General Capillary Endothelial Cells Undergo Reprogramming into Arterial Endothelial Cells in Pulmonary Hypertension through HIF-2α/Notch4 Pathway

Abstract

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase of pulmonary vascular resistance and obliterative pulmonary vascular remodeling that result in right heart hypertrophy, failure, and premature death. The underlying mechanisms of loss of distal capillary endothelial cells (ECs) and obliterative vascular lesion formation remain unclear. Our recent single-cell RNA sequencing, spatial transcriptomics analysis, RNASCOPE, and immunostaining analysis showed that arterial ECs accumulation and loss of capillary ECs were evident in human PAH patients and pulmonary hypertension (PH) rodents. Pseudotime trajectory analysis of the single-cell RNA sequencing data suggest that lung capillary ECs transit to arterial ECs during the development of PH. Our study also identified CXCL12 as the marker for arterial ECs in PH. Capillary EC lineage tracing approach using capillary specific-Dre;Tdtomato reporter mice demonstrated that capillary ECs gave rise to arterial ECs during PH development. Genetic deletion of HIF-2a or pharmacological inhibition of Notch4 normalized the arterial programming in PH. In conclusion, our study demonstrates that capillary endothelium transits to arterial endothelium through the HIF-2a-Notch4 pathway during the development of PAH. Thus, targeting arterial EC transition might be a novel approach for treating PAH patients.

Figure.. General Capillary Endothelial Cells Undergo Reprogramming into Arterial Endothelial Cells in Pulmonary Hypertension.

A and B, A representative UMAP plot showing the abnormal EC subpopulations change with accumulation of arterial ECs (green) in both male and female CKO mice at the age of 13 months. C, A representative heatmap showing the top markers selectively enriched for each EC cluster, the data was generated by integrated data from WT and CKO. D, Integration of scRNA-seq and Visium data reveals increased arterial ECs and decreased gCap ECs in the distal capillary bed of CKO mice. The visualization shows the predicted spatial distribution of arterial ECs and gCap ECs within the lungs. E, Increase of arterial ECs in distal vascular bed in PH mice. RNASCOPE analysis showing increase of Cxcl12⁺/CD31⁺ ECs in the distal capillary region of CKO mice. n=5 per group. F and G, A representative UMAP plot showing the lung EC subpopulations change with accumulation of arterial ECs in IPAH patients. H, Egln1^Cdh5-CreERT2 (iCKO) mice develops spontaneous PH with increase of RVSP and RV hypertrophy. n=3 (WT) or 8 (iCKO). I, Cxcl12-DsRed reporter studies showed that Cxcl12 co-localizes with gCap ECs but not aCap ECs in PH mice. More than 3 mice were examined. J, Pseudotime trajectory analysis suggested that arterial ECs might be derived from gCap ECs analyzed by Monocle3. K, Plvap-DreER^T2 lineage-tracing study demonstrated that gCap ECs give rise to Cxcl12⁺ arterial ECs in the distal microvascular bed under PH condition. gCap^R mice were treated with tamoxifen to label gCap ECs with Tdtomato, followed by SuHx treatment to induce PH development. Cxcl12 was labeled by RNASCOPE. Arrows point to the Cxcl12⁺/Tdtomato⁺ cells, indicative of ArtECs. L, Quantification of arterial ECs and gCap derived arterial ECs in PH. n=4 per group. M, Transcription factors prediction of lung endothelial subpopulation via SCENIC. SOX17 is one of the top candidates governing arterial EC gene signature. N, Pseudotime trajectory showing SOX17 expression across EC subpopulations. Lighter color indicates higher gene expression. O and P, Sox17 and arterial reprogramming is depended on HIF-2α. A representative UMAP demonstrating HIF-2α deletion (EH2) blocks arterial reprogramming related genes in CKO mice by scRNA-seq analysis. Q. Western blotting confirmed that Sox17 and Nicd4 expression was downregulated by HIF-2α deletion in CKO mice. R, Notch4 neutralized antibodies inhibited RVSP in iCKO mice. n=12 (Anti-IgG) or 13 (Anti-Notch4, 10mg/kg, intraperitoneal injection twice weekly for 2 weeks). S, Notch4 neutralized antibodies inhibited RV hypertrophy in iCKO mice. T, Notch4 neutralized antibodies reduced arterial gene expression. U, A diagram showing the hypothesis of this study. Student t analysis. **p<0.01. ***p<0.001, ****p<0.0001. Scale bar: 50 m.