Pressure-overload induced cardiac hypertrophy is associated with vascular malformations via up-regulation of Notch4 in endothelial cells

Pressure-overload induced by aortic stenosis leads to pathological cardiac hypertrophy with remodeling of the cardiac tissue and microvascular dysfunction. However, little is known about the long-term effects of cardiac hypertrophy on the coronary vasculature itself. Using single nuclei RNA sequencing of pressure-overload induced cardiac hypertrophic human tissue samples, we disclosed a dysregulation of vessel malformation-associated genes in endothelial cells. Morphologically, coronary angiograms of the sequenced patients demonstrated tortuous coronary vessels. Using lightsheet microscopy and µCT, similar vessel malformations as well as vascular convolutes were observed in latex-perfused murine hearts after pressure-induced overload by transverse aortic constriction (TAC). Finally, histological examinations revealed dilation and arterialization of microvessels in line with an overall increase in SMA+ cells in whole-heart staining. Among the regulated vessel malformation genes in endothelial cells, members of the NOTCH family and its downstream targets were profoundly up-regulated. Specifically, NOTCH4, a known inducer of developmental vascular malformation, was among the top upregulated genes. Importantly, endothelial cells with a high expression of Notch4 demonstrated a particular upregulation of vascular malformation associated genes, e.g. Vegfa, Efnb2, Dll4 and Hes1. Increased Notch4 expression was validated on protein level by immunohistochemistry of hypertrophic human and mouse cardiac tissue specimens. In order to address the role of Notch4 on cardiac vessels in vivo, we used an endothelial-specific Notch4 overexpression mouse model. Induction of Notch4 overexpression in endothelial cells resulted in profound coronary vascular abnormalities, as well as significant changes in single cell gene signatures not only in endothelial cells, but also in genes related to metabolic alterations in cardiomyocytes and extracellular matrix composition. A screen for upstream modulators of Notch4 identified hypoxia as inducer of Notch4 expression, which was validated by in-vitro experiments. In summary, chronic pressure-overload is associated with increased endothelial Notch4 expression and coronary vascular malformations in both human and murine hypertrophic cardiac tissue samples. Mechanistically, locally distributed inadequate oxygen supply due to microvascular dysfunction in cardiac hypertrophy may contribute to the development of coronary vascular malformations via hypoxia-induced upregulation of endothelial Notch4, which further propagate cardiac remodeling.