2. Academic Validation
  3. Human lung microvascular endothelial cell protein modification by 2-chlorohexadecanoic acid: RhoA mediates 2-chlorohexadecanoic acid-elicited endothelial activation

Human lung microvascular endothelial cell protein modification by 2-chlorohexadecanoic acid: RhoA mediates 2-chlorohexadecanoic acid-elicited endothelial activation

  • Redox Biol. 2025 May:82:103596. doi: 10.1016/j.redox.2025.103596.
Haley L Carlson 1,  David A Ford 2
Affiliations

Affiliations

  • 1 Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, 63104, USA; Center for Cardiovascular Research, Saint Louis University School of Medicine, St. Louis, MO, 63104, USA.
  • 2 Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, 63104, USA; Center for Cardiovascular Research, Saint Louis University School of Medicine, St. Louis, MO, 63104, USA. Electronic address: david.ford@health.slu.edu.
PMID: 40117888 DOI: 10.1016/j.redox.2025.103596
Abstract

Chlorolipids are produced during the neutrophil respiratory burst as a result of myeloperoxidase (MPO)-generated hypochlorous acid (HOCl) targeting the vinyl ether bond of plasmalogen Phospholipids. The initial products of this reaction are 2-chlorofatty aldehydes (2-ClFALDs), which are subsequently oxidized to 2-chlorofatty acids (2-ClFAs). 2-Chlorohexadecanoic acid (2-ClHA) is the 16-carbon 2-ClFA species, and previous studies have shown that increased levels of plasma 2-ClHA associate with acute respiratory distress syndrome (ARDS)-caused mortality in human sepsis. 2-ClHA causes endothelial barrier dysfunction and increases neutrophil and platelet adherence to the endothelium. In this study, click chemistry analogs of 2-ClHA and hexadecanoic acid (HA) were used to identify proteins covalently modified by 2-ClHA and HA in human lung microvascular endothelial cells (HLMVECs). Eleven proteins were specifically modified by 2-ClHA, and an additional one hundred and ninety-four proteins were modified by both 2-ClHA and HA. STRING analysis of 2-ClHA-modified proteins revealed a network of proteins with RhoA as a hub. RhoA is one of the proteins specifically modified by 2-ClHA and not HA. The RhoA inhibitors, Rhosin and C3, inhibited both 2-ClHA-elicited HLMVEC barrier dysfunction and angiopoietin-2 (ANG-2) release from HLMVEC. Further studies showed 2-ClHA activates HLMVEC RhoA activity. The specificity of the 2-ClHA-RhoA pathway for endothelial activation was further confirmed since HA did not cause HLMVEC barrier dysfunction, ANG-2 release and RhoA activation. Collectively, these studies have identified multiple proteins modified exclusively by 2-ClHA in HLMVECs, including RhoA. These proteomics studies led to the key finding that RhoA is an important mediator of 2-ClHA-caused endothelial barrier dysfunction.

Keywords

Chlorolipids; Endothelium; Protein modification; Proteomics; RhoA.

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