Exosomal LOX-1 Mediates Pulmonary Arterial Remodeling In Hypoxia-Induced Pulmonary Hypertension And The Underlying Mechanisms

[Background]Pulmonary hypertension（PH）is a progressive disease with the mean pulmonary arterial pressure（m PAP）>20 mm Hg and pulmonary vascular resistance≥3 Wood units at rest.Pulmonary vascular remodeling,a typical pathological feature of PH,is the main cause of elevated m PAP and pulmonary vascular resistance,but the underlying mechanisms have not been fully elucidated.As a kind of extracellular vesicles with a diameter range of about 40to 160 nm,exosomes contain a variety of proteins,lipids and nucleic acids which can mediate intercellular materials transfer and signals transduction,thereby regulating the pathological and physiological states.Though some interesting studies have suggested that exosomes are involved in PH by directly acting on pulmonary vascular cells or mediating the interaction of pulmonary vascular cells,the underlying mechanisms need to be further studied.Endothelial-mesenchymal transition（EndMT）is referred to as a process that the endothelial cells loss the typical characteristics and gain the phenotype and properties of myofibroblasts under the microenvironment in vivo and external stimuli（such as hypoxia,inflammation,endothelin-1,etc.）.At the same time,the intrinsic connection between monolayer endothelial cells is destroyed.During the pathological state of PH,pulmonary artery endothelial cells（PAECs）undergo EndMT and acquire stronger proliferation,migration and pro-inflammatory capabilities,thereby promoting pulmonary vascular remodeling and PH.The phenotypic switching of pulmonary artery smooth muscle cells（PASMCs）is also the main cause of the pulmonary vascular remodeling.PASMCs are transformed from contractile phenotype to synthetic phenotype,thereby acquiring the enhanced capabilities of proliferation,migration,secretion of active substances and extracellular matrix,thus promoting pulmonary vascular remodeling.Lectin-1ike oxidized low density lipoprotein receptor-1（LOX-1）is a membrane surface glycoprotein of the C-type lectin family,which is expressed in vascular endothelial cells,smooth muscle cells and other cell types.LOX-1 mediates the occurrence and development of various cardiovascular diseases by regulating oxidative stress and inflammatory,and may be a new target for the prevention and treatment of cardiovascular diseases.We have found that the expression of LOX-1 is markedly up-regulated in the pulmonary arteries of hypoxia-induced PH rats,in hypoxia-treated PAECs and PASMCs,and in activated platelets-treated PAECs and PASMCs.Our previous studies have shown that LOX-1 promotes PAECs-EndMT and PASMCs phenotypic switching under hypoxic conditions,and mediates the interaction of platelets with vascular cells,thereby accelerating pulmonary vascular remodeling in hypoxia-induced PH.The first step of exosomes formation is the endocytosis of cell membrane to form the precursors.Whether LOX-1,a membrane protein,can be loaded into plasma-derived exosomes in order to drive PAECs-EndMT and phenotypic modulation of PASMCs,thereby facilitating the pulmonary vascular remodeling of PH deserves to be deeply studied.In summary,this study aims to explore whether plasma-derived exosomes of hypoxia-induced PH rats can promote pulmonary vascular remodeling by regulating PAECs-EndMT and phenotypic switching of PASMCs through in vivo and in vitro experiments,and to further explore whether plasma-derived exosomes of hypoxia-induced PH rats promote pulmonary vascular remodeling by delivering LOX-1 to PAECs and PASMCs and activating the downstream signaling axis.