Role And Mechanism Of Endothelial Extracellular Vesicles In Smoking-induced Pulmonary Hypertension

ObjectivePrevious literature reports and laboratory studies have found that smoking can directly induce pulmonary hypertension（PH）without relying on alveolar hypoxia caused by chronic obstructive pulmonary disease,but the mechanism is still unclear.Several studies have found a significant increase in the number of endothelial extracellular vesicles（eEVs）in the plasma of smokers^[1-3].The purpose of this study was to investigate whether eEVs play a role in the pathophysiological process of smoking-induced PH and its mechanism.MethodExtracellular vesicles（EVs）were isolated by ultracentrifugation;eEVs were sorted by flow cytometry;[Ca²⁺]i was measured by Fura-2/AM calcium ion probe;CCK-8 cell proliferation assay The proliferation rate of hPASMCs was detected;UHPLC-Q-TOF-MS/MS technique was used for metabolomics detection;high performance liquid chromatography（HPLC）was used to detect spermine content;immunofluorescence staining was used to detect the expression and distribution of EVs and spermine;the hemodynamic parameters of rats were measured by Powerlab system;the cardiac output（CO）was measured by thermodilution method;the pathological changes of pulmonary artery were observed by HE staining.Result1.Smoking induces a significant increase in the number of eEVs in human and rat plasmaCompared with non-smokers,the number of eEVs in the plasma of smokers increased,and there was a statistical difference（p<0.05,n=6）;compared with the control rats,the plasma in the smoking model rats The number of CD31⁺/CD42b^-EVs（ie,eEVs）also increased and was statistically different（p<0.05,n=5）.2.eEVs mediate physiological function changes of human pulmonary artery smooth muscle cells（hPASMCs）,and this process is dependent on calcium-sensing receptor（CaSR）.Compared with non-smokers,eEVs isolated from plasma of smokers induced an increase in[Ca²⁺]i（n=24-27）and cell proliferation（n=4）in hPASMCs,with statistical differences（p<0.05）;Down-regulation of CaSR expression in hPASMCs significantly inhibited the increase in[Ca²⁺]ⁱ and cell proliferation induced by eEVs in hPASMCs.3.Metabolomics of eEVs isolated from plasma in smokers and non-smokersCompared with non-smokers,there were 25 metabolites in eEVs isolated from plasma in smokers,and spermine increased 4.38 times higher and statistically significant（p<0.05,n=6）,the content of spermidine and ornithine associated with its metabolism also increased,1.72 times,2.20 times,and statistically significant（p<0.05,n=6）.Further,this change was confirmed in animal experiments.Compared with the control group,the spermidine content of eEVs isolated from the plasma of the smoking model rats was significantly increased,and there was a statistical difference（p<0.05,n=5）.There was no significant difference in the content of spermine in other types of EVs compared with the control group.4.eEVs carry spermamine migration from the pulmonary artery to the smooth muscle layer of the pulmonary arteryCompared with the control group,the fluorescence of CD63（labeled EVs）,CD31（labeled pulmonary artery endothelium and endothelium-derived extracellular vesicles）,spermine,andα-actin（labeled pulmonary artery smooth muscle layer）were increased in the smoking group;smoking group CD31+EVs（fluorescence overlap of CD63 and CD31）accounted for（76.96±3.16）%of all EVs（CD63 total fluorescence）;spermine（CD63,CD31,spermine andα-actin）carried by CD31⁺EVs in pulmonary smooth muscle layer fluorescence overlap)（80.65±3.61）%of all spermine（spermidine andα-actin total fluorescence overlap）at the smooth muscle layer of the pulmonary artery;spermine（CD31,CD42b）carried by CD31⁺/CD42b⁺EVs at the smooth muscle layer of the pulmonary artery The fluorescence overlap of spermine andα-actin only accounted for（17.04±1.15）%of the spermine（CD31,spermine,andα-actin total fluorescence overlap）carried by CD31⁺EVs in the smooth muscle layer of the pulmonary artery.5.eEVs induce PH in rats,and this process is dependent on spermine/CaSR pathwayCigarette smoke extract（CSE）stimulates eEVs（eEV-CSE）produced by pulmonary artery endothelial cells（PAECs）,which can induce mean pulmonary artery pressure in rats after tail vein injection（mean pulmonary artery）.Pressure,mPAP),pulmonary vascular resistance（PVR）,right ventricular hypertrophy index（RV/（LV+S））,pulmonary wall thickness（wall thickness）were significantly increased,compared with the control group,significant Statistical differences（p<0.05,n=5-8）;eEVs（eEV-CSE-DFMO）produced by treatment of PAECs with CSE+DFMO,and CSE-stimulated over-spermidine N1-acetyltransferase 1（Spermidine N1-acetyltransferase）1,SAT1)PAECs of plasmids produced eEV（eEV-CSE-Ad_SAT1）tail vein injection,resulting in significantly decreased rat mPAP,PVR,RV/（LV+S）,wall thickness compared with eEV-CSE group And statistically significant（p<0.05,n=5-8）;compared with CaSR^+/+PTH^-/-rats,CaSR^-/-PTH^-/-rat tail vein injection of eEV-CSE,not Cause mPAP,PVR,RV/（LV+S）,wall thickness Significantly elevated（n=6-7）;eV-CSE-DFMO was injected into the tail vein,whether it was CaSR^+/+PTH^-/-rats or CaSR^-/-PTH^-/-rats,with mPAP,PVR,RV/（LV+S）and wall thickness were significantly lower than the eEV-CSE group,and there was a statistical difference（p<0.05,n=6-7）.ConclusionSmoking induces increased secretion of eEVs,and eEVs carry more spermine.eEVs enriched in spermine release from the pulmonary endothelium to the smooth muscle layer of the pulmonary artery,activate CaSR on the membrane of pulmonary artery smooth muscle cells,cause an increase in[Ca²⁺]i in pulmonary artery smooth muscle cells,and promote pulmonary artery Smooth muscle cell proliferation eventually leads to pulmonary hypertension.