Urban Particulate Matter Induces Vascular Endothelial Damage Through The Dysfunction Of Multiple Subcellular Structures

Aim:This study aims to explore the toxicity and potential mechanisms of urban particulate matter on vascular endothelial cells from the perspectives of subcellular structures.Taking multiple classical organelles(endoplasmic reticulum,mitochondria and lysosomes)as the checkpoints,combined with programmed cell death(autophagy and apoptosis),this study discusses the relevant mode of action underlying the urban particulates-induced vascular endothelial cell toxicity and vascular tissue damage by using in vitro and in vivo models.Method:(1)The urban atmospheric particulate matter standard material(No.1648a,PM SRM1648a)produced by NIST was used as the research object for subsequent assays.Initially,the composition,hydrodynamic size and surface potential of PM SRM1648a were characterized,respectively.And the endotoxin content of particulate matter was measured.Then,two types of widely used human umbilical vein vascular endothelial cell lines,EA.hy926 and HUVECs,were selected for in vitro experiments.For the cellular experiments,MTT and CCK8 methods were employed for cytotoxicity detection and served as the basis of dose screening for further in vitro experiments.The uptake of PM SRM 1648a by vascular endothelial cells was observed by transmission electron microscope(TEM)and recorded by flow cytometer(FCM).A series of indicators of GSH/GSSG,MDA,NADP+/NADPH,and C11-BODIPY581/591 were used to evaluate the state of oxidative stress in vascular endothelial cells under the stimulation of particulate matter.At the subcellular structure level,the immunofluorescence method was used to detect y-H2AX foci to measure DNA damage,and the TEM method was used to observe the uptake ability of vascular endothelial cells and to capture the changes of intracellular ultra-structures.(2)Focusing on the endoplasmic reticulum(ER),it is discussed that particulate matter induces ER stress in vascular endothelial cells,which leads to autophagosome accumulation and endothelial cell apoptosis.According to TEM observation,ER experiences abnormal expansion.ER-Tracker Blue-White DPX probe was used to label ER to discuss its structural and functional changes.DCFH-DA probe was employed to detect ROS,and FITC-Annexin V/PI double staining was used to detect cell apoptosis rate.The autophagic state was measured by using TEM observation and LC3B immunofluorescence,combined with LC3II/I ratio by western blot.Withal,western blot method was used to discuss the expression changes of multiple proteins responsible for ER stress(GRP78/BIP,CHOP and caspase 12),autophagy(Beclin1,LC3II/I and p62)and apoptosis(caspase9,caspase3,BCL-2 and BAX),respectively.Mechanistically,oxidative stress inhibitor GSH-MEE and ER stress broad-spectrum inhibitor 4-PBA were applied to explore the role of ROS/ER stress signal axis in PM SRM1648a-induced autophagy and apoptosis in vascular endothelial cells.Moreover,the relationship between autophagy and apoptosis under particulate matter exposure was also discussed.In addition,to explore the specific role of autophagy in PM SRM1648a-caused ER stress and apoptosis,different autophagy regulators of 3-MA,Rapamycin and Bafilomycin Al were used.(3)Considering lysosomal damage,the specific reasons for the accumulation of autophagosomes were explored in vascular endothelial cells caused by particulate matter.mRFP-GFP-LC3 adenovirus vector was employed for the estimation of the dynamic process of autophagy and autophagic flux in EA.hy926 and HUVECs vascular endothelial cells.Additionally,by using the saturated concentration of Bafilomycin A1,it is applicable to discuss the specific reasons for the accumulation of autophagosomes in vascular endothelial cells in response to PM SRM1648a.The LysoSensorTM Green DND-189 probe was planned to label lysosomes for fluorescence semi-quantification that can indirectly reflect the relative pH value of lysosomes under different circumstances.Meanwhile,the double-labeled immunofluorescence method(LAMP-2 and LC3B)helped determine the co-localization of lysosomes and autophagosomes(i.e.autolysosomes).Also,the activity of acid phosphatase ACP,and lysosomal hydrolase CTSB were determined for the evaluation of lysosomal function.