pulmonary vascular remodeling,causing a significant increase in pulmonary artery pressure,and this finally leading to heart failure.Underlying the pathogenesis,the imbalance between cell proliferation and apoptosis is the convergence point,which presents excessive vascular smooth muscle cell proliferation and endothelial cell damages.Chronic hypoxia exposure induces vascular smooth muscle cell proliferation,and vascular remodeling.Vascular smooth muscle cells is also regulated by other cell types.Endothelial cells constitute a monolayer of endothelium covering the vascular lumen and regulate vascular smooth muscle cell proliferation,aggregation and adhesion of platelets,and secretion of endogenous diastolic substances.Hypoxia could induce endothelial apoptosis,and direct NF-?B-mediated expression of adhesion molecules and pro-inflammatory cytokines,recruiting inflammatory cells infiltration.Meanwhile,the secretion of vasodilative sustances is reduced,and contractile substances increase.As a result,endothelial injury exacerbates hypoxia-induced vascular remodeling.Mitochondria serve not only as energy?producing machines but also as signalling hubs that orchestrate and activate a set of signals such as those associated with autophagy,inflammatory response,proliferation/apoptosis.During hypoxia,abnormal changes in mitochondrial structure and function are observed in both vascular endothelial cells and smooth muscle cells.Hypoxia could induce glycolysis in vascular smooth muscle cells,and inhibit glucose oxidation?GO?.A decrease in the level of mitochondrial Ca²⁺(Ca_m²⁺)is one of the predominant reasons underlying this impairment.As a result,the activity of Ca²⁺-dependent GO enzymes,including pyruvate dehydrogenase,isocitrate dehydrogenase,?-oxoglutarate dehydrogenase and ATP synthase,is compromised,leading to GO inhibition,accompanied with an imbalance of mitochondrial oxygen free radicals.Mitochondrial dysfunction direts the activation of downstream proliferative signaling pathways such as HIF-1?,NFATc and downregulated Kv1.5,thereby promoting cell proliferation.In addition,hypoxia induces endothelial mitochondrial rupture,swelling,activates mitophagy,and impairs mitochondrial biogenesis,thereby directing NF-?B-mediated inflammatory molecular expression,promoting the secretion of contractile factors and inhibiting NO synthesis.Mitochondria-associated Endoplasmic Reticulum membranes/mitochondria-associated membranes?MAMs?,the close contact between the endoplasmic reticulum and the mitochondrial membrane,involves in mitochondrial bioenergetics.The proteins anchored respectively at endoplasmic reticulum membranes and mitochondrial outer membranes interact and serve as a linkage to maintain membrane contacts.IP₃Rs/GRP75/VDAC1 is one of the endoplasmic reticulum-mitochondrial protein linkers that mediates endoplasmic reticulum-mitochondrial calcium transfer and mitochondrial function.Additionally,calcium release could be affected by the interation of IP₃Rs with chaperone protein or phosphorylation of IP₃Rs by kinase Akt in MAMs.Taken together,endoplasmic reticulum-mitochondrial calcium transfer is regulated by MAMs formation and IP₃Rs/Grp75/VDAC1 calcium transfer axis-related modification signals.In addition to regulating intracellular calcium transfer,the core proteins in multiple biological processes are presented at the interface of MAMs.Thus,MAMs widely involves in lipid metabolism,autophagy,mitochondrial division,immune response,apoptosis and endoplasmic reticulum stress.A series of studies have shown that through the proteomic analysis of liver and brain tissues,thousands of proteins and dozens of kinases and phosphatases are discovered at MAMs interface.Under pathological conditions,the expression,modification level of proteins and signaling pathways in MAMs are significantly changed,which coordinate the pathogenic processes of disease.It has recently been reported that silencing Nogo-B expression enhances the formation of MAMs during hypoxia,promotes endoplasmic reticulum-mitochondrial calcium transfer,in such that way vascular smooth muscle cells apoptosis is increased,and hypoxic pulmonary hypertension is prevented.In a congenital pulmonary hypertension model,Nogo-B receptor?NgBR?expression is inhibited,and