Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Endothelial Cells Drive Organ Fibrosis by Inducing The Transcription Factor Sox9

F. A. Trogisch1, M. Keles1, A. Birke1, A.-M. Abouissa1, M. Fuhrmann1, G. M. Dittrich1, N. Weinzierl1, E. Wink1, J. Cordero2, A. Martin Garrido1, S. Grein1, S. Hemanna1, L. Nicin3, S. Quanchao4, S. W. Kürschner5, M. Winkler5, N. Gretz4, C. Mogler6, T. Korff7, P.-S. Koch5, 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; 3Institute for Cardiovascular Regeneration & Cardiopulmonary Institute, Goethe University, Frankfurt am Main; 4Medical Research Center, Mannheim Faculty of Medicine, Heidelberg University, Mannheim; 5Department of Dermatology, Venereology and Allergology, ECAS (European Center for Angioscience), Adjunct Faculty & Center of Excellence in Dermatology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, Mannheim; 6Institute of Pathology, School of Medicine, Technical University of Munich, München; 7Department of Cardiovascular Physiology, ECAS (European Center for Angioscience), Adjunct Faculty, Medical Faculty Heidelberg, Heidelberg University, Heidelberg;

Introduction: Fibrotic remodeling is a critical maladaptive feature in several diseases. Previously, we suggested that endothelial cells (ECs) contribute to extracellular matrix (ECM) deposition during pressure overload-induced cardiac hypertrophic and fibrotic remodeling by transient induction of mesenchymal genes. However, the functional importance of mesenchymal activation of ECs for the development of organ fibrosis and disease remains elusive. Objective: Aim of the study was to investigate the contribution of endothelial cells to cardiac and other organs' fibrosis and function by analyzing the role of the fibrogenic transcription factor Sox9 in endothelial cells. Methods & Results: Adult mice were challenged either with transverse aortic constriction (TAC) or a combination of high fat diet and L-NAME in order to induce heart failure with moderately reduced (HFmrEF) or preserved (HFpEF) systolic function. Early during each fibrotic heart failure entity, we found a significant induction of Sox9 mRNA in isolated cardiac ECs. Similarly, Sox9 mRNA was induced in endothelial cells during fibrotic liver disease (through CDAA diet) and fibrotic lung disease (through Bleomycin application). EC-specific overexpression of Sox9 in transgenic mice (by Cdh5-promoted recombination of a Sox9-expressing Hprt locus) resulted in fibrotic and hypertrophic remodeling of heart, lung, liver and spleen. Bulk and single-cell RNA sequencing of isolated ECs showed that ECs were the origin of ECM deposition. On the functional level, we found cardiac diastolic dysfunction, followed by systolic functional impairment. In contrast, mice with an inducible, EC-restricted knock-out of Sox9 (Sox9EC-KO) were protected from both HFmrEF and HFpEF following TAC or specific diet, resp., as indicated by preserved cardiac function, reduced cardiac hypertrophy and fibrosis. Additionally, endothelial deletion of Sox9 in pre-existing TAC attenuated disease progression and ameliorated cardiac dysfunction. Likewise, fibrotic remodeling and organ failure of lung or liver, resp., were ameliorated in Sox9EC-KO mice. RNA-sequencing of isolated ECs in hearts, lungs and livers upon Sox9 overexpression or deletion in disease showed that Sox9 directly induced the expression of mesenchymal genes (e.g. Col1a1, Col2a1, Col3a1) in endothelial cells, but also triggered the expression of paracrine growth factors. This implied direct generation of extracellular matrix by ECs, but could also involve the paracrine activation of fibroblasts. Indeed, mono-cultured ECs showed mesenchymal gene activation upon adenoviral Sox9 overexpression, but in addition activated mesenchymal gene transcription and migration in co-cultured fibroblasts. Interestingly, single-cell sequencing of explanted human fibrotic hearts revealed significant induction of Sox9 mRNA in 4 distinct endothelial cell clusters. Furthermore, immune-histochemical staining of human tissue samples showed a strong correlation of endothelial Sox9 protein abundance and the extent of organ fibrosis (r=0.7677, p<0.0001, n=20 patients). This suggested that endothelial Sox9-dependent disease progression might also be involved in human heart failure. Conclusions:Endothelial Sox9 is a master regulator of mesenchymal activation of ECs, hence driving organ fibrosis and failure, by both distinct ECM deposition and fibroblast activation. Absence of Sox9 in ECs, in turn, ameliorates fibrotic remodeling and maintains organ function.

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