Functional Characterization of ALI-Associated MYLK SNPs

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): Acute lung injury (ALI) is a disorder affecting over 200,000 U.S. patients per year and has an unacceptably high mortality of 30-35%. The pathophysiology of ALI centers on inflammation-induced disruption of the endothelial cell (EC) barrier lining the pulmonary vasculature, causing leakage of fluid, protein, and cells into the airspaces. Pulmonary endothelial barrier maintenance and recovery from injury is dependent on EC cytoskeletal dynamics. Myosin light chain kinase (MLCK) is a multi-functional cytoskeletal effector protein that is a key regulator of barrier function and has been a focus of the Garcia lab for over 20 years. This work has clearly established that barrier-disrupting edemagenic agonists produce spatially localized MLC phosphorylation within cytoplasmic contractile stress fibers, resulting in actomyosin contraction, tension, and formation of paracellular gaps. In contrast, barrier-protective agonists induce nmMLCK translocation to cortical actin networks and into lamellipodial membrane protrusions designed to close paracellular gaps and restore barrier integrity. Additionally, nmMLCK regulates lung trafficking of inflammatory cells and is robustly activated by biophysical forces such as excessive mechanical stress, which underlies its implications in ventilator induced lung injury (VILI). Furthermore, levels of nmMLCK expression and enzymatic activity contribute to risk and severity of ALI and VILI in preclinical models and human samples. Previously, the Garcia lab has identified a haplotype of three coding SNPs (Pro21His, Ser147Pro, Val261Ala, "3XSNP") in the nmMLCK gene (MYLK) that is highly associated with increased ALI susceptibility and severe asthma. This three SNP haplotype is significantly more common in individuals of African descent (AD) and likely contributes to the significant health disparities in ALI incidence and mortality. We hypothesize that these ALI-associated MYLK SNPs alter the structure of nmMLCK and that these structural changes impair lamellipodial activity and paracellular gap closure in response to inflammatory injury. We plan to perform functional studies of known MYLK SNPs using sophisticated biophysical and ultrastructural techniques including trans-endothelial resistance (TER) measurement, confocal microscopy and co-localization, kymography and fluorescence resonance energy transfer (FRET). Subsequently, we plan to validate the clinical relevance of these SNPs in established preclinical models of ALI and VILI and clarify nmMLCK antagonism as a novel ALI therapeutic strategy.