Abstract 237 <P style="MARGIN-BOTTOM: 0pt">The fibrogenic transcription factor Sox9 controls mesenchymal activation of endothelial cells and drives fibrotic disease</P>

Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02180-w
The fibrogenic transcription factor Sox9 controls mesenchymal activation of endothelial cells and drives fibrotic disease
F. A. Trogisch¹, A. Abouissa¹, M. Keles¹, A. Birke¹, M. Fuhrmann¹, G. M. Dittrich¹, N. Weinzierl¹, E. Wink¹, J. Cordero², A. Elsherbiny², A. Martin Garrido¹, S. Grein¹, S. Hemanna¹, E. Hofmann¹, L. Nicin³, S. I. Bibli⁴, R. Airik⁵, A. Kispert⁵, R. Kist⁶, S. Quanchao⁷, S. W. Kürschner⁸, M. Winkler⁸, N. Gretz⁷, C. Mogler⁹, T. Korff¹⁰, P.-S. Koch⁸, S. Dimmeler³, G. Dobreva², J. Heineke¹
¹Department of Cardiovascular Physiology, ECAS (European Center for Angioscience), Mannheim Faculty of Medicine, Heidelberg University, Mannheim; ²Department of Anatomy and Developmental Biology, ECAS (European Center for Angioscience), Mannheim Faculty of Medicine, Heidelberg University, Mannheim; ³Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration, Goethe Universität Frankfurt am Main, Frankfurt am Main; ⁴Institut für Vascular Signalling, Universitätsklinikum Frankfurt, Frankfurt am Main; ⁵Institut für Molekularbiologie, Medizinische Hochschule Hannover, Hannover; ⁶Biosciences Institute, School of Dental Sciences, Faculty of Medical Sciences, Newcastle Upon Tyne, UK; ⁷Core Facility Preclinical Models, Mannheim Faculty of Medicine, Heidelberg University, Mannheim; ⁸Clinic for Dermatology, Venereology and Allergology, University Medical Centre Mannheim, University of Heidelberg, Mannheim; ⁹Institut für Allgemeine Pathologie und Pathologische Anatomie, Technische Universität München, München; ¹⁰Institute of Physiology & Pathophysiology, Heidelberg Medical Faculty, Heidelberg University, Heidelberg;
Introduction. Previously, we and others suggested that endothelial cells (ECs) contribute to extracellular matrix (ECM) deposition during fibrotic remodeling in the heart. Accordingly, we found an induction of the fibrogenic transcription factor Sox9 in ECs in several pre-clinic fibrotic models and human cardiac explants from heart failure patients. Objective. Aim of the study was to investigate the impact of ECs on the development of fibrotic disease. Methods & Results. Because Sox9 was upregulated in ECs in multiple pro-fibrotic conditions, we first evaluated the effects of its overexpression. Cdh5-Cre driven Sox9 overexpression in ECs from adult mice (Sox9^EC-OE) induced inflammatory, extracellular signaling-associated, and mesenchymal genes as shown by bulk-RNAseq of isolated cardiac, pulmonary and liver ECs. Single-cell RNAseq of isolated cardiac ECs from those animals revealed that clusters with mesenchymal and inflammatory genes increased in size by endothelial Sox9 overexpression, while clusters with angiogenic and neuronal genes were diminished. Interestingly, cultured human ECs demonstrated similar patterns upon overexpression of Sox9 by adenovirus. In-depth analysis of RNAseq data together with CUT&RUN peaks to detect genomic DNA regions bound by Sox9 in vivo and in vitro identified several direct Sox9 targets, e.g. Ccn2, which encodes for CTGF. On the functional level, Sox9^EC-OE mice developed massive fibrosis of the heart, lung, liver and spleen, accompanied by deteriorating organ function. On the other hand, deletion of Sox9 in ECs by Cdh5-Cre-targeted recombination (Sox9^EC-KO) protected from both systolic heart failure (induced by 2 weeks, W, transverse aortic constriction, TAC) and diastolic heart failure (induced by 10 W administration of high fat diet and L-Name) by maintaining organ function (as shown by echocardiography and catheterization), restricting cardiac hypertrophy and preventing fibrosis. Likewise, disease progression was reduced in these mice during pulmonary fibrosis (following bleomycin injection) and steato-hepatitis (following 15 weeks CDAA diet). Bulk RNAseq of isolated ECs from the respective organs demonstrated reduced mesenchymal and inflammatory gene-expression in Sox9^EC-KO mice. Cardiac EC single-cell RNAseq following TAC identified 9 clusters, wherein Sox9-expression was restricted to a distinct cluster which contained 735 genes, of which 360 were unique in this cluster. Within this cluster, expression of mesenchymal genes (e.g. Col1a1, Col3a1, Lox) and Ccn2 was markedly reduced in Sox9^EC-KO mice after TAC. In order to target those ECs during the progression of heart disease, we induced deletion of Sox9 in ECs 10 days after TAC and monitored Sox9^EC-KO mice for additional 30 days. Interestingly, both cardiac hypertrophy and systolic function were maintained at the level of 10 days post TAC, while they continuously worsened in control littermates. Co-culture studies showed that Sox9 not only led to increased matrix gene expression in ECs, but also triggered the paracrine activation of fibroblasts. Conclusions. Endothelial Sox9 serves as master regulator of fibrotic remodeling by both inducing mesenchymal genes and by enabling fibroblast activation through secretion of growth factors like CTGF. Sox9-deletion in ECs, in turn, protects the heart and other organs during fibrotic disease, suggesting endothelial Sox9 as therapeutic target.
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