Dissecting the role of myeloid and mesenchymal fibroblasts in age-dependent cardiac fibrosis

Abstract

Aging is associated with increased cardiac interstitial fibrosis and diastolic dysfunction. Our previous study has shown that mesenchymal fibroblasts in the C57BL/6J (B6J) aging mouse heart acquire an inflammatory phenotype and produce higher levels of chemokines. Monocyte chemoattractant protein-1 (MCP-1) secreted by these aged fibroblasts promotes leukocyte uptake into the heart. Some of the monocytes that migrate into the heart polarize into M2a macrophages/myeloid fibroblasts. The number of activated mesenchymal fibroblasts also increases with age, and consequently, both sources of fibroblasts contribute to fibrosis. Here, we further investigate mechanisms by which inflammation influences activation of myeloid and mesenchymal fibroblasts and their collagen synthesis. We examined cardiac fibrosis and heart function in three aged mouse strains; we compared C57BL/6J (B6J) with two other strains that have reduced inflammation via different mechanisms. Aged C57BL/6N (B6N) hearts are protected from oxidative stress and fibroblasts derived from them do not develop an inflammatory phenotype. Likewise, these mice have preserved diastolic function. Aged MCP-1 null mice on the B6J background (MCP-1KO) are protected from elevated leukocyte infiltration; they develop moderate but reduced fibrosis and diastolic dysfunction. Based on these studies, we further delineated the role of resident versus monocyte-derived M2a macrophages in myeloid-dependent fibrosis and found that the number of monocyte-derived M2a (but not resident) macrophages correlates with age-related fibrosis and diastolic dysfunction. In conclusion, we have found that ROS and inflammatory mediators are necessary for activation of fibroblasts of both developmental origins, and prevention of either led to better functional outcomes.

Keywords: Aging; Fibroblast; Fibrosis; Heart; Inflammation.

Fig. 1

The increased number of CD45⁺ leukocytes in the aging B6J hearts. A, Flow cytometry analysis of calcein⁺ (viable) non-myocytes isolated from young (3 month-old, B6J), and aged (21–25 month-old B6J, MCP-1KO, B6N) mice was performed using CD45-PE antibody. Upper panel shows superimposed CD45⁺ alive cells from representative histograms depicting all four experimental groups. The black double arrow illustrates the expansion of CD45^hi cell population that is abundant in cells isolated from aged B6J hearts. The lower panel portrays flow cytometry based quantification of CD45⁺ cells, cells that have low (CD45^lo) or high (CD45^hi) expression of CD45 in four experimental groups. Results are represented as mean ± SEM. N= 8, 12, 7, 7 for young B6J and aged B6J, MCP-1KO, and B6N respectively. B, Immunofluorescence staining of aged mouse paraffin heart sections or C, human frozen heart sections showing CD45 positive cells (red) within collagen type I deposition (green). Nuclei are stained with Dapi (blue). For mouse hearts, 21–24 month-old mice of an indicated genetic strain were used, N= 3, 2, 3 for biological repeats and N=3 for experimental repeats, scale bar=50 μm. For human heart N=1 per young (21 year-old) and aged (75 year-old), N=3 for experimental repeats, scale bar= 50 μm.

Fig. 2

The augmented participation from infiltrating monocytes into myeloid-dependent fibrosis in the aging B6J heart. A, Analysis of the contribution of resident (CD45^lo) and infiltrating (CD45^hi) leukocytes to the M2a macrophage/myeloid fibroblast pool by flow cytometry. B, An increased collagen expression by the M2a macrophages/myeloid fibroblasts in the aging B6J hearts. Upper panel shows representative images of M2a macrophage/myeloid fibroblasts (Col1a⁺CD301⁺CD45⁺) and the quantification of mean fluorescence intensity (MFI) of collagen type I in Col1⁺CD301⁺CD45⁺ M2a macrophage/myeloid fibroblasts. Results are represented as mean ± SEM. N= 6, 11, 6, 4 for young B6J and aged B6J, MCP-1KO, and B6N respectively.

