Endothelial Heterogeneity in the Response to Autophagy Drives Small Vessel Muscularization in Pulmonary Hypertension.

BACKGROUND: Endothelial cell (EC) apoptosis and proliferation of apoptosis-resistant cells is a hallmark of pulmonary hypertension (PH). Yet, why some ECs die and others proliferate and how this contributes to vascular remodeling is unclear. We hypothesized that this differential response may: (1) relate to different EC subsets, namely pulmonary artery (PAECs) versus microvascular ECs (MVECs); (2) be attributable to autophagic activation in both EC subtypes; and (3) cause replacement of MVECs by PAECs with subsequent distal vessel muscularization. METHODS: EC subset responses to chronic hypoxia were assessed by single-cell RNA sequencing of murine lungs. Proliferative versus apoptotic responses, activation, and role of autophagy were assessed in human and rat PAECs and MVECs, and in precision-cut lung slices of wild-type mice or mice with endothelial deficiency in the autophagy gene Atg7 (Atg7EN-KO). Abundance of PAECs versus MVECs in precapillary microvessels was assessed in lung tissue from patients with PH and animal models on the basis of structural or surface markers. RESULTS: In vitro and in vivo, PAECs proliferated in response to hypoxia, whereas MVECs underwent apoptosis. Single-cell RNA sequencing analyses support these findings in that hypoxia induced an antiapoptotic, proliferative phenotype in arterial ECs, whereas capillary ECs showed a propensity for cell death. These distinct responses were prevented in hypoxic Atg7EN-KO mice or after ATG7 silencing, yet replicated by autophagy stimulation. In lung tissue from mice, rats, or patients with PH, the abundance of PAECs in precapillary arterioles was increased, and that of MVECs reduced relative to controls, indicating replacement of microvascular by macrovascular ECs. EC replacement was prevented by genetic or pharmacological inhibition of autophagy in vivo. Conditioned medium from hypoxic PAECs yet not MVECs promoted pulmonary artery smooth muscle cell proliferation and migration in a platelet-derived growth factor-dependent manner. Autophagy inhibition attenuated PH development and distal vessel muscularization in preclinical models. CONCLUSIONS: Autophagic activation by hypoxia induces in parallel PAEC proliferation and MVEC apoptosis. These differential responses cause a progressive replacement of MVECs by PAECs in precapillary pulmonary arterioles, thus providing a macrovascular context that in turn promotes pulmonary artery smooth muscle cell proliferation and migration, ultimately driving distal vessel muscularization and the development of PH.

MIR503HG Loss Promotes Endothelial-to-Mesenchymal Transition in Vascular Disease.

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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 diseases 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.