Kathrin A. Stilz

Background and Aims: Aging significantly increases the risk of cardiovascular disease, characterized by progressive cardiac dysfunction. The vascular niche is crucial for maintaining cardiac homeostasis, yet endothelial cell (EC) impairment during aging remains poorly understood. This study investigates epigenetically regulated mechanisms underlying EC-dependent cardiac aging and identifies a critical role for zinc finger and BTB domain-containing protein 16 (ZBTB16). Methods: Chromatin accessibility (snATAC-seq) and transcriptomic (snRNA-seq) analyses of aged hearts were performed to define age-related regulatory changes. Functional studies using genetic models were performed to assess cardiac aging phenotypes. In vitro assays examined EC senescence, secretory profiles, and effects of ZBTB16-deficient EC supernatants on fibroblasts, cardiomyocytes, and neurons. Overexpression experiments in vitro and in vivo tested whether ZBTB16 mitigates aging-associated dysfunction. Results: Aged hearts exhibited decreased chromatin accessibility and reduced ZBTB16 expression in both humans and mice. Zbtb16 deletion in young mice, including Zbtb16-haploinsufficient and endothelial-specific knockout mice, led to premature aging, diastolic dysfunction, and increased secretion of pro-fibrotic and inflammatory factors. ZBTB16-deficient EC supernatants activated fibroblasts, induced cardiomyocyte hypertrophy, and impaired neuronal sprouting. Overexpression of ZBTB16 reversed these effects in senescent ECs and aged mice and reduced diastolic dysfunction. Mechanistic studies identified nuclear receptor-interacting protein 1 as a downstream target suppressed by ZBTB16, thereby limiting fibroblast activation and pro-fibrotic signalling. Conclusions: ZBTB16 preserves endothelial integrity and vascular niche homeostasis, protecting against aging-associated cardiac dysfunction. Its loss promotes EC senescence and fibrosis, whereas restoring its expression may represent a therapeutic strategy to improve cardiac function and reduce cardiovascular disease risk during aging.

BACKGROUND: Endothelial cells (ECs) play pivotal roles in maintaining cardiac blood supply and regulating inflammation by acting as gatekeepers for immune cell activity. This study unveils a novel immunomodulatory function of cardiac ECs following myocardial infarction. METHODS: We used single-cell RNA sequencing and spatial transcriptomics to identify EC states after acute myocardial infarction in mice. Subsequently, we mimicked the cytokine environment that was predicted to induce EC activation in cell culture studies and confirmed the results in an endothelial-specific deletion mouse model. RESULTS: Single-cell RNA sequencing analysis identified a transient myeloid CD45 ⁺ CD11b ⁺ Cdh5 ⁺ immunomodulatory EC phenotype (IMEC) emerging between days 1 and 3 after myocardial infarction. IMECs derived from Cdh5 ⁺ tissue resident cells as shown by bone marrow transplantation and lineage tracing experiment. Ligand-receptor interaction predictions indicated a cytokine-mediated activation of IMECs, which we validated through in vitro experiments in cultured ECs. Notably, while cytokine treatment with IL-1β and TGF-β (transforming growth factor β) induced mesenchymal gene expression, the addition of IFN-γ (interferon γ) facilitated the transition into the immunomodulatory phenotype. IMECs exhibited an upregulation of MHC-II (major histocompatibility complex class II) genes, along with the expression of RUNX1 (runt-related transcription factor-1) and proinflammatory cytokines, such as IL-6 and IL-12. IMECs induced T-cell activation through paracrine signaling and were colocalized with T cells in vivo. Inhibition of endothelial-specific IFN-γ-signaling in mice by IFN-γ receptor 1 deletion improved the recovery after myocardial infarction. CONCLUSIONS: These findings provide insight into the role of ECs regulating adaptive immune responses following myocardial infarction, offering potential insights into therapeutic interventions for postinfarction immunomodulation.

Aging is a major risk factor for impaired cardiovascular health. Because the aging myocardium is characterized by microcirculatory dysfunction, and because nerves align with vessels, we assessed the impact of aging on the cardiac neurovascular interface. We report that aging reduces nerve density in the ventricle and dysregulates vascular-derived neuroregulatory genes. Aging down-regulates microRNA 145 (miR-145) and derepresses the neurorepulsive factor semaphorin-3A. miR-145 deletion, which increased Sema3a expression or endothelial Sema3a overexpression, reduced axon density, mimicking the aged-heart phenotype. Removal of senescent cells, which accumulated with chronological age in parallel to the decline in nerve density, rescued age-induced denervation, reversed Sema3a expression, preserved heart rate patterns, and reduced electrical instability. These data suggest that senescence-mediated regulation of nerve density contributes to age-associated cardiac dysfunction.