Early Fibroblast Progenitor Cell Migration to the AngII-Exposed Myocardium Is Not CXCL12 or CCL2 Dependent as Previously Thought

Abstract

Fibroblast progenitor cells (fibrocytes) are important to the development of myocardial fibrosis and are suggested to migrate to the heart via CXCL12 and chemokine ligand (CCL) 2. We hypothesized that if these chemokines are recruiting fibrocytes, disrupting their signaling will reduce early (3-day) fibrocyte infiltration and, consequently, fibrosis in the myocardium. C57/Bl6 and CCR2(-/-) mice were infused with saline or angiotensin (Ang) II, with or without CXC receptor 4 blockade (AMD3100). Hearts were assessed for chemokine up-regulation, immunofluorescence, and histological features. AngII caused early myocardial up-regulation of CXCL12 and CCL2, which corresponded to significant myocardial infiltration and fibrosis compared with controls. Animals receiving AMD3100 and/or with the genotype CCR2(-/-) failed to demonstrate reductions in infiltrate or fibrosis after 3 days of AngII, and AngII + AMD3100 animals showed exacerbated fibrocyte infiltration and fibrosis compared with AngII alone. CCR2(-/-) mice demonstrated significant reductions in myocardial fibrosis relative to wild type, but this was after 28 days of AngII infusion and was the result of reduced infiltrating cell proliferation. An alternative CCR2 ligand, CCL12, was found to be increasing infiltrating cell proliferation in the heart after AngII infusion, which we confirmed in vitro. In conclusion, early fibrocyte recruitment cannot be inhibited through modulating CXCL12 or CCL2, as previously thought. Ablating CCR2 signaling did confer myocardial fibrosis reductions, but these benefits were not observed until much later and were likely the result of modulated proliferation through ablating the CCL12-CCR2 interaction.

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

“…Favoring the Ly6C low CX3CR1 þ macrophage phenotype is consistent with previous work in which Ccr2 À/À mice did not demonstrate a reduction in early cellular infiltrate or fibrosis in the myocardium early after AngII exposure. 21 Furthermore, our previous work with green fluorescent protein bone marrow chimeras indicated that these areas of cellular infiltrate are predominantly derived from circulating monocytes. 15 It followed that these macrophages could be depleted with i.v.…”

“…21 Mice were randomly assigned treatments of vehicle (saline) or 2.8 mg/kg per day AngII (SigmaAldrich, Oakville, ON, Canada). The pumps remained in for 3 days, during which time, the mice were observed for morbidity.…”

“…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%).…”

“…15,21,22 Primers were designed against mRNA sequences of chemokine (C-X3-C motif) ligand 1 (CX3CL1; forward, 5 0 -GCAACCC-…”

“…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.…”