Saturday, December 21, 2024
12/21/2024
|| ABSTRACT Breakdown of the endothelial cell barrier is considered a defining pathological hallmark of multiple diseases. Indeed, sepsis accounts for more hospital deaths per year than any other condition in the United States, and the disease is currently devoid of any targeted pharmacological intervention. Critical to understanding how inflammation affects vascular barrier function is that endothelial cells throughout the circulatory system are not homogenous. Inflammation specifically affects vascular permeability through effects on the venous endothelium, whereas the arterial endothelium is not susceptible to inflammation-induced permeability and instead primarily regulates blood pressure. Thus, a mechanistic view of how venous endothelial barrier function is regulated is essential to human health and disease. Our current understanding of vascular barrier function does not account for endothelial heterogeneity and the unique cell adhesion and signaling pathways specific to each endothelial cell type. Purinergic signaling has been identified as a key regulator of endothelial permeability; however the cellular pathway allowing for simultaneous regulation of the purine response and tight junctions has not been explored. We hypothesize that the purine release channel pannexin (Panx)1 in venous endothelium is a component of a dynamic signaling nexus activated upon inflammatory stimuli such as sepsis. We will use three aims to test this concept. In Aim 1, we will identify kinase signaling pathways targeting Panx1 that regulate venous permeability. This aim will use novel mouse models with point mutations in Panx1 for post-translational modification and measure how these key modifications alter pathological responses to cecal ligation puncture (CLP) sepsis model, venous permeability, and ATP release. Aim 2, we will measure the impact of claudin11 (cldn)11 on the intracellular distribution of Panx1 and organization of other components required for purinergic signaling in response TNFa stimulated venous permeability. Based on our previous work, we found cldn11 to be uniquely expressed across the venous endothelium that was selectively broken down in response to TNFa/sepsis; this contrasted with claudin5. Here was posit cldn11 is a unique signaling hub due to its ability to be regulated by changes in calcium, and that property of the tight junction regulates Panx1 function on venous endothelium. Last, in Aim 3 we will determine roles for internalization and turnover of components of the Panx1/Cldn11 hub in regulation of venous permeability. Turnover and reseting the signaling complex is an important component to the cellular response to inflammation. In this aim we provide evidence that caveolin1 facilitates recycling of both Panx1 and cldn11 after inflammatory stimuli, and probe the mechanisms of how this may occur. The feasibility of accomplishing these aims is underscored by all proposed knockout mice being in hand, coupled with strong preliminary data and a highly productive previous funding cycle between the two investigators.
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