Clinicopathologic Heterogeneity And Single-cell Transcriptomic Landscape Of Chronic Thromboembolic Pulmonary Hypertension

BackgroundObstuction of chronic organized fibrotic clots and secondary pulmonary vascular remodeling contribute to pathophysiology of chronic thromboembolic pulmonary hypertension(CTEPH),leading to disease progression.Assessing the morphological characteristics of lesions of pulmonary artery dissected by pulmonary endarterectomy(PEA)can promote a more comprehensive understanding of relevant pathological alterations and underlying pathogenesis.But few studies have been reported in the correlation between pathological manifestations of elastic pulmonary artery and clinical manifestations.Single-cell transcriptomics could elucidate the heterogeneity of cells and provide the cell landscape atlas of CTEPH.Therefore,this study aimed to describe the pathomorphology of the PEA specimens,comprehensively elaborate the heterogeneity of the pathological changes of CTEPH,and construct the cell atlas by combining the single-cell transcriptomics,to investigate the molecular mechanisms of the pathology of CTEPH.Methods1.CTEPH patients who underwent PEA surgery in China-Japan Friendship Hospital from December 2016 to March 2021 were included retrospectively.Clinical information was extracted from the electronic medical record,and pulmonary vascular lesions were identified and analyzed through various staining methods.2.patients with type Ⅰ or type Ⅱ lesions who underwent PEA surgery at the China-Japan Friendship Hospital from March 2021 to June 2022 were included prospectively.The proximal vessels originate from the main pulmonary artery or lobar artery,while the distal vessels include the segmental and subsegmental pulmonary arteries.Statistical analysis was conducted on the pathological differences between the proximal and distal pulmonary vascular lesions.3.Single-cell 10X Genomics transcriptome sequencing was performed on the proximal and distal vessels mentioned above,and cell classification and biological function enrichment analysis were performed through bioinformatics analysis.Comparative analysis of key signaling pathways and cellular interactions was conducted using proximal and distal self control comparisons.Results1.81 consecutive patients with CTEPH were included.The four basic lesions on the pulmonary vascular bed were thrombotic lesion,neointima,atherosclerosis and recanalization.Neointima formation was noticed in almost all patients,and extracellular matrix deposition was obvious.Atherosclerotic lesion of pulmonary artery is not correlated with the common risk factors of atherosclerosis in systemic circulation,but with the history of symptomatic embolism(88%vs 56%,P=0.012)and duration of disease(8.0±6.5 years vs 4.7± 4.2 years,P=0.039).2.34 patients with CTEPH were included prospectively.The proximal lesions(thrombotic area)presented eccentric thickening neointima,with fibrin components predominating in the thrombus.The distal leisons presented uneven thickening of neointima and recanalization,with focal irregular lumina.The neointimal thickness of proximal pulmonary artery was not related to the severity of the disease,while the angiogenesis of distal pulmonary artery was negatively correlated with the inflammatory situation before the operation through C reactive protein(r=-0.392,P=0.022).3.After quality control of the proximal and distal pulmonary arteries,there were a total of 53893 cells and 11 types of cells.Angiogenesis and glycolysis related signaling pathways in endothelial cells were significantly enhanced,while mTORC1 signaling pathway and NOTCH signaling pathway in smooth muscle cells were significantly upregulated.The hypoxia response process,TGF-β pathways,and endothelial mesenchymal transformation processes in the distal neointima were significantly activated.In contrast,the oxidative phosphorylation reaction and NOTCH pathway in the proximal neointima were significantly activated.It was corresponded to the histopathology of proximal and distal neointima,and explained the cytological alterations and related molecular mechanisms of the pathology in the CTEPH neointima.By combining multiple analysis methods such as trajectory analysis,cell interaction,and pathway enrichment,it was found that the JAG1-NOTCH signaling pathway is a key ligand receptor for neointimal formation and participated in CTEPH vascular lesions.ConclusionsThe formation of neointima of pulmonary artery of CTEPH is the main pathological alteration,with obvious deposition of extracellular matrix.The neointima of the proximal pulmonary artery consisted of few cellular components and significant collagen deposition.While the neointima of the distal pulmonary artery was significantly recanalized,with a focal distribution of collagen.JAG1-NOTCH signaling pathway was involved in angiogenesis and pulmonary vascular remodeling of CTEPH,which is expected to be a potential therapeutic target.Precise pathological classification of different sites could contribute to better understand the pathological lesions and pathogenesis of patients with different clinical characteristics.This research may provide a potential target for the treatment of CTEPH.