Study On The Mechanism Of Intercellular Communication In Urban Particulate Matter Induced Vascular Endothelial Injury And Ventricular Remodeling

AimsThe harmful cardiovascular effects caused by urban ambient particulate matter have received widespread attention.Endothelial cells,which are the first barriers of the cardiovascular system,have attained the primary status.In view of this,this paper intends to use the standard reference material 1648a（PM 1648a）of urban fine particulate matter to first explore the cardiovascular toxicity in male and female BALB/c mice,and further deepen the toxic effects in cardiovascular system under the urban atmospheric particulate matter exposure by combined with the in vitro mono-culture model of endothelium or cardiomyocytes,and and bi-culture model of endothelial-myocardial cells.On the basis of vascular endothelial cell damage,the extravascular effect of endothelial paracrine signal release on cardiac regulation is taken as the entry point,and its toxic effect on heart function is clarified.To explore the direct/indirect toxicity mechanism of urban particulate matter on vascular endothelial cells and cardiomyocytes,and to explore the cardiovascular toxicity of urban particulate matter on the vascular endothelial barrier and cardiomyocytes from the perspective of endothelial-cardiomyocyte communication,fine particulate matter-induced detrimental cardiovascular effects have been widely concerned.Methods（1）Human umbilical vein endothelial cells（HUVEC）were exposed to PM 1648a for 24hours.For the detection of oxidative stress indicators,we used DHE probes to detect intracellular superoxide anion levels and the cellular hydrogen peroxide levels were detected with a hydrogen peroxide kit.RT-q PCR screened the significantly expressed NOXs family subtypes in HUVEC cells and their protein-level expression levels were measured by western blot.For the detection of endothelial cell function-related indicators,we used nitric oxide（NO）kit to detect secretory NO levels.ELISA method used to detect endothelial nitric oxide synthase（e NOS）and inducible nitric oxide synthase（i NOS）levels,as well as endothelin-1（ET-1）and asymmetric dimethyl arginine（ADMA）levels,also,the proportion of tissue plasminogen activator（t PA）and plasminogen activator inhibitor 1（PAI-1）.For the detection of related indicators of endothelial cell inflammatory response,we used ELISA method detects changes in the expression of inflammatory factors（IL-1β,IL-10,IL-18）and adhesion molecules（ICAM-1,VCAM-1,P-selectin）.Regarding the role of NOXs in regulating endothelial dysfunction caused by PM 1648a,the NOX1/4 inhibitor GKT137831 was used to pretreat HUVEC before PM 1648a exposure.Western blot method was also used to detect AKT pathway and corresponding phosphorylated protein expression levels before and after pretreatment.（2）PM 1648a exposure was performed on adult BALB/c mice to explore subacute cardiotoxicity induced by fine particulate matter.By combining with realistic exposure concentrations,the dose conversion settings were set by extrapolating humans to mice according to the physiological respiratory parameters and body surface area.Male and female BALB/c mice are given PM 1648a weekly of 1.28（low-dose group）,5.5（medium-dose group）and 11 mg/kg（high-dose group）by oropharyngeal inhalation for 4 weeks.H&E staining of cardiac tissue was used to observe pathological changes in cardiac tissue;WGA staining was used to observe cardiac hypertrophy;TUNEL method was used to detect the apoptotic level in cardiac tissue;and ELISA and western blot were used to detect the changes in paracrine protein levels,calcium regulatory pathways,and protein expression levels related to mitochondrial dysfunction in heart tissue.Here,the mechanism of PM 1648a-induced cardiac hypertrophy through calcium overload and mitochondrial dysfunction was further investigated in vitro.Based on in vivo tests,a mono-culture model of human cardiomyocytes（AC16）was used and exposed to PM 1648a for 24 hours.Cell viability and apoptosis levels of cardiomyocytes after PM 1648a exposure were investigated.The effect of PM 1648a on the unit cell area of cardiomyocytes was detected by WGA staining.The production of mitochondrial superoxide anion,intracellular calcium ion production,mitochondrial membrane potential,and the opening of mitochondrial permeability transition pore were detected.Mitochondrial bioenergetics instrument was used to detect the changes of mitochondrial respiratory function and ATP production.To focus on the role of calcium ions in PM 1648a-induced mitochondrial damage in cardiomyocytes,cardiomyocytes were pretreated with calcium chelator BAPTA-3AM before PM 1648a exposure,and the aforementioned mitochondrial phenotype and function-related indicators were detected.The activation of m TOR/AKT/GSK-3βpathway before and after pretreatment was detected by western blot.