Endothelial cell (EC)-specific CTGF/CCN2 Expression Increases EC Reprogramming and Atherosclerosis

Feifei Li; Sandeep Kumar; Anastassia Pokutta-Paskaleva; Dong-Won Kang; Chanwoo Kim; Julia Raykin; Victor Omojola; Carson Hoffmann; Fujie Zhao; Maiko Teichmann; Christian Park; Kyung In Baek; Gloriani Sanchez Marrero; Jing Ma; Hiromi Yanigasawa; Andrew Leask; Lucas Timmins; Xiangqin Cui; Roy Sutliff; Rudy L Gleason Jr; Hanjoong Jo; Luke Brewster

doi:10.1016/j.matbio.2025.01.003

Endothelial cell (EC)-specific CTGF/CCN2 Expression Increases EC Reprogramming and Atherosclerosis

Matrix Biol. 2025 Jan 14:S0945-053X(25)00009-5. doi: 10.1016/j.matbio.2025.01.003. Online ahead of print.

Authors

Feifei Li¹, Sandeep Kumar², Anastassia Pokutta-Paskaleva¹, Dong-Won Kang², Chanwoo Kim³, Julia Raykin¹, Victor Omojola², Carson Hoffmann⁴, Fujie Zhao⁴, Maiko Teichmann⁴, Christian Park², Kyung In Baek², Gloriani Sanchez Marrero⁵, Jing Ma⁶, Hiromi Yanigasawa⁷, Andrew Leask⁸, Lucas Timmins⁹, Xiangqin Cui¹⁰, Roy Sutliff¹¹, Rudy L Gleason Jr¹², Hanjoong Jo², Luke Brewster¹³

Affiliations

¹ Department of Surgery, Emory University, Atlanta, GA, USA.
² Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA.
³ Department of Advanced Animal Model Development, Non-Clinical Center, Osong Medical Innovation Foundation (KBIO), Cheongju, Republic of Korea.
⁴ Department of Surgery, Emory University, Atlanta, GA, USA; Research Services, Atlanta VA Medical Center, Decatur, GA, USA.
⁵ Department of Surgery, Emory University, Atlanta, GA, USA; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA.
⁶ Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Department of Medicine, Emory University School of Medicine, Atlanta, GA.
⁷ Life Science Center, Tsukuba Advanced Research Alliance, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan.
⁸ College of Dentistry, University of Saskatchewan, 105 Wiggins Rd, Saskatoon, SK, Canada.
⁹ Department of Bioengineering, University of Utah, Salt Lake City, UT, USA.
¹⁰ Department of Biostatistics and Bioinformatics, Emory University, Atlanta, GA, USA.
¹¹ National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
¹² Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA; George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA.
¹³ Department of Surgery, Emory University, Atlanta, GA, USA; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA; Research Services, Atlanta VA Medical Center, Decatur, GA, USA. Electronic address: [email protected].

PMID: 39818254
DOI: 10.1016/j.matbio.2025.01.003

Abstract

Arterial endothelial cells (ECs) reside in a complex biomechanical environment. ECs sense and respond to wall shear stress. Low and oscillatory wall shear stress is characteristic of disturbed flow and commonly found at arterial bifurcations and around atherosclerotic plaques. Disturbed flow is pro-inflammatory to ECs. Arteries also stiffen with aging and/or the onset of vascular disease. ECs sense and respond to stiffening in a pro-fibrotic manner. Thus, flow and stiffening disturbances elicit EC responses that promote pathologic arterial remodeling. However, the pathways elicited by ECs under pathologic stiffening and disturbed flow are not well understood. The objective of this work was to discover and test the modifiability of key pathways in ECs. To do this we used the partial carotid ligation model to impose disturbed flow onto the precociously stiffened fibulin-5 knockout (Fbln5^-/-) mouse carotid arteries. Biomechanical testing demonstrated that Fbln5^-/- arteries under disturbed flow approximate the stiffness ratio of diseased human arteries, and the ECs in these Fbln5^-/- arteries underwent rapid reprogramming via endothelial to mesenchymal transition (EndMT). Under atherogenic conditions, disturbed flow Fbln5^-/- arteries developed more vulnerable plaques than the wild type (WT) mouse arteries. Connective tissue growth factor/cellular communication network factor 2 (Ctgf/Ccn2) was upregulated in vivo in ECs with aging, with stiffening in the Fbln5^-/- arteries, and increased again by disturbed flow under stiffened conditions, supporting CTGF as a key biomarker for flow and stiffening. This was validated by immunohistochemistry, which demonstrated increased CTGF deposition in areas of disturbed flow in patient carotid endarterectomy and peripheral artery disease (PAD) specimens. Finally, to test the role of CTGF in regulating and combining these processes, we created an EC-specific Ctgf knockout (Ctgf^ecko). We identified that carotid arteries under disturbed flow and atherogenic conditions in male Ctgf^ecko, but not female, mice had decreased plaque area compared to WT control mice. We then tested the Ctgf expression in the carotid endothelium exposed to disturbed or stable flow in WT and Fbln5^-/- mice. Here we found that under disturbed flow male mice had greater Ctgf expression than female mice. This work demonstrates that stiffened + disturbed flow conditions drive EC reprogramming, that CTGF is increased by these conditions, and that this increase is more prominent in male carotid arteries. Future exploration of sex-based differences in these fibrotic pathways are warranted to develop targeted therapeutics to limit pathologic arterial remodeling under pathologically stiffened + disturbed flow environments.

Keywords: Arterial stiffening; Atherosclerotic Plaque; Connective tissue growth factor/CCN2; Endothelial cells; Fibulin-5; Flow-mediated arterial remodeling; Sex-differences.