# Regulation of Peripheral EC Cytoskeletal Remodeling, Gap Closure and Barrier Restoration by nmMLCK/MYLK and Cortactin/CTTN

> **NIH NIH P01** · UNIVERSITY OF ARIZONA · 2022 · $417,085

## Abstract

ABSTRACT:
Project #1 system biology studies have highlighted 2 critical lung cytoskeletal effector proteins/genes as central
to addressing vascular inflammation, endothelial cell (EC) permeability and the multi-organ failure critical to
ARDS mortality and the ARDS vascular endotype. We have convincingly demonstrated the multi-functional
non-muscle myosin light chain kinase isoform (MYLK) and its cytoskeletal-binding partner, cortactin (CTTN),
are primary regulators of inflammation-induced vascular permeability, leukocyte trafficking, and vascular
responses to ventilator-derived mechanical stress. Furthermore, the genes encoding nmMLCK (MYLK) and
cortactin (CTTN) harbor genetic variants that confer increased risk of sepsis/trauma-induced ARDS and ARDS
mortality in Blacks. In sync with PPG thematic goals, Project #1 is designed to translate mechanistic insights
into nmMLCK and cortactin structure and function into novel, effective therapeutic opportunities to reduce ARDS
mortality. SA #1 will explore genetic/epigenetic regulation of the non-muscle MYLK and CTTN promoters by: i)
ROS–regulated (or sensing) transcription factors (hypoxia-induced factors HIF-1a/HIF-2a, and NRF2), ii)
MYLK/CTTN promoter SNPs, and by iii) MYLK/CTTN promoter DNA methylation (Core B). SA #2 will detail EC
barrier-responses elicited by S1PR1 and TLR4 receptor activation that are influenced by tyrosine
phosphorylation of nmMLCK1, nmMLCK2 (the pro-inflammatory MYLK splice variant) and cortactin; and by
MYLK/CTTN coding SNPs (over-represented in Blacks). SA #3 will functionally characterize the involvement of
novel nmMLCK-binding proteins (pyruvate kinase M2, kindlin-2) and cortactin-binding proteins (DOCK1/ELMO1)
in S1PR1/TLR4-mediated EC cytoskeletal dynamics and barrier regulation. Pyruvate kinase M2 (PKM2), a
central regulator of glycolysis and inflammation, selectively binds the nmMLCK1 IgGCAM3 domain to potentially
influence EC cytoskeletal-driven barrier restoration. The focal adhesion (FA) regulatory protein, kindlin2, is a
Project #3 target gene, and was recently identified as a nmMLCK binding partner likely crucial for linking the
cytoskeleton to integrin-mediated cell-ECM focal adhesion and signaling. DOCK1 and ELMO1 are key Rac
GTPase and cytoskeletal regulatory proteins and novel cortactin-binding proteins. SA #2 and SA #3 studies
utilize Core C/D proteomic and biophysical imaging modalities (super resolution, AFM) to define protein
interactions in S1PR1/TLR4-mediated EC spatially-specific cytoskeletal remodeling, gap formation/closure and
lamellipodia formation and barrier regulation. SA #4 utilize established Core C rat and porcine ARDS/VILI
models to assess a novel barrier-promoting liposome bearing the S1PR1 agonist, Tysiponate, on its outer
surface, encargoed with simvastatin (nmMLCK antagonist), bixin (NRF2 agonist, MYLK antagonist), or PIK
(nmMLCK peptide inhibitor). Thus, by leveraging the integrated interactions with each PPG Project and Core,
Project #1'...

## Key facts

- **NIH application ID:** 10496460
- **Project number:** 2P01HL126609-06A1
- **Recipient organization:** UNIVERSITY OF ARIZONA
- **Principal Investigator:** Joe G. N. Garcia
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $417,085
- **Award type:** 2
- **Project period:** 2016-09-20 → 2023-01-05

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10496460

## Citation

> US National Institutes of Health, RePORTER application 10496460, Regulation of Peripheral EC Cytoskeletal Remodeling, Gap Closure and Barrier Restoration by nmMLCK/MYLK and Cortactin/CTTN (2P01HL126609-06A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10496460. Licensed CC0.

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