(4)It aimed to discuss the potential mechanism of PM SRM1648a-induced inflammatory reaction in endothelial cells from the perspective of mitochondrial dynamics.Therefore,indicators of mitochondrial function,such as ATP,mtROS and mitochondrial membrane potential(MMP)were initially detected.Mitochondrial probe MitoTracker(?)Red CMXRos was used for the observation of mitochondrial morphological changes.qRT-PCR and western blot methods were employed to detect expression changes of mitochondrial dynamics-related molecules at gene and protein levels,respectively;Cytokines such as TNF-?,IL-1?,etc.were determined by the ELISA method.It is preliminarily to judge cell membrane damage through Hoechst 33258/PI double staining and LDH content detection in the supernatant.In addition,cell pyroptosis and caspasel activity were detected by caspasel/PI double staining and recorded by FCM;Mechanistically,si RNA lentiviral transfection was used to knock down mitochondrial fission regulation gene DNM1L(DRP1)in EA.hy926 cells to discuss the role of DRP1 in mitochondrial dysfunction and inflammatory response.Based on this knock-down cell model and the normal vehicle-transfected cells,it can be used to discuss the role of mitochondrial fission in PM SRM1648a-induced inflammation in vascular endothelial cells and to respond to the question of whether DRP-1 is involved in mitochondrial fission and inflammatory response under the exposure to PM SRM 1648a.Subsequently,mitochondrial fission,caspase1 enzyme activity,cellular inflammation and other indicators mentioned above were further detected in knock-down cells and vehicle cells under the same conditions of PM SRM1648a exposure to ascertain the involvement of DRP1/caspase1/IL-1? signaling pathway in PM SRM1648a-caused endothelial cell inflammation.(5)Male and female BALB/c mice were acutely(7d)or sub-acutely(28d)exposed to PM SRM 1648a by oropharyngeal aspiration.The exposure concentration included three dosage levels of 1.28,5.5 and 11 mg/kg·bw/w,and the control counterparts were exposed to 0.9%saline.Bronchoalveolar lavage fluid(BALF),blood samples,lung,aorta,and heart tissues were extracted after certain exposure periods for follow-up experiments.H&E,von kossa and Masson staining were deployed to observe the pathological changes,calcium ion deposition and collagen deposition of each tissue sample.ELISA method was used to detect inflammatory cytokines and oxidative stress-related indicators in BALF and blood samples.BCA method was employed to detect the protein content in the BALF.Additionally,side-scanning value combined with specific antibody staining was employed to detect the total cell number,monocyte number,lymphocyte number and neutrophil number in the BALF by flow cytometry.Also,F4-80/CD11b/CD86/CD206 antibody staining and screening were implemented by FCM for detecting the proportion of M1 and M2 macrophages.The qRT-PCR method was used to measure the gene expression of inflammatory factors and oxidative stress-related markers in aortic tissue.Moreover,changes in the expression of subcellular structure and function related indexes in aortic tissues were also detected by qRT-PCR,including hsp5,ddit3,dnm1l,fis1,mfn1,mfn2,opal,lamp1 and lamp2.Immunohistochemistry,immunofluorescence and western blot were applied to detect protein expressions of subcellular structure and function related markers in aortic tissue,which includes BIP,CHOP,DRP1,LAMP1 and LAMP2.Finally,the TUNEL method was used to detect cell apoptosis in aortic tissue.All the above experiments were used to decipher the hypothesis that structural and functional disorder at the subcellular level is a potential biological mechanism of atmospheric particulates-induced vascular damage from the in vivo level.Result:(1)In vitro experiments show that vascular endothelial cells are more sensitive than alveolar epithelial cells when exposed to particulate matter.PM SRM 1648a caused endothelial cell toxicity without causing a decrease in epithelial cell survival at relatively low dosage levels(0,5,10,and 20 ?g/cm2,equivalent to 0,16,32,and 64 ?g/mL).Endothelial cells have the ability to uptake PM SRM1648a,and this uptake precedes the occurrence of cytotoxicity.Intriguingly,PM SRM1648a is more toxic to EA.hy926 than HUVECs,which may be because EA.hy926 cells have a stronger ability of particle uptake than HUVECs.In addition to cytotoxicity,PM SRM1648a can induce DNA damage and disrupt multiple organelles.(2)PM SRM 1648a causes intracellular ROS overproduction and oxidative stress in human vascular endothelial cells,which further damages subcellular structural functions,such as ER stress,thereby inducing the accumulation of autophagosomes and cell apoptosis.Normal autophagy induction can protect vascular endothelial cells from apoptosis caused by PM SRM 1648a.However,inhibiting autophagy or disrupting the degradation process of autophagy will aggravate endothelial cell damage in response to PM SRM1648a.The crosstalk between ER stress and autophagy indicates that ER stress is related to the up-regulation of LC3II expression,but the autophagy process has no significant effect on ER damage caused by PM SRM1648a.To a certain extent,the relationship between autophagy and apoptosis under ER stress was discussed.Among them,autophagy itself serves as a protective factor for vascular endothelial cell damage.On the contrary,ROS/ER stress pathway and dysfunction of autophagy degradation facilitated endothelial cell apoptosis under the exposure to PM SRM 1648a.