Study On The Role And Mechanism Of The Novel Anti-inflammatory Mediator Maresin-1 In Pulmonary Hypertensio

BackgroundPulmonary arterial hypertension(PAH)is a progressive and life-threatening disease characterized by severe pulmonary arterial remodeling,leading to increased pulmonary vascular resistance and pulmonary artery pressure,and ultimately,to right ventricular failure.Increasing evidence of PAH pathophysiology has contributed to several effective therapies including drugs targeting prostacyclin,endothelin and nitric oxide pathways with selective pulmonary vasodilator properties.Although the current therapeutic drugs significantly improved symptoms,they are limited in reversing pulmonary vascular remodeling and decreasing mortality.Thus identifying novel treatments targeted pulmonary vascular remodeling in PAH is still critically needed.Growing evidence has illustrated that pulmonary artery smooth muscle cells(PASMCs)overproliferation and mitochondrial dysfunction are important elements contributing to pulmonary vascular remodeling and the pathophysiology of PAH.Inhibition of excessive proliferation of PASMCs,improve mitochondrial homeostasis may be new therapeutic targets for reversing pulmonary vascular remodeling and PAH.Macrophage mediators in resolving inflammation(Maresins)are a family of specialized pro-resolving lipid mediators(SPMs)derived from the ω-3 fatty acid docosahexaenoic acid(DHA).MaR1 is the first member of Maresins family and plays critical role in protecting against many cardiovascular diseases,such as atherosclerosis,abdominal aortic aneurysm,hypertension,diabetes,myocardail infarction and arrhythmia etc.However,the effect of MaR1 on the PAH development are not clear.And the relationship of MaR1 and mitochondrial homeostasis in the pathogenesis of PAH has not been investigated as well.Here we aimed to explore the role and mechanism of MaR1 in the development of PAH and provide a new therapeutic target for the treatment of PAH.ObjectiveThe aim of this study was to clarify the role of MaR1 in the progression of PAH and investigate the underlying mechanisms.MethodsFor in vivo experiments,monocrotaline(MCT)-induced rat and hypoxia+SU5416(HySu)-induced mouse models of pulmonary hypertension(PH)were used.MaR1(100mg/day)and/or BOC-2(a specific antagonist of ALXR,50μg/kg)were used to investigate the role of MaR1 and its receptor ALXR in the development and therapeutic effect of PH.The right ventricular systolic pressure(RVSP)was measured directly by right ventricular intubation.The heart was collected and the ventriculars were carefully dissected and weighed.RV hypertrophy was assessed by Fulton index measurements(weight of RV/weight of left ventricle plus septum [RV/LV+S]).HE and α-SMA staining of lung tissue sections were used to detect the vascular remodeling index and the degree of vascular muscularization.DHE and Mit SOX Red staining were performed on lung tissue sections to detect reactive oxygen species(ROS)production.The ATP assay and total SOD assay kits were used to measure ATP levels and SOD activity in lung tissues.Lung tissue proteins were extracted,and the expressions of proliferation-related proteins PCNA,CyclinD1 and mitochondrial homeostasis related proteins were detected by Western blot.For in vitro cell culture experiment,primary extracted rat pulmonary artery smooth muscle cells(PASMCs)were used.After 3% hypoxia treatment,cells were given MaR1and/or LGR6,ALXR,RORα specific antagonists or shRNA adenovirus treatment to clarify the role of MaR1 and the three receptors in hypoxy-induced PASMCs proliferation and mitochondrial oxidative stress damage.RNA-seq high-throughput sequencing was performed to explore the possible molecular mechanism.CCK-8 kit was used to detecte cell viability,Western blot was used to detect the expression of CyclinD1 and mitochondrial homeostasis related proteins.DHE and Mito SOX Red fluorescent probes were used to to detect ROS production in PASMCs,mitochondrial membrane potential probes JC-1 and TMRM were used to detect mitochondrial membrane potential.The ATP assay and total SOD assay kits were used to measure the ATP levels and SOD activity in PASMCs.ResultsIn the present study,we demonstrated that MaR1 content was decreased in serum from both MCT-induced PH rats and HySu-induced PH mice,MaR1 treatment delayed the development of experimental PH and attenuated pulmonary vascular remodeling in the two models.Mechanistically,MaR1 treatment significantly decreased mitochondrial injury,inhibited ROS production,increased ATP and SOD levels,and rescued the impaired signaling of mitochondrial dynamics and mitophagy.In primary cultured hypoxia-exposed PASMCs,MaR1 also profoundly suppressed cell proliferation,decreased oxidative stress,increased mitochondrial membrane potential,restored disturbed mitochondrial dynamics,and decreased mitochondrial autophagy.Our data strongly revealed that MaR1 may contribute to PAH prevention by protecting mitochondria.Moreover,we found that abrogating the function of MaR1 receptor ALXR,but not LGR6 or RORα,with BOC-2,significantly abolished the protective effect of MaR1 against PAH development and reduced its therapeutic potential,indicating that ALXR functions as the receptor of MaR1 action on PH.To further decipher the underlying mechanisms of MaR1/ALXR axis on PASMCs proliferation and mitochondrial homeostasis,we compared RNA-seq data of hypoxia-challenged PASMCs treated or not with MaR1 and proved that the MaR1/ALXR axis suppressed hypoxia-induced PASMCs proliferation and pulmonary vascular remodeling through improving HSP90-mediated mitochondrial homeostasis.These observations broaden our in-depth understanding of the role of MaR1 in PAH.Targeting MaR1/ALXR/HSP90 signaling may be used therapeutically in PAH treatment.ConclusionsOur present study provides strong evidence that MaR1 exerts a protective role in both the development and the progression of PAH.Mechanistically,MaR1 inhibits PASMCs proliferation and pulmonary vascular remodeling through improving mitochondrial homeostasis via the MaR1/ALXR/HSP90 axis.Targeting the MaR1/ALXR/HSP90 axis is expected to provide new therapeutic strategy for the prevention and treatment of PAH.