Deciphering endothelial heterogeneity in health and disease at single cell resolution: progress and perspectives

Endothelial cells (ECs) constitute the inner lining of vascular beds in mammals and are crucial for homeostatic regulation of blood vessel physiology, but also play a key role in pathogenesis of many diseases, thereby representing realistic therapeutic targets. However, it has become evident that ECs are heterogeneous, encompassing several subtypes with distinct functions, which makes EC targeting and modulation in the disease-context challenging. The rise of the new single cell era has led to an emergence of studies aimed at interrogating transcriptome diversity along the vascular tree, and has revolutionized our understanding of EC heterogeneity from both a physiological and pathophysiological context. Here, we discuss recent landmark studies aimed at teasing apart the heterogeneous nature of ECs. We cover driving (epi)genetic, transcriptomic and metabolic forces underlying EC heterogeneity in health and disease, as well as current strategies used to combat disease-enriched EC phenotypes, and propose strategies to transcend largely descriptive heterogeneity towards prioritization and functional validation of therapeutically targetable drivers of EC diversity. Lastly, we provide an overview of the most recent advances and hurdles in single EC OMICs

Investigating murine endothelial cell injury in pulmonary arterial hypertension using single-cell RNA sequencing

Endothelial cells (ECs) are highly heterogenous across different organs and within the vascular bed, in both health and disease. However, EC heterogeneity has not been extensively dissected in the context of pulmonary arterial hypertension (PAH). It is a rare and progressive disease characterised by remodelling of the distal pulmonary vasculature. Using an EC lineage-tracing mouse model and single-cell RNA-sequencing (scRNA-seq), I first investigated how enriched mouse ECs respond to Sugen 5416/Hypoxia-induced (SuHx) PAH. Globally, ECs adopt an immunophenotype in PAH, with upregulation of genes relating to the major histocompatibility class II (MHC-II) complex. The strongest upregulation is observed in Artery and one of the two capillary ECs (Capillary A/gCap). The other capillary EC cluster (Capillary B/aerocyte) had a distinct response to PAH, with upregulation of genes regulating apoptosis, migration, and angiogenesis. Comparison with whole-lung scRNA-seq in rat and human PAH identified several candidate genes conserved across all species. Following this, I further examined heterogeneity within Artery and Capillary A ECs, the pulmonary vasculature predominantly affected in PAH. Artery ECs can be subdivided into Proximal and Distal Artery ECs, with both groups having distinct biomarkers. In PAH, a loss of Proximal Artery EC identity and a gain in Distal Artery EC identity is observed. As for Capillary A ECs, three subpopulations were identified. In Control, two of these subclusters (CapillaryA_0, CapillaryA_1) were marked by high expression of Distal Artery markers, and low expression of Proximal Artery markers, whilst the reverse was true for the third CapillaryA_2 subcluster. There was also significantly more CapillaryA_2 ECs in PAH. Differential gene expression analysis found some genes uniquely upregulated in each CapillaryA subcluster: senescence-associated genes in CapillaryA_0 (Cd9, H3f3b, Neat1), signalling genes in CapillaryA_1 (Rab5a, Slc6a6, Tmem252), and both inflammatory (H2-Q4, Irgm1, Tgtp2) and angiogenesis regulatory genes (Cldn5, Cxcl12, Esam, Rhoj) in CapillaryA_2. Overall, this work has revealed in high-resolution, how ECs in different vascular beds distinctly respond to PAH

DECIPHERING ENDOTHELIAL HETEROGENEITY IN HEALTH AND DISEASE AT SINGLE CELL RESOLUTION: PROGRESS AND PERSPECTIVES

Endothelial cells (ECs) constitute the inner lining of vascular beds in mammals and are crucial for homeostatic regulation of blood vessel physiology, but also play a key role in pathogenesis of many diseases, thereby representing realistic therapeutic targets. However, it has become evident that ECs are heterogeneous, encompassing several subtypes with distinct functions, which makes EC targeting and modulation in the disease-context challenging. The rise of the new single cell era has led to an emergence of studies aimed at interrogating transcriptome diversity along the vascular tree, and has revolutionized our understanding of EC heterogeneity from both a physiological and pathophysiological context. Here, we discuss recent landmark studies aimed at teasing apart the heterogeneous nature of ECs. We cover driving (epi)genetic, transcriptomic and metabolic forces underlying EC heterogeneity in health and disease, as well as current strategies used to combat disease-enriched EC phenotypes, and propose strategies to transcend largely descriptive heterogeneity towards prioritization and functional validation of therapeutically targetable drivers of EC diversity. Lastly, we provide an overview of the most recent advances and hurdles in single EC OMICs

Single-cell RNA sequencing profiling of mouse endothelial cells in response to pulmonary arterial hypertension.

AIMS: Endothelial cell (EC) dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterize EC dynamics in PAH at single-cell resolution. METHODS AND RESULTS: We carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility, and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and microvasculature. CONCLUSION: This study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development