（3）The sub-chronic cardiotoxicity in BALB/c mice of fine particulate matter was explored using an in vivo model by the same method described above.Male and female BALB/c mice were given 1.28,5.5,and 11 mg PM 1648a/kg bodyweight weekly through oropharyngeal inhalation for 8 weeks and were assigned to low,medium,and high dose groups,respectively.Pathological changes in heart tissue were observed via MASSON staining,and the status of cardiac tissue remodeling was observed by immunofluorescence staining,abnormal contractile and diastolic function of mice was detected by cardiac ultrasound,and alterations in paracrine protein levels and autophagy-related protein expression levels in heart tissues were detected by ELISA,western blot and immunofluorescence assay.Based on in vivo tests,a bi-culture model using HUVEC and AC16 was exposed to PM 1648a for 24 hours to explore.RTCA method was used to detect the tight junctions of HUVEC endothelial barrier and the cytotoxic effect of PM 1648a on endothelial cell-cardiomyocyte bi-culture system in real time.The phagocytosis of PM 1648a by HUVECs,and the autophagy and subcellular organelle damage of co-cultured AC16 cells by PM 1648a were investigated under transmission electron microscopy.The transcription levels of paracrine factors（ET-1,ETAR,NRG-1,Erb B2 and VEGF）were detected by RT-q PCR,and the expression levels of COL1A1,COL3A1 and autophagy-related proteins LC3Ⅱ/Ⅰand p62 were detected by western blot.Tom 20+LC3 fluorescence co-localization was used to observe the mitophagy of AC16 cells in the endothelial-myocardial co-culture system under a fluorescence microscope.To focus on the indirect toxic effects on mitophagy in PM1648a-induced co-culture model,cells were transfected with a small interfering lentivirus to knock down EDN1 in HUVECs in the co-culture system,with Tom 20+LC3 fluorescent colocalization labeling and observation of AC16 mitochondrial morphology and mitophagy level.Results（1）Regarding to the toxic effect of fine particulate matter on endothelial barrier,HUVEC cells were exposed to PM 1648a for 24h,which resulted in NOX-sourced oxidative stress,endothelial dysfunction,and inflammation induction.These are manifested by the up-regulation of NOX,increase of superoxide anion and hydrogen peroxide,elevated ET-1 and ADMA level,reduced NO production,and down-regulation of phosphorylation of e NOS with increased levels of i NOS,as well as the imbalance of t PA and PAI-1,and changes in the levels of pro-inflammatory and anti-inflammatory factors.However,administration of NOX1/4 inhibitor GKT137831 alleviated PM_2.5-induced elevated endothelial dysfunction biomarkers（NO,ET-1,ADMA,i NOS,t PA/PAI-1）,inflammatory factors（IL-1β,IL-10,IL-18）and adhesion molecules（ICAM-1,VCAM-1,P-selectin）,also passivated NOX-dependent AKT and e NOS phosphorylation that involved in endothelial activation.In summary,PM 1648a-induced NOX upregulation is the source of ROS in HUVEC,which activated AKT/e NOS/NO signal response leading to endothelial dysfunction and inflammatory damage in HUVEC cells.（2）Regarding to the direct toxic effect of fine particulate matter on myocardium,PM1648a-induced myocardial edema and cardiac hypertrophy were detected in the high-dose group.Mitochondria were scattered and ruptured with abnormal ultrastructural morphology.In vitro experiments on human cardiomyocyte AC16 showed that exposure to PM 1648a for 24 h caused opened mitochondrial permeability transition pore,leading to excessive calcium production,decreased mitochondrial membrane potential,weakened mitochondrial respiratory metabolism capacity,and decreased ATP production.Nevertheless,the administration of calcium chelator ameliorated the mitochondrial damage in the PM 1648a-treated group.Our in vivo and in vitro results confirmed that calcium overload under PM 1648a exposure triggered m TOR/AKT/GSK-3βactivation,leading to mitochondrial bioenergetics dysfunction and cardiac hypertrophy.