[Method]1.Construction of PH model:Wild type（WT）male SD rats were randomly divided into normoxia group and hypoxia group.The PH model was induced by hypoxia（10%O₂）for 3 weeks.The test indicators include:（1）echocardiography to assay hemodynamics,heart and lung function of rats;（2）right heart catheterization to detect right ventricular systolic pressure（RVSP）;（3）body weight,right ventricle weight/（left ventricle+interventricular septum）weight[RV/（LV+S）],right ventricle weight/tibia length（RV/tibia length）;（4）HE staining to detect pulmonary vascular remodeling.2.Isolation and identification of plasma-derived exosomes:Ultra-high speed differential centrifugation was used to obtain the plasma-derived exosomes of normoxic WT rats(Exos-WT^Normo.)and the plasma-derived exosomes of hypoxic WT rats(Exos-WT^Hypo.),the test indicators include:（1）observation of the morphology of exosomes by transmission electron microscope;（2）observation of the particle size distribution and concentration of exosomes by nanoparticle tracing analysis;（3）detection of the expression of exosomes biomarker CD9 by Western Blotting.3.Effects of WT rats plasma-derived exosomes on the PAECs-EndMT and phenotypic switching of PASMCs:Exos-WT^Normo.and Exos-WT^Hypo.were used to treat primary normoxic cultured PAECs（PAECs-WT）and PASMCs（PASMCs-WT）derived from the pulmonary arteries of WT rats,the test indicators include:（1）laser confocal microscopy to detect the PKH67-labeled exosomes in PAECs-WT and PASMCs-WT;（2）immunofluorescence and Western Blotting to detect the expression of EndMT markers CD31,VE-cadherin,vimentin,α-SMA in PAECs-WT,and to detect the expression of phenotypic switching markers SM22α,α-SMA and PCNA in PASMCs-WT;（3）flow cytometry and Ed U assay to detect the proliferation of PASMCs-WT;（4）scratch wound healing assay to detect the migration of PAECs-WT and PASMCs-WT.4.LOX-1 expression in plasma-derived exosomes of WT rats and the effect of exosomes on LOX-1 expression in PAECs-WT and PASMCs-WT:the test indicators include:（1）immuno-electron microscopy and Western Blotting to detect the expression of LOX-1 in Exos-WT^Normo.and Exos-WT^Hypo.;（2）immunofluorescence and Western Blotting to detect the expression of LOX-1 in exosomes-treated PAECs-WT and PASMCs-WT.5.Effects of hypoxic LOX-1 gene knockout(Olr-1^-/-)rats plasma-derived exosomes on PAECs-EndMT and phenotypic switching of PASMCs:Exos-WT^Hypo.and plasma-derived exosomes of hypoxic Olr-1^-/-rats(Exos-KO^Hypo.)were used to treat primary normoxic cultured PAECs-WT and PASMCs-WT,the test indicators include:（1）immunofluorescence and Western Blotting to detect the expression of VE-cadherin,α-SMA in PAECs-WT,and the expression of SM22α,α-SMA,PCNA in PASMCs-WT;（2）flow cytometry and Ed U assay to detect the proliferation of PASMCs-WT;（3）scratch wound healing assay to detect the migration of PAECs-WT and PASMCs-WT.6.Effects of exosomal LOX-1 on pulmonary vascular remodeling in hypoxic Olr-1^-/-rats:The experimental groups include PBS-treated hypoxic WT rats,PBS-treated hypoxic Olr-1^-/-rats,Exos-WT^Hypo.treated hypoxic Olr-1^-/-rats,and Exos-KO^Hypo.treated hypoxic Olr-1^-/-rats.The test indicators include:（1）echocardiography to detect hemodynamics,heart and lung function of rats;（2）right heart catheterization to detect RVSP;（3）rat body weight,RV/（LV+S）,RV/tibia length;（4）HE staining to detect pulmonary vascular remodeling;（5）transmission electron microscope to detect the EndMT of pulmonary arteries in rats;（6）immunofluorescence and Western Blotting to detect the phenotypic switching of pulmonary arteries in rats.7.The effects of endogenous LOX-1 in PAECs and PASMCs on Exos-WT^Hypo.induced PAECs-EndMT and phenotypic switching of PASMCs:Exos-WT^Hypo.was used to treat primary normoxic cultured PAECs-WT and PAECs-KO;Exos-WT^Hypo.were also used to treat primary normoxic cultured PASMCs-WT and PASMCs-KO;the test indicators include:（1）immunofluorescence and Western Blotting to detect the expression of CD31,VE-cadherin andα-SMA in PAECs,and to detect the expression of SM22α,α-SMA