Introduction. Even though heart failure with reduced (HFrEF) and preserved ejection fraction (HFpEF) differ in their clinical appearance, both entities feature fibrotic cardiac remodeling as shared hallmark. We recently demonstrated that endothelial cells (ECs) transiently contribute to extracellular matrix (ECM) deposition following transverse aortic constriction (TAC). The functional importance for fibrotic remodeling and the underlying mechanisms, however, remain elusive.

Objective. Aim of the study was to investigate the contribution of endothelial cells to fibrosis and dysfunction in the heart and identify potential master regulators.

Methods & Results. Following both TAC and administration of high fat diet together with NOS inhibitor L-NAME in adult mice, we found significant induction of the fibrogenic transcription factor Sox9 in isolated cardiac ECs by RT-qPCR. Interestingly, both single-cell RNA sequencing (RNAseq) and immune-histochemical staining of human cardiac explants showed endothelial SOX9 mRNA induction as well as a strong correlation of SOX9 protein abundance and the extent of organ fibrosis, suggesting that endothelial Sox9-dependent disease progression might also be involved in human heart failure.

Endothelial cell-specific, Cdh5-promoted overexpression of Sox9 in transgenic mice resulted in fibrotic and hypertrophic remodeling of heart, lung, liver and spleen, accompanied by left-ventricular diastolic followed by systolic cardiac dysfunction. Both bulk and single-cell RNAseq of isolated ECs as well as immunofluorescence staining showed that ECs were the origin of ECM.

In contrast, mice with an inducible, EC-restricted knock-out of Sox9 were protected from both HFrEF and HFpEF following TAC or specific diet, resp., as indicated by preserved cardiac function, reduced cardiac hypertrophy and fibrosis.

Bulk RNAseq of cardiac ECs from both TAC-operated or high fat diet/L-NAME-fed mice with endothelial Sox9 deletion, intersected with cardiac ECs after EC-restricted Sox9 overexpression, revealed that Sox9 is responsible for induction of mesenchymal, inflammatory and cell-to-cell communication-associated gene ontologies. Cardiac single-cell RNAseq of isolated ECs following TAC showed a specific endothelial cell cluster, in which Sox9 and ECM genes were co-expressed. Endothelial Sox9 deletion, in turn, completely ablated ECM genes expression in ECs from this cluster.  As activated ECs could also recruit fibroblasts, we co-cultivated NRFBs with SOX9-overexpressing HUVECs (via adenoviral transfection). Indeed, co-cultured NRFBs displayed increased levels of mesenchymal genes and improved migration when co-cultivated with SOX9-overexpressing HUVECs. CCN2 (CTGF), a known stimulant of mesenchymal cells, was induced on mRNA level in ECs but not in co-cultured fibroblasts, suggesting that CTGF signals from ECs to fibroblasts. Indeed, knock-down of CCN2 by siRNA in parallel to SOX9 transfection in HUVECs reduced the activation of co-cultured NRFBs.

Conclusions. Endothelial cells are critical drivers of fibrotic cardiac remodeling during both HFrEF and HFpEF by upregulating the fibrogenic transcription factor Sox9. Sox9 directs endothelial-to-mesenchymal activation in ECs, which leads to ECM secretion from ECs as well as paracrine fibroblast activation via CTGF.