Mechanisms Of Smooth Muscle Hypertrophy Induced By Atmospheric PM Exposure Through Activation Of PDGFRβ Gene

Objective:Particulate matter(PM)exposure has been shown to be a serious health risk factor in recent years and has been associated with several types of vascular disease,although the exact mechanisms remain unclear.Vascular smooth muscle cells(VSMCs)are the main cells that constitute the middle layer of the artery and play a key role in the response to vascular growth and injury.Platelet derived growth factor receptor β(PDGFRβ)is a mitogen for VSMCs.It is essential for normal blood vessel formation.In this study,the potential role of the PDGFRβ signalling pathway in vascular toxicity was elucidated using an individually ventilated cage(IVC)-based mouse model of a real-ambient PM exposure system and a mouse model with high PDGFRβ expression,and using in vitro PM-exposed VSMCs.Methods:1.Construction of a real-ambient PM exposure mouse modelThis study utilised a real-ambient PM exposure mouse model constructed in Shijiazhuang,Hebei Province during two PM collections.7-week-old male SPF-grade C57/B6 mice were randomly divided into control and PM-exposed groups(30 mice per group for the 2018 sample and 1 5 mice per group for the 2022 sample),and the two groups were exposed to PM for 6 and 15 weeks,respectively.During the exposure period,mice were placed in an IVC and exposed to filtered air or unfiltered air for approximately 15 hours per day.PM concentrations in indoor and ambient air were measured daily using an aerosol detector DUSTTRAKTM Ⅱ 8530(TSI Incorporated,Shoreview,MN,USA).Mice were executed at the end of PM exposure and their aortas were collected.(1)PM collection:PM was collected from November 2018 to January 2019,and from November 2021 to March 2022,respectively,at Hebei Medical University using Teflon filter membranes(PTFE membrane,Whatman,Pittsburgh,PA,USA)at a flow rate of 1.05 L/min for 24 hours per day.The PM extracted from the membrane was characterised and identified by scanning electron microscope(SEM).The organic and ionic components and metal components of the PM were extracted for compositional analysis.(2)HE staining was used to observe the pathological changes of aortic tissues,and immunofluorescence staining was used to observe the pathological changes of aortic smooth muscle layer.(3)The expression of PDGFRβ and its ligand PDGFB in aortic tissues was observed by immunohistochemistry.Real-time Fluorescence Quantitative PCR(RTqPCR)and Western Blot(WB)were used to detect PDGFRβ and its downstream protein Janus kinase 2/Signal transducer and activator of transcription 3(JAK2/STAT3),hypertrophy related genes include α smooth muscle actin(αSMA),Matrix metalloproteinase 2(MMP2),and Matrix metalloproteinase 9(MMP9),proliferation-associated genes include Ki6 7,Proliferating cell nuclear antigen(PCNA),and MYC proto-oncogene(c-Myc)mRNA and protein expression levels.2.Construction of PM exposure model of smooth muscle cells(1)Optical microscopy and Hematoxylin-eosin staining(HE)were used to observe cell morphology,and CCK8 experiment was used to detect the vitality of VSMCs.The entry of PM into VSMCs was observed by transmission electron microscopy(TEM).(2)The mRNA and protein levels of PDGFRβ and its downstream JAK2/STAT3 pathway,hypertrophy-related genes and proliferation-related genes were examined by RT-qPCR and WB assay.3.Construction of smooth muscle cell inhibitor modelConstruct cell inhibitor models.Cell experiments were divided into four groups:control group(Con),PM exposure group(Exp),control+inhibitor group(Con-1),and PM exposure+inhibitor group(Exp-1).Smooth muscle cells were treated with PDGFRβ inhibitor(Imatinib),JAK/STAT pathway inhibitor(AG490)and Angiotensin type I receptor(AT1R)inhibitor(Losartan),respectively.The mRNA and protein expression levels of PDGFRβ,downstream JAK2/STAT3 genes and smooth muscle hypertrophy related genes in VSMCs were detected in each group.4.Construction of smooth muscle cell transfection modelHighly expressed PDGFRβ/AT1R(hPDGFRβ/hAT1R)recombinant enhanced green fluorescent protein(EGFP)plasmid and empty plasmid(hPDGFRβ-con/hAT1R-con)were transfected into VSMCs to construct a smooth muscle cell PDGFRβ/AT1R high expression model.The experiment was divided into four groups:control group(Con),PM-exposed group(Exp),PDGFRβ/AT1R high expression group(hPDGFRβ/hAT1R),and PM