Myocardial fibrosis in response to Angiotensin II is preceded by the recruitment of mesenchymal progenitor cells

Abstract

Myocardial fibrosis is characterized by significant extracellular matrix (ECM) deposition. The specific cellular mediators that contribute to the development of fibrosis are not well understood. Using a model of fibrosis with Angiotensin II (AngII) infusion, our aim was to characterize the cellular elements involved in the development of myocardial fibrosis. Male C57Bl/6 and Tie2-GFP mice were given AngII (2.0 mg/kg/min) or saline (control) via mini osmotic pumps for up to 7 days. Hearts were harvested, weighed and processed for analysis. Cellular infiltration and collagen deposition were quantified. Immunostaining was performed for specific markers of leukocytes (CD45, CD11b), myofibroblasts (SMA), endothelial cells (vWF) and hematopoietic progenitor cells (CD133). Bone marrow (BM) origin of infiltrating cells was assessed using GFP+ chimeric animals. Relative qRT–PCR was performed for pro-fibrotic cytokines (transforming growth factor (TGF)-β1, CTGF) as well as the chemokine stromal-derived factor (SDF)-1α. Myocardial-infiltrating cells were grown in vitro. AngII exposure resulted in multifocal myocardial cellular infiltration, which preceded extensive ECM deposition. A limited number of myocardial-infiltrating cells were positive for leukocyte markers but were significantly positive for myofibroblast (SMA) and endothelial cell (vWF) markers. However, using Tie2-GFP mice, where endothelial cells are GFP+, myocardial-infiltrating cells were not GFP+. Transcript levels for SDF-1α were significantly elevated at 1 day of AngII exposure suggesting that hematopoietic progenitor cells may be recruited. This was confirmed by positive CD133 staining of infiltrating cells and evident GFP+ cellular infiltration when exposing GFP+ BM chimeras to AngII. Furthermore, a significant number of CD133+/SMA+ cells were grown in vitro from the myocardium of AngII-exposed animals (P<0.01). Myocardial ECM deposition is preceded by the infiltration of the myocardium with hematopoietic cells that express mesenchymal markers. These data suggest that mesenchymal progenitor cells are recruited, and may have a primary role, in the initiation of myocardial fibrosis.