Single‐Cell Sequencing in the Genetic Model of Mitochondrial Dysfunction Reveals Heterogeneity of Lung Endothelial Cells and Novel Targets in Pulmonary Hypertension
Authors: James Joel , Valuparampil Varghese Mathews , Niihori Maki , Yi Dan , Dai Zhiyu , Rafikova Olga , Rafikov Ruslan 
Published: May 2022

Introduction

Pulmonary hypertension (PH) is a complex, lethal vascular disease and currently has very limited treatment options with no cure. Severe pulmonary vascular remodeling and right ventricular (RV) hypertrophy in PH ultimately lead to RV failure. Endothelial dysfunction (ED) is demonstrated to play a key role in the initiation of PH. However, the pathogenic mechanisms leading up to ED remain unclear.

Hypothesis

We hypothesized that mitochondrial dysfunction (MD) in endothelial cells (EC) would produce a specific gene expression pattern in each EC subpopulation, revealing pathogenic pathways that trigger ED.

Methods

Rats with the NFU1G206C mutation spontaneously develop PH, recapitulating the human disease in patients with multiple mitochondrial dysfunctions syndrome and PH. Transcriptomics was performed with the 10X Genomics kit on isolated single cell EC’s from WT and NFU1G206C rats. EC clustering was done with the Seurat V4 R package. The Database for Annotation, Visualization and Integrated Discovery (DAVID) identified enriched biological pathways. RV function was determined by echocardiography imaging (Vevo 3100 system).

Results

Echo results confirmed that NFU1G206C rats showed a spontaneous PH phenotype. Thirteen distinctive subpopulations of EC’s were identified in WT and NFU1G206C rats with UMAP and tSNE analysis, based on specific marker expressions such as alveolar capillary ECs (expressing ca4), small capillary ECs (fabp4), lymphatic ECs (prox1) and ECs regulating arteriogenesis (gja5) to ECs undergoing endothelial mesenchymal transition (EndMT) (acta2, encoding for α‐SMA). DAVID analysis of genes expressed in individual EC clusters showed heterogenous functions such as cell adhesion, inflammatory response, migration, and angiogenesis, highlighting the physiological relevance of EC’s. Cells were then classified as either senescent, quiescent, or proliferative, based on genes enriched in the respective pathways. We found that EC clusters of NFU1G206C rats significantly deviated from conventional gene expression profiles of WT clusters. The cluster1 of NFU1G206C rats showed an increase in senescence marker, lamb1 and decreased mki67. On the other hand, genes associated with proliferation were significantly increased in cluster2. We also identified EC’s with increased association of EndMT genes in cluster7. Most remarkably, in clusters 3, 5 and 9 of the NFU1G206C rats, we discovered a significant decrease in genes controlling cell adhesion and junction proteins, which could potentiate EC barrier dysfunction, an important contributor to PH pathobiology. These global aberrations from conventional EC functions could initiate pathobiological transformation.

Conclusion

We demonstrate that EC functions are diverse and crucial for normal vascular function and maintenance. We also highlight that MD can shift the EC’s from their native to pathological states. Therefore, treatment approaches that attenuate MD or targets the specific changes leading up to ED would effectively control PH.