|| 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 and angiogenesis. 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 means by which purine nucleotides are brought into and affect the local environment has never been identified. We hypothesize that venous endothelial barrier function is regulated by a localized purinergic signaling cascade that controls the stability and expression of tight junction proteins. We will use three aims to test this concept.
In Aim 1, we will determine roles for Pannexin 1 in regulating venous endothelial permeability.
This aim will use novel methods for ex vivo vein isolation and measure transendothelial resistance and dye movement from endothelial cell specific Panx1 knockout mice and endothelial cell specific Panx1 over-expressing mice, as well in vivo testing using the cecal ligation puncture (CLP) septic model.
In Aim 2, we will measure the relative contributions of different Adenosine Receptors (ARs) on endothelial barrier function. We will determine the differential role of CD39 and CD73 on adenosine receptor activation, using endothelial cell specific A2A and A2B floxed mice, as well as the relative contribution of PKA or PKC upon activation. Lastly, in Aim 3, we will define roles for claudin-11 in venous endothelial barrier function and as a target for TRPV4-mediated disruption of tight junctions.
This aim will utilize state of the art calcium imaging to determine a role for TRPV4 in regulation of claudin-11 production, trafficking, assembly and stability. Claudin-11 is a novel claudin isoform whose role in barrier function is only beginning to be elucidated. Molecular manipulation of claudins will be used to demonstrate that claudin-11 is required for veins to be sensitive to calcium fluxes because it has the unique capacity to bind calmodulin, whereas claudin-5 does not. The feasibility of accomplishing these aims is underscored by all proposed knockout mice being in hand, an IRB in place for human samples, and strong preliminary data.
Endothelial cells line the inside of blood vessels and provide barrier integrity to regulate the amount of fluid that moves across blood vessel barriers. Fluid leakage out of the vasculature is a major complication of inflammation that often leads to a lethal systemic bacterial infection known as sepsis. Fluid leak occurs in large part through veins, where the vast majority of blood resides. We will study how inflammatory signals induce changes in proteins that regulate the veinous endothelial barrier to identify targets with the ability to control sepsis.
| Aasen, Trond; Johnstone, Scott; Vidal-Brime, Laia et al. (2018) Connexins: Synthesis, Post-Translational Modifications, and Trafficking in Health and Disease. Int J Mol Sci 19: |