this promotes ROS-mediated vascular smooth muscle cells proliferation.However,the molecular mechanism needs to be further clarified.In addition,the role of MAMs in hypoxic endothelial cells is still unclear.Therefore,this study was undertaken to observe the roles of MAMs formation and MAMs signaling pathways in endothelial cells and vascular smooth muscle cells during hypoxia,and explored the factors contributing to MAMs changes.As such,targeting MAMs enhances our understanding on the pathogenesis of hypoxia-induced vascular remodeling.Methods:1.A hypoxia-induced vascular smooth muscle cell proliferation model was established.Then the formation of MAMs was determined during hypoxia by detections of MAMs constituent proteins and electron microscopy.Vascular smooth muscle cells were transfected with endoplasmic reticulum-mitochondrial linker to promote MAMs formation.And its effect in mitochondrial respiration and proliferation/apoptosis were explored under hypoxia.2.Sufficient MAMs protein of vascular smooth muscle cells were obtained.Then the proteomic changes of MAMs in hypoxia and its role in endoplasmic reticulum-mitochondrial calcium transfer were analyzed.3.The role of Nogo-B/NgBR in MAMs formation and endoplasmic reticulum-mitochondrial calcium transfer signaling in vascular smooth muscle cells were investigated.The expression of Nogo-B and NgBR were detected by a specific spontaneous regression model of hypoxic pulmonary hypertension in vivo and hypoxia-reoxygenation model in vitro.Treated with overexpression and inhibition of Nogo-B or NgBR,MAMs constituent proteins and endoplasmic reticulum-mitochondrial calcium transfer pAkt-IP3R3pathway in cells were detected by WB.Mitochondrial calcium,mitochondrial respiration,mitochondrial membrane potential,mitochondrial oxygen free radicals and cells proliferation were determined with oxygen consumption rate,laser confocal and WB.4.A hypoxia-induced endothelial cell injury model was established.Then the formation of MAMs was determined during hypoxia by detections of MAMs constituent proteins and endoplasmic reticulum-mitochondrial marker fluorescence labeling.To attenuate the formation of MAMs,MAMs constituent protein PACS-2 or IP₃R1 was downregulated by siRNA.Then,its protective effect on hypoxia-induced endothelial cell injury was explored.The items on mitochondrial function including mitochondrial calcium,mitochondrial respiration,mitochondrial membrane potential and oxygen free radicals,apoptosis including flow cytometry detection,PARP,PCNA expression and supernatant LDH enzyme activity,inflammatory molecular protein and mRNA expressions,and eNOS-NO pathway were checked.Results:1.The expression of proliferation-related protein PCNA was increased in hypoxic vascular smooth muscle cells by WB detections,while the expression of apoptosis-related protein cleaved-caspase-3 decreased.Meanwhile,an increase in the percentage of positive cells labeled with Edu was appreciated by IF.The expression of MAMs constituent proteins were decreased in hypoxia.The shortest distance between endoplasmic reticulum and mitochondrial membrane increased in electron microscopy observations,reflecting the destruction of MAMs formation.Transfected with endoplasmic reticulum-mitochondrial linker,mitochondrial respiration mounted in vascular smooth musle cells,pAMPK?and PCNA protein expression increased in hypoxia,and the cleaved PARP decreased.2.Through GO analysis,the differential proteins at MAMs interface in hypoxia were mainly located in endoplasmic reticulum,which had binding and catalytic activity,and participated in biological processes such as signal processing,biological regulation,stimulation response and protein localization.Enrichment analysis revealed that differential proteins were mainly involved in amide synthesis and inhibitions of cell differentiation.The KEGG pathway enrichment analysis indicated that endoplasmic reticulum protein processing and sphingolipid metabolism were significantly activated during hypoxia,and Nogo-B protein was increased at this interface.3.Nogo-B protein expression was increased in hypoxia-induced vascular remodeling by IF,whereas NgBR decreased.During the reversal of vascular remodeling induced by reoxygenation,Nogo-B expression was downregulated,and