(3)PM SRM 1648a leads to accumulation of autophagosomes and high expression of LC3? in human vascular endothelial cells.The specific reasons for LC3? up-regulation in EA.hy926 cells at different time points are different.Within a short period of 6 hours under the exposure to 20 ?g/cm2 of PM SRM 1648a,the increase in autophagy activity leads to the rapid conversion of LC3? from LC3?,thereby participating in the synthesis of autophagosomal membranes.As the exposure time is extended to 24 hours,the increase in LC3II is due to the lysosomal dysfunction,which brings about failure in waste degradation and clearance(Defective autophagy).Lysosomal rupture and hydrolase inactivation caused by PM SRM 1648a are the key factors for the blockade of autophagic flux,and the weakened ability of degradation and waste disposal.The recovery of oxidative stress can hardly alleviate lysosomal damage or recover the autophagy flux,but can help reduce the expression of LC3II and rescue the expression of lysosomal membrane structural protein,LAMP-2.Therefore,PM SRM 1648a causes the inactivation of lysosomal hydrolase through a non-oxidative stress-dependent pathway,which in turn leads to defective autophagy and subsequent endothelial cell damage;(4)Mitochondria are the subcellular targets of PM SRM 1648a in human vascular endothelial cells.Internalization of particles can disrupt mitochondrial functions,including increased mtROS,decreased MMP,and ATP inhibition.At the same time,the decrease of PGC-1?indicates that PM SRM 1648a impairs mitochondrial biogenesis.In addition,mitochondrial morphology was adversely affected by PM SRM1648a to show mitochondrial fission-like changes with abnormal up-regulation of molecules responsible for mitochondrial fission.PM SRM 1648a activates DRP1 to cause mitochondrial fission,and activates caspasel activity to trigger inflammatory cascade reaction.Caspasel inhibitor Z-YVAD-FMK can effectively decrease caspasel activity and inflammatory responses.Furthermore,after knocking down the fission-related gene of DNMIL(DRP1),PM SRM1648a-induced mitochondrial dysfunction can be effectively alleviated.Plus,caspasel activity and IL-1? release can also be effectively suppressed.Therefore,the DRP1/caspasel/IL-1? signaling transduction is involved in the mitochondrial fission and inflammatory damage in vascular endothelial cells caused by PM SRM1648a.Interestingly,under the same exposure scenario,the apoptosis rate in EA.hy926 cells is 23.43±1.76%(P<0.001)while the pyroptosis rate is 0.38±0.08%(P>0.05).Similarly,the apoptosis rate in HUVECs is 17.30+1.61%(P<0.01)while the pyroptosis rate is 0.41±0.07%(P>0.05).Combined with the cell morphology analysis,and FSC value,pyroptosis is not the main mode of cell death in PM SRM1648a-caused vascular endothelial damage.(5)After acute or subacute exposure to PM SRM 1648a,oxidative stress and inflammation are the main pathological changes in mice.There was no obvious lipid accumulation or plaque deposition in aortic tissues in BALB/c mice under the exposure to different concentrations(1.28,5.5 and 11 mg/kg-bw/w)of PM SRM1648a through oropharyngeal aspiration.However,in the subacute group(28 days),the wall of the aorta became wider and the collagen at the aortic root slightly increased.Without significant pathological damage,the expression of protein markers responsible for the equilibrium of subcellular structures significantly changed after subacute exposure,including the ER stress and mitochondrial function related molecules.Moreover,the result from the TUNEL assay suggested that cell apoptosis rate increased in the aortic tissue from mice after subacute exposure to PM SRM 1648a.The above results occurred both in males and females,and there is no gender difference in pathological states in BALB/c mice under the same exposure scenario.Conclusion:After discussing the structural dysfunction of the ER,lysosomes and mitochondria,it is reasonable to combine the cellular level with the subcellular structures when assessing the risk of ambient particulate matter.Among them,assessment at the subcellular level can help a more comprehensive understanding of the vascular toxicity caused by atmospheric particles.Focusing on early checkpoints,such as the observation and evaluation of organelles,helps provide timely and objective basic data for safety assessment,so as to better implement risk management to protect public health.