Acute respiratory distress syndrome (ARDS) is a vexing critical care illness in patients with respiratory failure. A fundamental pathobiological feature of ARDS is an increase in blood vessel permeability, and the permeability of its lining, resulting in excessive fluid leakage and the infiltration of inflammatory cells into the functional parts of the lung or parenchyma. Work from our laboratory has shown that each of these processes are tightly linked to the central cytoskeletal bioregulatory molecule, non-muscle isoform of myosin light chain kinase (nmMLCK).
The MYLK gene (sequence coding for a protein) is located on chromosome 3 and encodes both the aforementioned non-muscle and smooth muscle (smMLCK). nmMLCK is a protein of 1,914 amino-acids and is expressed predominantly in blood vessel cells while the smMLCK isoform is present in smooth muscle exclusively. We were the first to identify and clone nmMLCK and to demonstrate its key role in regulating cell shape, cell contractile events (acto-myosin) and formation of gaps between cells (paracellular), events which directly contribute to loss of cell barrier integrity and the increases in permeability. nmMLCK is also involved in the processes of barrier restoration via its modulation of cell peripheral rearrangement and cell wall protrusions to enhance interactions between cells and the surrounding medium.
One goal of our laboratory’s work has been to understand the genetic causes of health disparities among various ethnic populations, especially in patients of African American descent who are at increased risk for inflammatory disorders such as severe asthma and ARDS. To investigate these differences, we carried out extensive MYLK sequencing and identified key genetic variants between ARDS subjects and normal healthy individuals in the MYLK control element (promoter region) and the coding region which affect the expression and structure/function respectively. Of particular interest to us were changes to three specific residues within the nmMLCK protein that were present in ARDS and severe asthma subjects of African American descent. Alteration to these three residues within the protein sequence caused changes in the interaction of nmMLCK with other proteins and, using a bioinformatics approach, we predicted that these changes altered the stability of the coding instructions – or messenger RNA – with subsequent effects on nmMLCK expression. Since nmMLCK is critical to vascular barrier regulation, we hypothesized that the triple mutant alters nmMLCK function through a mechanism that remains unknown.
To understand the function of these changes in vascular barrier regulation we challenged endothelial cells with a lipid molecule (Sphingosine-1-Phosphate, S1P) that promotes cell-cell adhesion, and analyzed changes using live cell imaging. We observed that movement of cells carrying nmMLCK with the modified residues (as in the susceptible individuals we had been studying) was slower in response to treatment with the lipid molecule. That was accompanied by a reduction in protein modification, or phosphorylation, at a site which we have previously shown to affect nmMLCK function. These changes in protein modification were reflected in lower movement speed and reduced translocation of nmMLCK to the cell periphery where nmMLCK exerts its function in vascular barrier regulation.
In conclusion, we have identified a mechanism for the genetic component of susceptibility to mortality-inducing diseases such as ARDS, which are critical in addressing the susceptibility of patients of African-American patients to mortality.
Joseph Mascarenhas, PhD is Research Assistant Professor of Medicine at the University of Arizona College of Medicine, Tucson.
Joe G. N. ‘Skip’ Garcia, MD is a world-renowned pulmonary physician-scientist, an endowed professor of medicine at the University of Arizona College of Medicine, Tucson, and an elected member of the Institute of Medicine of the National Academies.
Ting Wang, Mary E. Brown, Gabriel T. Kelly, Sara M. Camp, Joseph B. Mascarenhas, Xiaoguang Sun, Steven M. Dudek, Joe G.N. Garcia
First Published: March 9, 2018