Molecular mechanisms controlling lung capillary speciation, plasticity and function during early postnatal development

The National Heart, Lung, and Blood Institute awarded The Regents of the University of California, San Francisco $778,504 on July 1, 2026, under the Cardiovascular Diseases Research program (CFDA 93.837) to investigate molecular mechanisms controlling lung capillary speciation, plasticity, and function during early postnatal development.

The research examines how distinct subtypes of lung capillary endothelial cells—CAP1 and CAP2—contribute to alveolar formation and repair after injury during early postnatal life. The project employs genetic lineage tracing to characterize the roles of these capillary subtypes in lung development, their responses to hyperoxia-induced injury, and their regenerative capacity. The work specifically investigates an intermediate capillary endothelial cell state (CAPINT) that expresses both KIT and TBX2 markers and appears to precede CAP2 expansion and terminal differentiation, as well as AP-1 transcription factor pathways that promote tip cell phenotype in response to hyperoxia.

The award funds research performed in San Francisco, California, with a period of performance through February 28, 2030. This is a project grant, a standard assistance type under the NHLBI's cardiovascular diseases research portfolio, which supports competitive hypothesis-driven biomedical research reviewed for scientific merit and program relevance by peer review panels and the National Heart, Lung, and Blood Advisory Council.

Abstract

During early postnatal life, septation of distal lung saccules into millions of alveoli and rapid expansion of lung capillaries markedly increases gas exchange surface area. Angiocrine signals from lung endothelial cells (EC) help promote alveolar formation and repair after injury. The lung capillary network is comprised of two distinct subtypes. Capillary 1 EC (CAP1) are smaller and more proliferative, and highly express kit. In contrast, CAP2 are larger, highly specialized EC with multiple filopodia (reminiscent of tip cells), that closely associate with alveolar epithelial cells and highly express TBX2. However, the separate roles of these distinct capillary EC on informing the alveolar niche, their sensitivity to injury, and their regenerative capacity remain unclear. CAP2 rapidly expand after birth and progress toward terminal differentiation across early postnatal life. Postnatal hyperoxia, an injury that suppresses CAP1 proliferation, further expands CAP2. Using genetic lineage tracing, we showed that the majority of expanded CAP2 in hyperoxia are derived from TBX2+ EC present at birth. This exciting finding contrasts with the adult lung where CAP1 EC appear to serve as the main progenitors for new CAP2. Additional data identified the unique presence of capillary EC that CO-express kit and TBX2 in the immature lung. These intermediate capillary EC (capint) were abundant at P3 but rare by P7 and comprised a significant portion of proliferating EC in the late saccular lung. Hyperoxia increased capint abundance and persistence, suggesting that this cell state precedes CAP2 expansion and terminal differentiation. Hyperoxia also increased chromatin accessibility for activator protein-1 (AP-1) transcription factors, a pathway that promotes tip cell phenotype in EC of other organs. Although IT is known that VEGFR2 signaling is required for CAP2 differentiation, the VEGFR2 mediated downstream mechanisms that regulate CAP2 differentiation are not known. Further, embryonic loss of epithelial-derived vegf blocks CAP2 differentiation and disrupts alveolarization, however, how CAP2 inform the alveolar niche has not been studied. Using a novel mouse model, we show that inducible abrogation of VEGFR2 in TBX2+ EC after birth: (i) blocked the hyperoxia-induced expansion of CAP2 EC; (II) prevented CAP2 terminal differentiation; (III) increased capint EC; and (IV) heightened expression of fibrotic mediators in the alveolus. Taken together, our data suggest the overall hypothesis that postnatal expansion of CAP2 is preceded by entry of CAP1 progenitors into an intermediate cell state; and requires VGFR2-mediated activation of AP-1 to promote CAP2 terminal differentiation to maintain a homeostatic alveolar capillary niche, which will be tested through three specific aims. Aim 1 will employ hyperoxia, in vivo EDU labeling, genetic lineage tracing and paired single cell RNA- and atac-SEQ to identify the molecular regulation of the capint cell state in development and disease. Aim 2 will employ mass spectroscopy and chip-sequencing in our novel mouse model to identify VEGFR2- downstream signaling pathways that promote CAP2 EC terminal differentiation. Aim 3 will induce abrogation of CAP2 VEGFR2 signaling to determine how impaired CAP2 terminal differentiation alters the alveolar niche.