| Background and Objective:Systemic sclerosis(SSc)is a chronic systemic autoimmune disease characterized by fibrosis of the skin,blood vessels and internal organs.SSc often affects the pulmonary vasculature,manifesting as thickening of the vessel walls,narrowing of the lumen or even occlusion,resulting in a progressive increase in pulmonary vascular pressure and resistance,which eventually leads to pulmonary arterial hypertension(PAH).PAH is a serious complication of SSc,which can lead to heart failure and even sudden death,and is one of the main causes of death in SSc patients.The pathogenesis of SSc-PAH is not yet fully understood and involves mechani-sms such as vascular endothelial injury,smooth muscle proliferation remodeling,and chronic inflammation,while vascular smooth muscle cell proliferation and vascular remodeling are central to the progressive development of SSc-PAH.Early studies suggested that endothelial cell mesenchymal transition is the source of myofibroblasts in organ fibrosis,but several studies in recent years have shown that pericytes can differentiate into myofibroblasts and are the main source of myofibroblasts in pathological fibrosis.Pericytes themselves have diastolic function,can maintain the integrity of vascular structures,regulate capillary blood flow,and also have high plasticity and can differentiate into cartilage,bone,fat and fibrous tissue structures under certain conditions.Several experiments investigating liver,kidney and SSc fibrosis have revealed that pericytes can differentiate into myofibroblasts,thereby repairing tissue damage.However,long-term chronic inflammation can lead to disruption and dysregulation of the normal fibrotic process,resulting in excessive deposition of extracellular matrix and scar formation,which impairs organ function.It has been shown that sphingosine 1-phosphate interacts with platelet-derived growth factor-β after binding to its receptor and is involved in the regulation of pericyte proliferation,migration and angiogenesis.In addition,the presence of a large number of activated macrophages in the peripheral blood of SSc patients and the significantly higher concentration of inflammatory cytokines than in healthy controls suggest that,in addition to pericytes,macrophages also play an important role in the inflammatory mechanism of SSc.Macrophages are intrinsic immune cells that originate from the differentiation of monocytes in peripheral tissues and upon activation can form two classical subtypes:the pro-inflammatory M1-type macrophages and the anti-inflammatory M2-type macrophages.It has been demonstrated that M1-type macrophages can induce differentiation of peripheral cells into myofibroblasts,thereby promoting vascular repair of injured tissues.It has also been reported that M1-type macrophages mediate active vasculopathy,whereas M2-type macrophages play a pathological role in chronic vasculopathy or in the stable phase.The results of several studies suggest that activated macrophages are particularly abundant in the blood and skin of SSc patients and suggest that they may be a potential source of cytokines that induce fibrosis in tissue dysfunction.Therefore,it is particularly important to elucidate the role of macrophages and pericytes in SSc-PAH and the link between macrophage polarization and pericytes.There are few basic studies on the role of macrophage polarization and pericytes in SSc-PAH,and the altered signaling pathways between macrophages and pericytes are not clear.For this reason,this study intends to investigate the link between macrophage polarization and pericytes by establishing a mouse model of SSc-PAH,and provide new clues and directions for the clinical treatment of SSc-PAH.Materials and Methods:We used a combination of drugs(bleomycin & monocrotaline)and hypoxia to construct SSc-PAH disease model mice,and the mice were observed by HE staining and Masson staining for dorsal skin,HE staining,EVG staining and immunofluoresc-ence staining for lung histopathology,and ELISA,flow cytometry,and q-PCR methods for peritoneal Macrophages were identified;pericytes extracted from lung tissue using magnetic bead sorting method were identified by Western blot and immunofluorescence staining,and the proliferation ability and