and PCNA in PASMCs;（2）flow cytometry and Ed U assay to detect the proliferation of PASMCs;（3）scratch wound healing assay to detect the migration of PAECs and PASMCs.8.The downstream mechanisms of exosomal LOX-1 mediated PAECs-EndMT:（1）DCFH-DA probe and Griess testing kit to detect the production of ROS and NO;（2）Western Blotting to detect the expression of NOX2,NOX4,e NOS,p-e NOS ser1177,p-e NOS thr495,TGF-β1,p-Smad2/3,Snail,Twist;（3）the small interference RNA of NOX2 and NOX4 were used to inhibit the expression of NOX2 and NOX4;the expression of CD31,α-SMA,vimentin,NOX2,NOX4,ROS,e NOS,p-e NOS ser1177,p-e NOS thr495,TGF-β1,p-Smad2/3,Snail were then detected by Western Blotting or DCFH-DA probe.9.The downstream mechanisms of exosomal LOX-1 mediated phenotypic switching of PASMCs:（1）immunofluorescence and Western Blotting to detect the expression of p-ERK1/2,ERK1/2,KLF4 expression in PASMCs;（2）ERK1/2 inhibitor SCH772984 was used to inhibit the expression of p-ERK1/2 and ERK1/2,the expression of SM22α,α-SMA,PCNA,p-ERK1/2,ERK1/2,KLF4 in PASMCs was then detected by immunofluorescence and Western Blotting;（3）the small interference RNA of KLF4 was used to inhibit the expression of KLF4.Then,immunofluorescence and Western Blotting to detect the expression of SM22α,α-SMA,PCNA,KLF4 in PASMCs;flow cytometry to detect the proliferation of PASMCs;scratch wound healing assay to detect the migration of PASMCs.[Results]1.Compared to the normoxic rats,RVSP,pulmonary artery wall thickness（PAWT）,right ventricular wall thickness（RVWT）,RV/（LV+S）,RV/tibia length were significantly increased;the body weight,the pulmonary artery acceleration time/pulmonary artery ejection time（PAT/PET）and tricuspid annular plane systolic excursion（TAPSE）were significantly reduced;the pulmonary vascular remodeling was aggravated in hypoxic rats.2.The plasma-derived exosomes were cup-shaped membranous vesicles with a diameter of about 100 nm and robust CD9 expression.Hypoxia promoted the secretion and CD9 expression of exosomes.3.Exos-WT^Hypo.delivered LOX-1 to PAECs-WT,and promoted EndMT and migration of PAECs-WT.4.Exos-WT^Hypo.delivered LOX-1 to PASMCs-WT,and promoted phenotypic switching,proliferation and migration of PASMCs-WT.5.Compared with Exos-WT^Hypo.,Exos-KO^Hypo.exhibited weakened effects on EndMT and migration of PAECs-WT.Compared with Exos-WT^Hypo.,Exos-KO^Hypo.also have weakened effects on the phenotypic switching,proliferation and migration of PASMCs-WT.6.Treatment of hypoxic Olr-1^-/-rats with Exos-WT^Hypo.significantly increased RVSP,PAWT,RVWT,RV/（LV+IS）,RV/tibial length and pulmonary vascular remodeling,and meanwhile decreased PAT/PET and TAPSE in hypoxic Olr1^-/-rats.In contrast,treatment of hypoxic Olr-1^-/-rats with Exos-KO^Hypo.had the signficant reduced effect on the above indicators.7.Exos-WT^Hypo.had no significant difference on the enhancing effects of EndMT and migration between PAECs-WT and PAECs-KO;Exos-WT^Hypo.also had no significant difference on the enhancing effects of phenotypic transformation,proliferation and migration between PASMCs-WT and PASMCs-KO.8.Exosomal LOX-1 induced the EndMT and migration of PAECs by regulating the expression of NOX2、NOX4、ROS、p-e NOS ser1177、p-e NOS thr495、NO、TGF-β1、p-Smad2/3、Snail and Twist.9.Exosomal LOX-1 induced phenotypic switching,proliferation and migration of PASMCs by regulating the expression of p-ERK1/2 and KLF4.[Conclusion]1.Exos-WT^Hypo.by delivering LOX-1 into PAECs powerfully promoted the EndMT and migration of PAECs by activating the NOX/ROS/e NOS/NO/TGF-β1/p-Smad2/3/Snail signaling axis,thereby leading to pulmonary vascular remodeling.2.Exos-WT^Hypo.by delivering LOX-1 into PASMCs powerfully promoted the phenotypic transformation,proliferation and migration of PASMCs by activating the p-ERK1/2/KLF4 signaling axis,thereby leading to pulmonary vascular remodeling.