Aicar treatment reduces interstitial fibrosis in aging mice: Suppression of the inflammatory fibroblast

Abstract

In 2030, elderly people will represent 20% of the United States population. Even now, chronic cardiac diseases, especially heart failure with preserved systolic function (HFpEF), are the most expensive DRGs for Medicare. Progressive interstitial fibrosis in the aging heart is well recognized as an important component of HFpEF. Our recent studies suggested an important pathophysiologic role for reduced TGF-β receptor 1 (TGFβR1) signaling in mesenchymal stem cells (MSCs) and their mesenchymal fibroblast progeny in the development of interstitial fibrosis. This report arises from our previous studies, which suggest that an inflammatory phenotype exists in these mesenchymal fibroblasts as a result of a reduced TGF-β-Smad-dependent pathway but upregulated farnesyltransferase (FTase)-Ras-Erk signaling. In this report we provide evidence for a therapeutic approach that downregulates Erk activation through an adenosine monophosphate-activated kinase (AMPK) pathway. Aging C57BL/6J mice were treated with AICAR (an AMPK activator) for a 30-day period. This treatment suppressed excessive monocyte chemoattractant protein-1 (MCP-1) generation, which diminished leukocyte infiltration and in consequence suppressed the formation of macrophage-derived myeloid fibroblasts. Interestingly, the number of mesenchymal fibroblasts was also reduced. In addition, we observed changes in extracellular matrix (ECM) deposition, specifically that collagen type I and the alternatively spliced variant of fibronectin (EDA) expressions were reduced. These data suggest that the upregulation of AMPK activity is a potential therapeutic approach to fibrosis in the aging heart.

Keywords: AMPK; Fibroblast; Fibrosis; Heart; Macrophage.

Figure 1. AICAR treatment reduces levels of injury markers and fibrosis

A. Western blot analysis of phosphorylated AMPK in heart lysates isolated from saline and AICAR-injected mice. B, QPCR analysis of RNA isolated from whole hearts derived from 21 month-old mice injected with saline or AICAR for 4 weeks. Results are represented as mean ± SEM. * denotes p<0.05. C, EDA is present in the aged uninjured heart as shown by immunofluorescence staining. Young denotes 3 month-old and aged denotes 24 month-old mice Scale bar = 50 μm. D, 7 days AICAR treatment reduces the presence of EDA in the aged heart. 14 month-old mice were injected with saline or AICAR for 7 days. Heart sections were stained with anti-EDA antibody (green) or Dapi (blue). Scale bar = 50 μm. E, 14 month-old mice were injected with saline or AICAR for 7 days and 30 days after the last injection hearts were analyzed using anti-collagen type I antibody (red) or Dapi (blue). Scale bar = 50μm. N=3 (for A, C), 5 (for D and E), and for B, N = 8, 4 for saline and AICAR treated mice respectively.

Figure 2. AICAR treatment reduces the number of fibroblasts of two developmental origins in the aged mouse heart

A, Flow cytometry analysis of calcein⁺ (viable) non-myocytes isolated from 21 month-old hearts using CD45-PE antibody. The graph depicts quantification of all CD45⁺ cells that have low (CD45^lo), high (CD45^hi) or total expression of CD45. B, Analysis of the contribution of resident (CD45^lo) and infiltrating (CD45^hi) leukocytes to the M2a macrophage/myeloid fibroblast pool (Col1a⁺CD301⁺CD45⁺) by flow cytometry. C, The percentage of mesenchymal fibroblasts (as collagen producing cells within the CD44⁺CD45⁻ pool) is reduced with AICAR treatment as quantified by flow cytometry analysis. D, AICAR treatment reduces Erk phosphorylation. Quiescent mesenchymal fibroblasts derived from 24–30 month-old hearts were treated with 0.5 mM of AICAR for indicated period of time. C denotes control, A denotes AICAR. E, QPCR analysis of MCP-1 expression in quiescent mesenchymal fibroblasts derived from 24–30 month-old hearts subjected to 1 μM PD0325901 (Erk inhibitor) for 24 h. ^* denotes p<0.05. F, Schema (see Conclusions). Results are represented as mean ± SEM. N = 6 and 12 (for A, B); N= 6 and 4 (for C) for saline and AICAR treated mice respectively and N=4 (for D and E).