exposure group after PDGFRβ/AT1R high expression(hPDGFRβ+Exp/hAT1R+Exp).24 hours later,the smooth muscle cells of each group were examined for PDGFRβ and downstream JAK2/STAT3,smooth muscle hypertrophy-related genes at mRNA and protein levels.5.Construction of a mouse model with high expression of PDGFRβA model of PDGFRβ overexpression in mouse aortic tissue was constructed by tail vein injection of adeno associated virus 9(AAV9).Thirty-two 7-week-old C57/B6 male mice were randomly divided into four groups:control group(Con),PM exposure group(Exp),PDGFRβ overexpression group(hPDGFRβ)and PM exposure group(hPDGFRβ+Exp)after PDGFRβ overexpression.Tracheal drip was performed once a week for four weeks after 2 0 days of tail vein inject AAV9.HE staining was used to observe the pathological changes of aorta tissues.The mRNA expression levels of PDGFRβ and its downstream JAK2/STAT3 genes and smooth muscle hypertrophy related genes were detected by RT-qPCR.Results:1.Physical properties of PMThe SEM results indicated that the PM samples collected in 2018 and 2022 were less than 2.5 μm in diameter and irregular in shape.Analysis of PM components showed that the chemical composition of PM,mainly including metals,poly aromatic hydrocarbons(PAHs)and ions,was similar between the two samples.The trend of PM concentration change in the IVC-based real-ambient PM exposure system was consistent with that in the external environment.2.Aortic wall thickening caused by real-ambient PM exposure in miceMouse aortic pathology revealed that PM exposure at 6 and 15 weeks resulted in a thickening of the mouse aortic wall,especially the smooth muscle layer.3.Effects of real-ambient PM exposure on PDGFRβ pathway and proliferationrelated genesImmunohistochemistry of tissue PDGFRβ and its ligand PDGFB showed that PM exposure resulted in increased levels of both in the aorta.Compared with Con group,mRNA levels and protein levels of PDGFRβ pathway and smooth muscle hypertrophy related genes in aorta of Exp group were increased.However,there was no significant difference in mRNA expression levels of proliferation-related genes.4.PM exposure induced smooth muscle cell hypertrophy and its effects on PDGFRβ pathway and proliferation-related genes(1)The effect of PM toxicity on VSMCs activity did not decrease significantly after 2 4 h exposure to different concentrations of PM.Optical microscopy showed that the morphology of VSMCs increased in the PM exposure group compared with the control group.After the cells were exposed to PM at a concentration of 6.25 μg/mL for 24 h,PM particles were observed to enter the cells by TEM and hyperplasia was observed by HE staining.(2)The mRNA and protein expression levels of PDGFRβ pathway and smooth muscle hypertrophy related genes were significantly increased,but the mRNA and protein levels of proliferation-related genes were not significantly changed.5.Effects of smooth muscle pathway inhibition on PDGFRβ signalPM-induced cell hypertrophy improved after exposure to VSMCs with the inhibitors Imatinib and AG490.However,mRNA and protein levels of JAK2/STAT3 were not significantly changed after Losartan treatment.6.Effects of smooth muscle cell plasmid on JAK2/STAT3 after transfectionAfter the high expression of PDGFRβ in cells,the protein levels of JAK2/STAT3 and hypertrophy related genes were increased.After high expression of AT1R,there was no significant difference in JAK2/STAT3 protein level in hAT1R group compared with Con group.7.Effects of high expression of PDGFRβ in aorta of mice exposed to real-ambient PM on vascular wallIn the tail vein injection PDGFRβ high expression mouse model,the results of HE staining showed that compared with Exp group,the aorta wall of mice in hPDGFRβ+Exp group was thickened.In addition,mRNA expression levels of genes associated with smooth muscle hypertrophy were increased.Conclusion:1.In vitro PM exposure induced hypertrophy of VSMCs,and IVC-based PM exposure induced thickening of mouse aortic wall,especially smooth muscle layer thickness.2.Overexpression of PDGFRβ in vivo and in vitro aggravated PM-induced smooth muscle hypertrophy.Imatinib inhibited PDGFRβ expression and AG490 inhibited JAK2/STAT3 signaling pathway to reduce VSMCs hypertrophy.PDGFRβ induced VSMCs hypertrophy by activating JAK2/STAT3 pathway.3.AngⅡ receptors may not directly activate the JAK2/STAT3 pathway under PM exposure.