Modification of extracellular matrix by the product of DHA oxidation promotes retention of macrophages and progression of chronic inflammation

Authors' Affiliations

Jared L. Casteel 1 , Kasey R. Keever 1,2, Christopher L. Ardell 1 , David L. Williams 2,3, Detao Gao4 , Eugene A. Podrez 4 , Tatiana V. Byzova5 and Valentin P. Yakubenko1,2* 1 Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States, 2 Center of Excellence in Inflammation, Infectious Disease and Immunity, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States, 3 Department of Surgery, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States, 4 Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, United States, 5 Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, United States

Location

Culp Center Rm. 311

Start Date

4-25-2023 1:00 PM

End Date

4-25-2023 1:20 PM

Faculty Sponsor’s Department

Biomedical Sciences

Name of Project's Faculty Sponsor

Valentin Yakubenko

Classification of First Author

Graduate Student-Doctoral

Competition Type

Competitive

Type

Oral Presentation

Project's Category

Cardiovascular System, Immune System

Abstract or Artist's Statement

Oxidation of polyunsaturated fatty acids contributes to different aspects of the inflammatory response due to the variety of products generated. Specifically, the oxidation of DHA produces the end-product, carboxyethylpyrrole (CEP), which forms a covalent adduct with proteins via an ϵ-amino group of lysines. Previously, we found that CEP formation is dramatically increased in inflamed tissue and CEP-modified albumin and fibrinogen became ligands for αDß2 (CD11d/CD18) and αMß2 (CD11b/CD18) integrins. In this study, we evaluated the effect of extracellular matrix (ECM) modification with CEP on the adhesive properties of M1-polarized macrophages, particularly during chronic inflammation. Using digested atherosclerotic lesions and in vitro oxidation assays, we demonstrated the ability of ECM proteins to form adducts with CEP, particularly, DHA oxidation leads to the formation of CEP adducts with collagen IV and laminin, but not with collagen I. Using integrin αDß2-transfected HEK293 cells, WT, and αD-/- mouse M1- polarized macrophages, we revealed that CEP-modified proteins support stronger cell adhesion and spreading when compared with natural ECM ligands such as collagen IV, laminin, and fibrinogen. Integrin αDß2 is critical for M1 macrophage adhesion to CEP. Based on biolayer interferometry results, the isolated αD I-domain demonstrates markedly higher binding affinity to CEP compared to the “natural” αDß2 ligand fibrinogen. Finally, the presence of CEP-modified proteins in a 3D fibrin matrix significantly increased M1 macrophage retention. Therefore, CEP modification converts ECM proteins to αDß2- recognition ligands by changing a positively charged lysine to negatively charged CEP, which increases M1 macrophage adhesion to ECM and promotes macrophage retention during detrimental inflammation, autoimmunity, and chronic inflammation.

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Apr 25th, 1:00 PM Apr 25th, 1:20 PM

Modification of extracellular matrix by the product of DHA oxidation promotes retention of macrophages and progression of chronic inflammation

Culp Center Rm. 311

Oxidation of polyunsaturated fatty acids contributes to different aspects of the inflammatory response due to the variety of products generated. Specifically, the oxidation of DHA produces the end-product, carboxyethylpyrrole (CEP), which forms a covalent adduct with proteins via an ϵ-amino group of lysines. Previously, we found that CEP formation is dramatically increased in inflamed tissue and CEP-modified albumin and fibrinogen became ligands for αDß2 (CD11d/CD18) and αMß2 (CD11b/CD18) integrins. In this study, we evaluated the effect of extracellular matrix (ECM) modification with CEP on the adhesive properties of M1-polarized macrophages, particularly during chronic inflammation. Using digested atherosclerotic lesions and in vitro oxidation assays, we demonstrated the ability of ECM proteins to form adducts with CEP, particularly, DHA oxidation leads to the formation of CEP adducts with collagen IV and laminin, but not with collagen I. Using integrin αDß2-transfected HEK293 cells, WT, and αD-/- mouse M1- polarized macrophages, we revealed that CEP-modified proteins support stronger cell adhesion and spreading when compared with natural ECM ligands such as collagen IV, laminin, and fibrinogen. Integrin αDß2 is critical for M1 macrophage adhesion to CEP. Based on biolayer interferometry results, the isolated αD I-domain demonstrates markedly higher binding affinity to CEP compared to the “natural” αDß2 ligand fibrinogen. Finally, the presence of CEP-modified proteins in a 3D fibrin matrix significantly increased M1 macrophage retention. Therefore, CEP modification converts ECM proteins to αDß2- recognition ligands by changing a positively charged lysine to negatively charged CEP, which increases M1 macrophage adhesion to ECM and promotes macrophage retention during detrimental inflammation, autoimmunity, and chronic inflammation.