Yuru Liu, Ph.D. - US grants

Title: Regulation of type II cells in the repair of alveolar epithelial injury ABSTRACT Each year in the United States, there are ~ 200,000 cases of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) . Repair of the injured alveolar epithelial barrier is essential for the resolution of ALI/ARDS. However, there is currently a lack of targeted therapies aimed at promoting the repair of the epithelium to restore barrier function. The goal of this project is to understand the signaling events contributing to the endogenous alveolar repair process and identify potential drug targets to accelerate repair. Lung alveoli are lined with two types of epithelial cells: Alveolar Type I cells (AT1) and Type II cells (AT2). AT1 cover ~95% of the surface area because of their thin and squamous shape and are responsible for blood-gas exchange. AT2 have multiple functions including the secretion of surfactant and are also able to act as progenitor cells, proliferating and converting to AT1 after lung injury to restore the alveolar epithelial barrier. However, the signaling events responsible for regulating AT2-mediated repair remain poorly understood. Our preliminary data demonstrated the requirement of a non-canonical Notch ligand Dlk1 (For delta-like 1 homolog) in the AT2 to AT1 differentiation. We found that a dynamic change in Dlk1 expression is correlated with the AT2 to AT1 transition during repair. Using a mouse model in which Dlk1 was specifically disrupted in AT2, we found that the mutant cells were unable to differentiate into AT1 and at mean time had abnormally elevated Notch signaling. Based on these data, we hypothesize that dynamically regulated Notch signaling is essential for the proper progenitor function of AT2 and that Dlk1 plays an essential role in mediating AT2 to AT1 transition during alveolar repair by inhibiting Notch signaling in a population of intermediate cells. We intend to pursue the following specific aims: Aim 1: To test the hypothesis that Dlk1 regulates AT2 to AT1 cell transition and thereby mediates alveolar repair. We will determine the function of Dlk1 in restoring epithelial barrier integrity and gas-exchange function post-injury. We will define the detailed steps and molecular mechanisms in the Dlk1 regulated AT2 to AT1 transition. Aim 2: To test the hypothesis that Dlk1- dependent dynamic regulation of the Notch signaling pathway mediates AT2-dependent alveolar repair. We will determine the role of Notch signaling activity in AT2 at different phases of alveolar repair. Aim 3: To test the hypothesis that Dlk1 dependent Notch signaling promotes the differentiation of human AT2 into AT1. At the conclusion of these studies, we will have filled an important gap in knowledge about the molecular mechanisms underlying alveolar repair. We believe that these studies will lead to the discovery of therapeutic targets to accelerate lung repair and prevent chronic pathological conditions resulting from improper recovery.

Title: Regulation of type II cells in the repair of alveolar epithelial injury ABSTRACT Each year in the United States, there are ~ 200,000 cases of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) . Repair of the injured alveolar epithelial barrier is essential for the resolution of ALI/ARDS. However, there is currently a lack of targeted therapies aimed at promoting the repair of the epithelium to restore barrier function. The goal of this project is to understand the signaling events contributing to the endogenous alveolar repair process and identify potential drug targets to accelerate repair. Lung alveoli are lined with two types of epithelial cells: Alveolar Type I cells (AT1) and Type II cells (AT2). AT1 cover ~95% of the surface area because of their thin and squamous shape and are responsible for blood-gas exchange. AT2 have multiple functions including the secretion of surfactant and are also able to act as progenitor cells, proliferating and converting to AT1 after lung injury to restore the alveolar epithelial barrier. However, the signaling events responsible for regulating AT2-mediated repair remain poorly understood. Our preliminary data demonstrated the requirement of a non-canonical Notch ligand Dlk1 (For delta-like 1 homolog) in the AT2 to AT1 differentiation. We found that a dynamic change in Dlk1 expression is correlated with the AT2 to AT1 transition during repair. Using a mouse model in which Dlk1 was specifically disrupted in AT2, we found that the mutant cells were unable to differentiate into AT1 and at mean time had abnormally elevated Notch signaling. Based on these data, we hypothesize that dynamically regulated Notch signaling is essential for the proper progenitor function of AT2 and that Dlk1 plays an essential role in the AT2 to AT1 transition during alveolar repair by inhibiting Notch signaling. We intend to pursue the following specific aims: Aim 1: To test the hypothesis that Dlk1 regulates the AT2 to AT1 transition required for alveolar repair. We will determine the function of Dlk1 in restoring epithelial barrier integrity and gas-exchange function post-injury. We will define the detailed steps in the Dlk1 regulated AT2 to AT1 transition and identifying factors downstream and upstream of Dlk1. Aim 2: To test the hypothesis that the Dlk1-dependent dynamic regulation of the canonical Notch signaling pathway is essential during AT2-mediated alveolar repair. We will determine the role of Notch signaling activity in AT2 at different phases of alveolar repair and investigate the molecular interactions between Dlk1 and Notch receptors and ligands. Finally, we will investigate the effects of improving alveolar repair through the introduction of Dlk1 and other Notch-regulating molecules into alveoli and will further test the roles of Notch/Dlk1 in human AT2 and human iPSC derived AT2. At the conclusion of these studies, we will have filled an important gap in knowledge about the molecular mechanisms underlying alveolar repair. We believe that these studies will lead to the discovery of therapeutic targets to accelerate lung repair and prevent chronic pathological conditions resulting from improper recovery.

