Fibroblast progenitor cells are recruited into the myocardium prior to the development of myocardial fibrosis

Abstract

Using an established model of myocardial hypertrophy and fibrosis after angiotensin II (AngII) infusion, our aim was to characterize the early cellular element involved in the development of myocardial fibrosis in detail. Male Lewis rats were infused with saline or AngII (0.7 mg/kg per day) for up to seven days. Collagen deposition and cellular infiltration were identified by histology stains. Infiltrating cells were grown in vitro and examined by flow cytometry and immunostaining. Chemokine expression was measured using qRT-PCR. AngII infusion resulted in multifocal myocardial cellular infiltration (peak at three days) that preceded collagen deposition. Monocyte chemotactic protein (MCP)-1 transcripts peaked after one day of AngII exposure. Using a triple-labelling technique, the infiltrating cells were found to express markers of leucocyte (ED1(+)), mesenchymal [α-smooth muscle actin (SMA)(+)] and haematopeotic progenitor cells (CD133(+)) suggesting a fibroblast progenitor phenotype. In vitro, ED1(+)/SMA(+)/CD133(+) cells were isolated and grown from AngII-exposed animals. Comparatively few cells were cultured from untreated control hearts, and they were found to be ED1(-)/SMA(+)/CD133(-). We provide evidence that myocardial ECM deposition is preceded by infiltration into the myocardium by cells that express a combination of haematopoietic (ED1, CD133) and mesenchymal (SMA) cell markers, which is a characteristic of the phenotype of fibroblast precursor cells, termed fibrocytes. This suggests that fibrocytes rather than (as is often presumed) leucocytes may have effector functions in the initiation of myocardial fibrosis.

Nonclassical Resident Macrophages Are Important Determinants in the Development of Myocardial Fibrosis

“…In the wellestablished AngII-dependent model of hypertension, we and others have demonstrated an absence of tissue necrosis and associated polymorphonuclear cell infiltrates, suggesting that this model may not require the mass influx of classical macrophages. 7,15,16,21,23 We have also characterized that early myocardial infiltration in AngII-exposed hearts is largely unaffected in Ccr2 À/À mice, suggesting redundancy in chemokines or implicating Cx3cr1-dependent nonclassical macrophage recruitment. 21 Despite this body of evidence it remained to be demonstrated whether macrophages were mediating the cellular changes observed in the myocardium and the ECM remodeling after AngII infusion.…”

“…15,16,21e23 The AngII model is characterized by significant macrophage infiltrate, a profibrotic milieu, and significant ECM deposition without the loss of healthy tissue as seen in ischemic injury. 15,16,24 Furthermore, our work with Ccr2 À/À mice in this model suggests that the nonclassical Cx3cr1 pathway may be important to nonischemic myocardial healing. 21 This model offers the unique opportunity to study myocardial fibrosis without conflicting ischemia that is seen after myocardial infarction and to focus the depletion to the fibrotic stage of the response.…”

“…16,21 In brief, hearts were mechanically and enzymatically digested in a collagenase solution (0.652 mg/mL collagenase II; Cedarlane Laboratories, Ltd., Burlington, ON, Canada) in DMEM-C at 37 C, with agitation for 45 minutes. Cell isolates were twice washed in DMEM-C and purified over a Percoll gradient (30% and 70%).…”

“…3,12e14 Work in our laboratory by using a hypertension model of myocardial fibrosis has shown that myocardial fibrosis is preceded by a mass influx of circulating mononuclear cells characteristic of macrophages, expressing markers such as ED1, CD11b, and F4/80. 15,16 Infiltrating monocytes can dramatically expand the macrophage population in the heart.…”

Downregulation of MicroRNA-19b Contributes to Angiotensin II-Induced Overexpression of Connective Tissue Growth Factor in Cardiomyocytes

“…Using an Ang II infusion model of myocardial fibrosis, Sopel et al [24] demonstrated that Ang II infusion resulted in rapid infiltration of fibroblast progenitor cells, termed fibrocytes. Fibrocytes are capable of producing both proinflammatory mediators and fibrogenic factors, such as CTGF, which is mainly synthesized and released by cardiac fibroblasts in the heart.…”