Fig. 3

Aging affects splenic monocyte migration. A, The sub-capsular red pulp and the marginal zone in the old (spleens) are depleted as visualized by hematoxylin and eosin staining of spleen sections (white arrows). B, MCP-1 dependent migration of splenic monocytes isolated from aged mice is increased. Results are represented as mean ± SEM, * denotes P<0.05. N=3 per age group. Scale bar= 100 μm. Young denotes 3 month-old and aged denotes 30 month-old.

Fig. 4

Mesenchymal fibroblasts derived from B6N hearts are protected from age-related ROS production and inflammation. A, Conditioned medium from mesenchymal fibroblast cultures containing 100 μg of protein was incubated with cytokine antibody array membranes. Panels depict representative membranes. B, Quiescent mesenchymal fibroblasts were stained with MitoSox (red) to determine ROS production. Scale bar = 20 μm. N= 3 per each strain and age group, young denotes 3 month-old and aged denotes 21–25 month-old mice.

Fig. 5

An increased number of mesenchymal fibroblast precursors in the aging heart. A, Quantification of flow cytometry analysis of calcein⁺ non-myocytes that are stained with CD44 and CD45 antibodies to distinguish CD44⁺CD45⁻ mesenchymal fibroblasts from other non-myocytes. Results are represented as mean ± SEM. N= 4, 4, 8, 8 for young B6J and aged B6J, MCP-1KO, and B6N respectively. B, Immunofluorescence staining of aged mouse paraffin heart sections or C, human frozen heart sections showing CD44 positive cells (red) within collagen type I deposition (green) and Dapi stained nuclei (blue). 21–24 month-old mice of an indicated genetic strain were used, and young denotes 3 month-old. N= 3, 2, 3, scale bar=50 μm. Human heart sections obtained from 21 and 75 year-old donors were used, scale bar= 50 μm.

Fig. 6

Augmented differentiation of mesenchymal fibroblast precursors into fibroblasts in B6J heart. A, Representative histograms of flow cytometry analysis of mesenchymal fibroblasts gated on the CD45⁻ cell population. B, Percentage of collagen producing cells within the CD44⁺CD45⁻ pool. C, Quantification of total mesenchymal fibroblasts in hearts. Results are represented as mean ± SEM. N= 3, 3, 6, 3 for young B6J and aged B6J, MCP1KO, and B6N respectively. D, Immunofluorescence staining of aged mouse paraffin heart sections showing CD44 positive cells (red) within procollagen type I deposition (green) and Dapi stained nuclei (blue). Yellow frame depicts enlarged view of double positive cells. 21–24 month-old mice of an indicated genetic strain were used, and young denotes 3 month-old. N= 3, 2, 3, scale bar=50 μm. Human heart sections obtained from 21 and 75 year-old donors were used, scale bar= 50 μm.

Fig. 7

The spring stiffness coefficient of mitral inflow for aged B6J, MCP-1KO and B6N hearts. A, Representative images from aged B6J and B6N hearts showing E/A ratio for aged B6J and B6N hearts. N=10 per each group. B, Kovacs analysis of Doppler mitral early flow predicts left ventricular diastolic stiffness. N= 7, 4, 5 for aged B6J, aged MCP-1KO, and aged B6N respectively. 21–24 month-old mice were used. Data are presented as mean ± SEM.

Fig. 8

The two way interaction between infiltrating leukocytes and mesenchymal fibroblasts in the aging mouse heart. The dysregulated mesenchymal fibroblasts produce higher levels of ROS that promote expression of MCP-1 and IL-6. Secreted MCP-1 attracts leukocyte infiltration into the heart. Infiltrating monocytes polarize into alternatively activated M2a macrophages/myeloid fibroblasts in response to Th2 products (IL-4 and IL-13) and IL-6 (derived at least partially from dysregulated mesenchymal fibroblasts). Both types of fibroblasts (mesenchymal and myeloid) secrete collagen. Excessive collagen production by mesenchymal fibroblasts is controlled by the infiltrating leukocytes; without input from leukocytes/monocyte-derived macrophages, mesenchymal fibroblasts remain unactivated and do not overproduce collagen. Augmented collagen production contributes to fibrosis and diastolic dysfunction. Resident macrophages do not play an apparent role in this process.