Abstract The lung alveolar epithelium is exposed to the environment and pathogens and is thus highly susceptible to injury. Repair of the alveolar epithelium requires the activation of alveolar epithelial stem cells by signals from their surrounding niche. Significantly, the role of lung microvascular endothelial cells (LMVECs) in mediating this repair is poorly understood. In this proposal we address this gap in knowledge by focusing on a novel mechanism of bioactive lipid mediated interaction between lung microvascular endothelial cells (LMVEC) and alveoli epithelial cells (AEC) in the reparative niche. LMVECs account for >40% of total lung cells. They are juxtaposed to AECs (both alveolar type I cells (AT1) and type II cells (AT2)) and play essential roles in regulating their repair, although the underlining mechanisms remain unclear. AT1s have a thin and extended squamous shape, occupy > 95% of the alveoli surface area and mediate O2?CO2 exchange. AT2 occupy only 5% of the surface area, but play multiples roles, including producing surfactant and, importantly, acting as adult tissue stem cells to repair injured alveoli. Studies, including ours, have shown that while AT2s are normally quiescent, they can respond to signals released by surrounding niches and initiate a repair program by differentiating into AT1. However, the signals that regulate AT2 stem cell function(s) remain unclear. Recent studies suggest that AT1 also exhibit a certain degree of plasticity, but it is almost completely unknown whether and how AT1s participate in lung repair after injury. In preliminary studies, we generated a mouse model with endothelial cell (EC)-specific deletion of sphingosine kinase 1 (Sphk1), the enzyme responsible for spinhgosine-1-phosphate (S1P) production. These mutant mice manifest a significantly defective repair of AECs in the standardized Pseudomonas aeruginosa bacterial lung injury model. We further showed that S1P functions through its receptor S1PR2 expressed in AT2, leading to nuclear translocation of the transcriptional regulator Yes-Associated Protein (YAP), which mediates the differentiation of AT2 to AT1 and repair of injured alveoli. Furthermore, we observed that in response to S1P, AT1s undergo substantive alteration which likely contribute to the repair process. These fundamental observations led to our central hypothesis: that LMVECs constitute a niche that, when activated by alveolar injury, releases the bioactive lipid factor, S1P, which acts via S1PRs on both AT2 and AT1 to promote their reparative capacity required for the restoration of alveolar epithelium. To test this hypothesis, we propose three specific aims: Aim 1: To determine S1P-mediated interactions between the EC niche and AEC required for lung repair. Aim2: To define the functional significance of S1P-S1PR2-YAP signaling axis in regulating AT2 to AT1 transition and mediating alveolar repair. Aim 3: To test the hypothesis that S1P induces AT1 alteration leading to alveolar repair. This research program will throw new light on the fundamental mechanisms driving lung repair after injury, with the long term potential for innovative therapeutic approaches.