Post-transcriptional regulation of vascular homeostasis by RNA-binding proteins - PROJECT SUMMARY Vascular homeostasis is essential for human health as endothelial cells (EC) perform important physiological functions such as formation of vascular barriers. EC activation occurs when hypoxic or inflammatory signals lead to EC sprouting, inflammatory gene expression, and vascular leakage, which disrupts vascular homeostasis and causes vascular dysfunction and cardiovascular diseases. Therefore, this application focuses on identifying key regulators of vascular homeostasis and understanding how they maintain normal vascular function, which forms the basis for our long-term objectives to understand how the identified key homeostasis factors confer resilience to the vasculature to prevent loss of vascular homeostasis in cardiovascular diseases. Here we focus on RNA- binding proteins (RBP) as they are essential regulators of gene expression but highly understudied in vascular biology. To identify key RBPs for maintenance of vascular homeostasis and suppression of EC activation, we performed an in silico screen (single-cell RNAseq) followed by an in vitro screen, and identified the RBP PABPC1, a critical polyA binding protein in cells, as a key suppressor of EC activation. To determine how PABPC1 regulates vascular homeostasis, we generated induced EC-specific knockout (iECKO) mice of Pabpc1. Within two weeks, these animals demonstrated robust EC activation and loss of vascular homeostasis characterized by systemic inflammation, immune cell infiltration, and vascular leakage, culminating in death after 2-3 weeks. Mechanistically, PABPC1 depletion without inflammatory stimulus led to increased inflammatory mRNAs in EC such as chemokines, suggesting that PABPC1 normally suppresses these mRNAs. PABPC1 likely achieves the suppression through binding another RBP that is well-established to mediate decay of inflammatory mRNAs. Based on our findings, we hypothesize that PABPC1 is required for suppression of EC activation and maintenance of vascular homeostasis by regulating turnover of inflammatory mRNAs. We will test this hypothesis with two aims: 1) determine if identified chemokine is a critical mediator of vascular phenotypes in Pabpc1iECKO mice, and 2) define the molecular mechanism by which PABPC1 suppresses chemokine mRNAs in EC. We will use single-cell RNAseq to investigate EC gene expression changes and EC-immune cell crosstalk in Pabpc1iECKO mice. We will test if blocking of the identified chemokine rescues phenotypes in Pabpc1iECKO mice. We will also use site-directed mutagenesis to test if PABPC1 binding to its RBP patterner is required for suppressing inflammatory RNAs in EC. The proposal is conceptually innovative by suggesting an unexpected mechanistic role of PABPC1 in maintaining vascular homeostasis by preventing EC activation through continuous suppression of inflammatory mRNAs in EC. Since PABPC1 levels in EC decrease with aging and chronic inflammation is a hallmark of aging, this mechanism is likely critical to age-related cardiovascular disease risk. Our work will address whether PABPC1 and its downstream genes are potential drug targets to confer resilience to the vasculature and prevent age-related cardiovascular diseases.