expression of PDGFR-β and α-SMA of pericytes in SSc-PAH group and normal control group were detected by CCK8 and Western blot,respectively.Mouse mononuclear macrophage leukemia cell line RAW264.7 was divided into5 groups and cultured for 24 h after normal complete medium,LPS complete medium containing 10 ng/m L,LPS complete medium containing 100 ng/m L,LPS complete medium containing 1000 ng/m L,IL-4 complete medium containing 25 ng/m L,using q-PCR method To detect the m RMA expression of IL-1β,i NOS,TNF-α,IL-18,IL-23,IL-6,MRC1,CD163 in each group of macrophages;after 24 h induction of RAW264.7 by LPS and IL-4,we replaced the medium with DMEM/F-12 serum-free medium without induction factors,and collected the conditioned medium from each group after 48 h The proliferation,migration and angiogenic ability of pericytes were verified by CCK8,scratch assay,compartment migration assay and angiogenic assay,and the proteins of pericytes cultured in each group of conditioned media for 48 h were extracted and detected by Western blot.The changes of PDGFR-β,α-SMA,S1PR1,S1PR2,P16,JAK-2,P-JAK-2 protein expression in pericytes;meanwhile,we also co-cultured human endothelial cells(HUVEC)with five groups of conditioned media,and extracted endothelial cell proteins after 48 h to detect the expression ofα-SMA,S1PR1,S1PR2,P16 in endothelial cells Changes.Three m L each of peripheral blood from SSc-PAH patients and healthy individuals meeting the diagnostic criteria were collected using anticoagulation tubes containing EDTA-K2,and PBMCs were obtained using human peripheral blood lymphocyte isolate and density gradient centrifugation.After extracting total RNA from PBMCs,the tRFs,tiRNAs and miRNAs expression in PBMCs from SSc-PAH patients were sequenced to compare the differences with those of healthy individuals.Results:1.In the mouse model established with bleomycin,monocrotaline and hypoxia,there was significant thickening of the skin dermis,marked proliferation of collagen fibers,reduction and disappearance of subcutaneous fat and appendages,swelling of the walls of small pulmonary arteries,thickening of the middle membrane,narrowing of the lumen,marked thickening of both the internal and external elastic layers,infiltration of inflammatory cells,more pronounced tissue damage and increased numbers of myofibroblasts and endothelial cells around small pulmonary vessels.2.In the mouse model established with bleomycin,monocrotaline and hypoxia,about 60.3% of mouse peritoneal macrophages were M1 subtype,and the proliferation and differentiation ability of pulmonary pericytes were increased.3.M1-type macrophage conditioned medium induced by different concentrations of LPS had less effect on pericyte proliferation ability,probably through P16 and JAK-2 proteins to enhance pericyte migration,differentiation and angiogenesis;M2-type macrophage conditioned medium probably promoted pericyte proliferation,migration,differentiation and angiogenesis through S1 P,P16 and JAK-2proteins.4.After tRF and tiRNA sequencing and data analysis,compared with healthy individuals,there were 1304 tRFs and tiRNAs expressions up-regulated,1761 tRFs and tiRNAs expressions down-regulated and 740 tRFs and tiRNAs expressions not significantly different in PBMC of SSc-PAH patients;459 miRNAs expressions up-regulated,374 miRNAs expressions down-regulated and 236 miRNAs expressions not significantly different in PBMC of SSc-PAH patients.Conclusion:1.The use of bleomycin,wild lily base and hypoxia can successfully establish a mouse model of SSc-PAH disease in which mice show an early inflammatory state in vivo,with the majority of peritoneal macrophages showing M1 type and increased proliferation and differentiation capacity of pericytes in the lung;2.In in vitro experiments,polarized macrophages may indicate effects on pericyte proliferation,differentiation,migration and angiogenic capacity through regulation of S1 P,P16 and JAK2 protein expression.differentiation,migration,and angiogenic capacity.3.tRFs,tiRNAs such as Other-1:33-tRNA-Glu-CTC-1-M2,hsa-let-7c-5p and hsa-miR-203a-3p in PBMC from SSc-PAH patients,and miRNAs are up-or down-regulated and may have an impact on the development of the disease. |
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