Sustained vascular leakage due to prolonged or exacerbated inflammation leads to organ damage, lasting sequelae and increased mortality. The mechanisms that mediate long-term loss of endothelial barrier function are poorly understood. We propose that transcriptional changes in the endothelium, and in particular STAT3- dependent gene regulation, may explain these long-term changes. STAT3 can promote opposing effects on the endothelium. While inhibition of its phosphorylation (mediated by JAK kinases) is anti-inflammatory and reduces endothelial permeability, endothelial STAT3 knockout actually increases vascular leakage. The central hypothesis of this grant is that prolonged STAT3 phosphorylation promotes a pro-inflammatory STAT3 transcriptional response that involves changes in the glycocalyx, including degradation of hyaluronic acid, and drastic alterations in the actin cytoskeleton. This hypothesis challenges the current understanding of the role of STAT3 in the endothelial inflammatory response by linking cytokine signaling to sustained vascular leakage via a novel mechanism. A better understanding of this pathway not only can explain multiple clinical observations, but also is expected to have a significant positive impact on future anti- inflammatory treatments by identifying novel pharmacological targets to prevent and/or revert the systemic vascular leakage without affecting other arms of the immune response that may be required for pathogen clearing or tissue healing. We will combine in vitro and in vivo experimentation in the three separate aims to: Identify the specific role of STAT3-dependent transcriptional mediators in endothelial barrier loss. We identified novel STAT3-dependent genes downstream of IL-6, including endothelial glycocalyx catabolic enzymes and modulators of the actin cytoskeleton. Loss of hyaluronan and actin stress fibers temporally correlate with IL-6- induced loss of barrier function in HUVEC. We will determine any causal roles for these candidate genes. Determine the requirement of endothelial STAT3 phosphorylation in promoting barrier function loss in vivo. Phosphorylated and non-phosphorylated STAT3 may have different and even opposing roles. We will use established models of sepsis to assess mice survival, vascular leakage and endothelial glycocalyx and cytoskeletal regulation of STAT3 (STAT3iEKO) and gp130iEKO knockout mice, as well as mice harboring an inducible, endothelial-specific knockin Y705F mutation (STAT3iE-Y705F). Determine the role of feedback loops leading to STAT3 sustained activation in barrier function. We will determine how an IL-6-induced sustained activation of STAT3 can lead to prolonged vascular leakage by interfering with negative feedback loops involving SOCS3 and TC45. We will identify potential post- translational modifications of the negative regulator SOCS3 that could lead to sustained STAT3 activation. To assess the role of these loops in vivo, we will create endothelial-specific SOCS3 knockouts by breeding SOCS3fl mice to cdh5-CreERT2 mice.

Public Health Relevance

Vascular barrier function is governed by endothelial cell-cell interactions. Excessive inflammation leads to vascular leakage, organ damage and long-term sequelae, including death. This project addresses a potential mechanism within endothelial cells regulating the extent and duration of the vascular leakage. Targeting endothelial-specific mechanisms regulating barrier function could provide a new therapeutic strategy to prevent vascular leakage while maintaining the immune response intact.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM124133-02
Application #
9788493
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Zhao, Xiaoli
Project Start
2018-09-20
Project End
2023-07-31
Budget Start
2019-08-01
Budget End
2020-07-31
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Albany Medical College
Department
Physiology
Type
Schools of Medicine
DUNS #
190592162
City
Albany
State
NY
Country
United States
Zip Code
12208