Bone marrow-derived fibroblast precursors mediate ischemic cardiomyopathy in mice - PubMed (original) (raw)

Bone marrow-derived fibroblast precursors mediate ischemic cardiomyopathy in mice

Sandra B Haudek et al. Proc Natl Acad Sci U S A. 2006.

Abstract

We previously described a mouse model of fibrotic ischemia/reperfusion cardiomyopathy (I/RC) arising from daily, brief coronary occlusion. One characteristic of I/RC was the prolonged elevation of monocyte chemoattractant protein 1 (MCP-1), which was obligate to its phenotype and may contribute to the uptake of bloodborne cells. Here we describe in I/RC hearts a population of small spindle-shaped fibroblasts that were highly proliferative and expressed collagen I and alpha-smooth muscle actin (myofibroblast markers), CD34 (a precursor marker), and CD45 (a hematopoietic marker). These cells represented 3% of all nonmyocyte live cells. To confirm the cells' bone marrow origin, chimeric mice were created by the rescue of irradiated C57BL/6 mice with marrow from ROSA26, a congenic line expressing lacZ. I/RC resulted in a large population of spindle-shaped fibroblasts containing lacZ. We postulated that the fibroblast precursors represented a developmental path for a subset of monocytes, whose phenotype we have shown to be influenced by serum amyloid P (SAP). Thus, we administered SAP in vivo, which markedly reduced the number of proliferative spindle-shaped fibroblasts and completely prevented I/RC-induced fibrosis and global ventricular dysfunction. By contrast, SAP did not suppress the inflammation or chemokine expression seen in I/RC. SAP, a member of the pentraxin family, binds to Fcgamma receptors and modifies the pathophysiological function of monocytes. Our data suggest that SAP interferes with assumption of a fibroblast phenotype in a subset of monocytes and that SAP may be an important regulator in the linkage between inflammation and nonadaptive fibrosis in the heart.

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Conflict of interest statement

Conflict of interest statement: Rice University has patent applications on the use of SAP to inhibit fibrosis, and this intellectual property has been licensed to Medior Biopharm. D.P. and R.H.G. are founding members of, have equity in, and receive royalties from Medior Biopharm.

Figures

Fig. 1.

Fig. 1.

Characterization of cardiac fibroblasts after 5-d I/RC. (A) Isolated fibroblasts cultured in vitro (panel 1, magnification: ×100) were tested for the presence/absence of cell markers by immunocytochemistry (panels 2–6, magnification: ×200). (B) Freshly isolated CD34+ or CD45+ cells (red) were sorted by immunoabsorption, plated on coverslips, and stained for collagen I (green; blue indicates DAPI-stained nuclei). (C) Proliferation of cardiac fibroblasts in vitro was determined by BrdU incorporation. Values represent the fold induction of growth rate induced by 10% serum compared with serum-free medium (∗, P < 0.01; n = 6 per group).

Fig. 2.

Fig. 2.

CD34 and CD45 expression. (A) Perfusion-fixed heart tissue was stained for α-SMA (green) and CD45 (red) to identify myofibroblasts. The arrowhead indicates a cell positive for both markers, whereas the arrow indicates an α-SMA+ smooth muscle cell in the wall of an adjacent venule. Also present are CD45+ α-SMA− cells that represent infiltrating macrophages. (B) Cytometric analysis of freshly dispersed nonmyocyte cardiac cells for CD34 and CD45 expression of viable (calcein+) cells in sham and I/RC animals (∗, P < 0.001; n = 8 per sham and 10 per I/RC). (C) Representative cytometric diagram for three-color staining (x axis, PE/Cy-5-CD45; y axis, PE-CD34; gated on calcein+ cells).

Fig. 3.

Fig. 3.

Cardiac fibroblasts from chimeric mice (ROSA26 bone marrow donor cells to C57BL/6 recipient mice) after 5-d I/RC. (A) Cultured fibroblasts were stained with X-gal to visualize bone marrow-originating cells (lacZ+ cells). (Magnification: Left and Center, ×100; Right, ×200.) After I/RC, many small spindle-shaped cells stained positive (blue) for lacZ expression, whereas no positive cells were found in sham animals. (B and C) Further immunocytochemistry analysis using a specific antibody against β-galactosidase demonstrated that only small spindle-shaped cells (arrowheads) but not large flat cells (arrows) were positive for lacZ (red), although both cell populations expressed the fibroblast markers α-SMA (green; B; scale bar, 40 μm) and collagen I (green; C). LacZ+ cells also expressed CD34 (cyan; C), whereas large flat cells did not. Blue indicates DAPI-stained nuclei; yellow results from merging images.

Fig. 4.

Fig. 4.

Cellular effects of SAP. (A) Isolated cardiac fibroblasts after 5-d I/RC with SAP treatment cultured in vitro were large and flat, similar to sham fibroblasts (see Fig. 1_A_). (Magnification: ×100.) (B) Proliferation of these cells was determined by BrdU incorporation in response to 10% serum compared with serum-free medium. Note that fibroblasts after I/RC with SAP treatment proliferated significantly more slowly than fibroblasts without SAP and were not different from sham fibroblasts (see Fig. 1_C_) (∗, P < 0.02; _n_ = 6 per group). (_C_) Cytometric analysis of dispersed nonmyocyte cardiac cells for CD34 and CD45 expression of viable (calcein+) cells in 5-d I/RC animals with and without SAP treatment. Note that SAP significantly reduced the number of CD34+ cells (∗, _P_ < 0.001; _n_ = 10 per group) and CD34+/CD45+ cells (∗, _P_ < 0.003; _n_ = 6 per group) but not the number of CD45+ cells (_P_ > 0.05; n = 10 per group). (D–F) Quantitative analysis of macrophage (D) and myofibroblast density (E), as well as collagen content (F), in the ischemic myocardium was performed in perfusion-fixed hearts. The posterior septum region served as an internal control because this region should not be affected by I/RC. Note that SAP did not diminish macrophage infiltration (P > 0.05, n = 5 per group), indicating that I/RC-induced inflammation was not inhibited by SAP. However, treatment with SAP significantly reduced the number of myofibroblasts in tissue (∗, P < 0.05; n = 4 per group) and blunted the development of fibrosis (∗, P < 0.001; n = 6 per group) after I/RC.

Fig. 5.

Fig. 5.

Effects of SAP on ventricular function and mRNA expression. SAP treatment improved fractional shortening (∗, P < 0.05; _n_ = 7 per group) (_A_) and preserved anterior wall thickening (∗, _P_ < 0.001; _n_ = 7 per group) (_B_) in mice undergoing I/RC as measured by M-mode echocardiography and were similar to results for sham-operated animals (_P_ > 0.05, n = 7–8 per group). SAP treatment did not alter I/RC-induced mRNA expression of MCP-1 (P = 0.15; n = 8 per group) and/or other chemokines (C) or cytokines (D). (See section RNA in Materials and Methods for abbreviations.)

References

    1. Brilla CG, Weber KT. Cardiovasc Res. 1992;26:671–677. - PubMed
    1. Weber KT, Brilla CG, Janicki JS. Cardiovasc Res. 1993;27:341–348. - PubMed
    1. Weber KT, Pick R, Jalil JE, Janicki JS, Carroll EP. J Mol Cell Cardiol. 1989;21(Suppl 5):121–131. - PubMed
    1. Weber KT, Janicki JS, Pick R, Capasso J, Anversa P. Am J Cardiol. 1990;65:1G–7G. - PubMed
    1. Jalil JE, Janicki JS, Pick R, Abrahams C, Weber KT. Circ Res. 1989;65:258–264. - PubMed

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