NgBR was upregulated.Nogo-B expression in intact cell homogenates was unchanged in hypoxia in vitro.However,its distribution in MAMs increased by WB and immunoprecipitation detections.NgBR expression was decreased in hypoxic vascular smooth muscle cells in vitro.Overexpression of Nogo-B or inhibition of NgBR inhibited the formation of MAMs,which were characterized by down-regulation of MAMs constituent proteins or decreased coefficient index of endoplasmic reticulum-mitochondrial labeling and the shortest distance between endoplasmic reticulum and mitochondrial outer membrane observed by electron microscopy.In addition,endoplasmic reticulum-mitochondrial calcium transfer pAkt-IP₃R3 sigaling pathway were activated by WB detections.A decrease in mitochondrial calcium level was detected by Rhod-2 am probe.A inhibition of mitochondrial respiration was detected by oxygen consumption measurements.At the same time,an increase in mitochondrial membrane potential,a decrease in mitochondrial oxygen free radicals,and an increase in the percentage of positive cells in which HIF-1?enters the nucleus were appreciated by IF.The detections on MTT,PCNA,Cyclin D1 protein expression or the percentage of Edu positive nucleus showed that overexpression of Nogo-B or inhibition of NgBR induced vascular smooth muscle cell proliferation under normoxia.Inhibition of Nogo-B or overexpression of NgBR reversed the above effects,and inhibited hypoxia-induced vascular smooth muscle cell proliferation.4.Through WB and IF,the expression of MAMs constituent proteins in HPAEC and HUVEC and the endoplasmic reticulum-mitochondrial marker fluorescence coefficient index increased.MAMs formation was inhibited with siRNA targeting MAMs constituent protein PACS-2 or IP₃R1.After that,IF detections suggested that mitochondrial calcium signal decreased,mitochondrial membrane potential increased,and ROS production decreased under hypoxia.The oxygen consumption rate measurements showed an increase in basal respiration and ATP production.Flow cytometry detections revealed that increased percentage of early apoptosis,late apoptosis,and total apoptosis were diminished under hypoxia.A decrease in cleaved PARP and an increase in PCNA expression were detected by WB,accompanied with the lowered LDH activity in cell culture supernatant.In addition,hypoxia-induced NF-?B phosphorylation was significantly inhibited.The expression of proinflammatory molecules ICAM-1,VCAM-1,E-selectin,MCP-1,IL-6,IL-1?were decreased by Realtime PCR,WB and Elisa detections.In the end,WB and Elisa assays showed that hypoxia-suppressed eNOS S1177 phosphorylation was rescued,with the increased eNOS activity.As a result,NO concentration in cell supernatant increased.Conclusions:1.Hypoxia could induce vascular smooth muscle cell proliferation,and inhibit MAMs formation.Promoting the formation of MAMs induces cells apoptosis and inhibits proliferation during hypoxia.2.At MAMs interface,the endoplasmic reticulum protein processing and sphingolipid metabolism are significantly activated during hypoxia,and the expression of Nogo-B protein increases.As such,they involve in the endoplasmic reticulum stress response,ceramide,sphingosine,and sphingosine 1-phosphate metabolism and regulation of endoplasmic reticulum-mitochondrial calcium transfer,to induce vascular smooth muscle cell proliferation.3.The protein expression pattern of Nogo-B and NgBR is separated in hypoxia-induced vascular smooth muscle cell proliferation.The distribution of Nogo-B at MAMs interface increase,and remaines unchanged in the total homogenate under hypoxia.NgBR expression is reduced during hypoxia.Overexpression of Nogo-B or inhibition of NgBR inhibits MAMs formation and directs the activation of endoplasmic reticulum-mitochondrial calcium transfer pAkt-IP₃R3 pathway,thereby reduces mitochondrial calcium,impairs mitochondrial function,and promotes vascular smooth muscle cell proliferation.4.The formation of MAMs increases in hypoxic endothelial cells.MAMs destruction attenuates hypoxia-induced mitochondrial impairment,cell apoptosis and inflammatory responses,and increases NO production during hypoxia,.The above studies indicate that MAMs are expected to be an effective strategy against hypoxic pulmonary hypertension.