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Transcriptional heterogeneity of fibroblasts is a hallmark of the aging heart
Ramon Vidal, … , Stefanie Dimmeler, Sascha Sauer
Ramon Vidal, … , Stefanie Dimmeler, Sascha Sauer
Published November 14, 2019
Citation Information: JCI Insight. 2019;4(22):e131092. https://doi.org/10.1172/jci.insight.131092.
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Categories: Research Article Aging Cardiology

Transcriptional heterogeneity of fibroblasts is a hallmark of the aging heart

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Abstract

Aging is a major risk factor for cardiovascular disease. Although the impact of aging has been extensively studied, little is known regarding the aging processes in cells of the heart. Here we analyzed the transcriptomes of hearts of 12-week-old and 18-month-old mice by single-nucleus RNA-sequencing. Among all cell types, aged fibroblasts showed most significant differential gene expression, increased RNA dynamics, and network entropy. Aged fibroblasts exhibited significantly changed expression patterns of inflammatory, extracellular matrix organization angiogenesis, and osteogenic genes. Functional analyses indicated deterioration of paracrine signatures between fibroblasts and endothelial cells in old hearts. Aged heart-derived fibroblasts had impaired endothelial cell angiogenesis and autophagy and augmented proinflammatory response. In particular, expression of Serpine1 and Serpine2 were significantly increased and secreted by old fibroblasts to exert antiangiogenic effects on endothelial cells, an effect that could be significantly prevented by using neutralizing antibodies. Moreover, we found an enlarged subpopulation of aged fibroblasts expressing osteoblast genes in the epicardial layer associated with increased calcification. Taken together this study provides system-wide insights and identifies molecular changes of aging cardiac fibroblasts, which may contribute to declined heart function.

Authors

Ramon Vidal, Julian Uwe Gabriel Wagner, Caroline Braeuning, Cornelius Fischer, Ralph Patrick, Lukas Tombor, Marion Muhly-Reinholz, David John, Magdalena Kliem, Thomas Conrad, Nuno Guimarães-Camboa, Richard Harvey, Stefanie Dimmeler, Sascha Sauer

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Figure 6

Age-dependent changes of fibroblast function on endothelial cells.

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Age-dependent changes of fibroblast function on endothelial cells.
(A an...
(A and B) Experimental outline and tube formation assay of human umbilical vein endothelial cells (HUVECs) that were cultured in conditioned medium received from young (12 weeks) and aged (20 months) cardiac mouse fibroblasts. Accumulated tube length was measured in 5 randomly chosen microscopic fields with a computer-assisted microscope using Axiovision 4.5 (Zeiss) (scale bar: 100 μm; n = 4). (C) Capillary density of random healthy areas of young (12 weeks; n = 6) and old (n = 8) heart sections versus capillary density of fibrotic (n = 8) areas of aged hearts (20 months). Shown is the quantification of the Isolectin B4–positive area versus the total area (%). (D) Expression of Serpine1, Serpine2, and Efemp1 in fibroblasts displayed in log scale in trajectory plots. (E) Secretion of SerpinE1 in isolated young and aged cardiac fibroblasts cultured for 24 hours in serum-free medium. Data were derived using a mouse angiogenesis array (R&D Systems) of culture supernatants of isolated cardiac fibroblasts (n = 4). (F) Tube formation assay of HUVECs that were cultured in conditioned medium received from young (12 weeks) and aged (20 months) cardiac mouse fibroblasts. The aged phenotype was rescued by supplementing the aged fibroblast medium with 4 μg of an anti-Serpin antibody. Accumulated tube length was measured in 5 randomly chosen microscopic fields with a computer-assisted microscope using Axiovision 4.5 (Zeiss) (scale bar: 200 μm; n = 4). (G) RNA expression of IL-6 in HUVECs cultured in conditioned medium received from young (12 weeks) and aged (18 months) cardiac mouse fibroblasts and in the presence and absence of 10 ng/mL TNF-α for 24 hours (n = 5). (H) Quantification of autophagosomes in HUVECs cultured with conditioned medium received from young (12 weeks) and aged (18 months) cardiac mouse fibroblasts. Autophagosomes were detected by immunostainings against p62 and LC3, whereby autophagosomes were considered p62/LC3–double positive (n = 5). Data are shown as mean ± SEM (B, C, E, F, G, and H). After passing Gaussian distribution, statistical analysis was performed using the unpaired, 2-sided t test (C, E, G, and H). For comparisons of more than 2 groups, multiple-group 1-way ANOVA with a post hoc Bonferroni’s test was used (B and F). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
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