（3）Regarding to the indirect toxic effect of fine particulate matter on myocardium,under the sub-chronic exposure of PM 1648a,MASSON staining showed severe myocardial tissue oedema and myocardial fiber rupture induced by PM1648a,more peri-vessel collagen deposition,and more collagen deposition in the myocardial tissue space in male mice,suggesting pericardial pre-fibrosis.Histoimoleciothesis results showed that the number of neovascular vessels labeled CD31 in female/male mice in the high-dose group was not significant,and the positive markers ofα-SMA increased,suggesting cardiac remodeling.Myocardial tissues COL1A1 and COL3A1 expression were significantly upregulated.Mouse Doppler echocardiography results showed that the sub-chronic toxic effect was concentrated in the effect on the diastolic function of the left ventricle,and the significant E/A ratio abnormality.Transmission electron microscopy can see muscle fiber rupture,vascular elastic structure damage,mitochondrial rupture,crest disappearance,ultrastructural abnormalities,liposcin concentration in myocardial tissue,indicating possible autophagy.The significant decrease in the TEER value of HUVEC cells under the exposure of PM 1648a suggests that the endothelial barrier is damaged and the permeability is increased.The results of RTCA showed that PM1648a treatment co-culture system produced cytotoxicity,and compared with the direct effect of PM 1648a on AC16,HUVEC cells played a barrier protection role.In endothelial-myocardial communication,the expression level of endothelial secretion paracrine factor ET-1increased,the expression of nitric oxide synthase i NOS increased,e NOS decreased,and the expression of Myocardial secretion VEGF paracrine factor increased.In the co-culture system,the uptake of PM 1648a by the HUVEC endothelial barrier,mitochondrial damage and autophagy body generation can be seen in AC16 under transmission electron microscopy.Tom20+LC3 co-localization results in AC16 cells showed that in the co-culture system group constructed by lentiviral knockdown si-EDN1 HUVEC endothelial cell line,compared with the non-knockdown si-EDN1 HUVEC endothelial cell line.Compared with the endothelium-cardiomyocyte co-culture system group（si-control）transfected with EDN1,the mitochondrial morphology showed a continuous rod-shaped network structure,and there was no obvious change.Under the exposure of 20μg/cm² PM 1648a,the reticular structure of mitochondria in the si-control group was discontinuous,and the positive expression of LC3 in green was higher than that in the si-control group,which was consistent with the results of western blot.The MERGE co-localization results showed that compared with si-control,the co-localization with LC3 and Tom 20 increased,indicating that the level of mitophagy was increased.The si-EDN1group showed significant damage to mitochondrial morphology after exposure to 20μg/cm²PM 1648a,and rod-shaped mitochondria were fragmented,the network structure disappeared.the green fluorescence intensity of LC3 increased,indicating that autophagy occurred,and the autophagy flux was blocked and blocked.The MERGE co-localization results show that the co-localization of Tom 20 and LC3 is higher than that of si-EDN1.It suggested that the level of mitophagy was increased,indicating that ET-1 had a certain regulatory effect on mitochondrial biosynthesis and autophagy.ConclusionsCombined with in vitro and in vivo experiments,we systematically explored the direct and indirect cardiotoxic effects of urban atmospheric particulate matter from the paracrine signaling level of multicellular interactions,and confirmed that urban atmospheric particulate matter can directly induce the cardiovascular system.Vascular endothelial barrier damage and endothelial dysfunction led to cardiac systolic and diastolic dysfunction by directly acting on mitochondria and calcium ion homeostasis,resulting in myocardial hypertrophy.It also activated the endothelin ET-1 signal by activating the endothelial barrier to act on the corresponding endothelial receptors in distal cardiomyocytes.regulates mitophagy,and participates in the toxic effects and paracrine molecular mechanisms affecting ventricular remodeling.