Depict the cell fate map of pulmonary angioblast - ABSTRACT Endothelial cells are vital for maintaining homeostasis and facilitating repair following vascular injury or disease. Vascular regeneration is increasingly recognized as critical for treating vascular diseases, with recent evidence highlighting the role of resident angioblasts. Despite their importance, the cellular characteristics and regulatory mechanisms governing angioblasts remain poorly understood. This project seeks to bridge this gap by leveraging existing scRNA-seq data to explore the trajectory of pulmonary angioblasts (PACs), their derived cells, and the molecular markers and signaling pathways that drive their differentiation and function. As such, this proposal aligns perfectly with the NIH PAR-23-036 funding opportunity, which supports the reanalysis of existing data to address critical gaps in knowledge. By reanalyzing these datasets, we will uncover novel insights into PAC identity, fate, and the regulatory mechanisms driving their function. This approach allows us to address important questions in pulmonary vascular biology with minimal experimental burden, maximizing the utility of existing data while advancing our understanding of vascular development. Our preliminary analyses of available scRNA-seq datasets have identified PACs with significant self-renewal capacity in mouse lungs, peaking between embryonic day 12.5 (E12.5) and postnatal day 7 (P7). Intriguingly, trajectory analysis reveals that PACs do not directly differentiate into mature endothelial cells (ECs) such as arterial ECs (aECs), venous ECs (vECs), or capillary ECs (C2-ECs). Instead, PACs differentiate into an intermediary population of CD34high cells (C1-ECs), subsequently generating other EC subtypes. Underscoring their importance, PACs and their progeny were significantly reduced in hyperoxia-induced bronchopulmonary dysplasia (BPD) mice, which have arrested pulmonary vascular development and alveolarization. We have identified several major knowledge gaps in our understanding of pulmonary vascular development. a) How vasculogenesis, intussusceptive and sprouting angiogenesis are coordinated with lung development remains unclear. b) The spatial-temporal lineages of EC- related cells during lung development and regeneration are unexplored. c) The functional roles of different lineages in lung development and homeostasis are not well defined. d) The specificity of the PACs vs angioblasts from blood island during the embryo stage in mice. e) The regulatory mechanisms guiding PAC cell fate and localization are largely unknown. The previous findings about pulmonary vascular development and our preliminary data lead us to hypothesize that PACs are crucial for pulmonary vasculature development by residing in specific niches that facilitate lung development. PACs have the most robust self-renew ability. In proximal regions, PACs localize along arterial and venous vessel walls to contribute to intussusceptive angiogenesis. In distal regions, PACs differentiate into CD34high cells and lead the protruding into secondary septa, which form neovasculature and promote the alveolar-capillary structure. (Fig. 1). To test this hypothesis, we propose two specific aims. Aim 1: Define PAC cell identity, cell fates, and regulatory mechanisms in mice and humans. Aim 2: Visualize PAC and PAC-derived cell lineages during development and vessel injury.
