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. 2018 Jul 1;315(1):H92-H100.
doi: 10.1152/ajpheart.00719.2017. Epub 2018 Mar 9.

Increased macrophage-derived SPARC precedes collagen deposition in myocardial fibrosis

Lindsay T McDonald  1   2 , Michael R Zile  3   2 , Yuhua Zhang  3 , An O Van Laer  3   2 , Catalin F Baicu  3   2 , Robert E Stroud  2   4 , Jeffrey A Jones  2   4 , Amanda C LaRue  1   2 , Amy D Bradshaw  3   2
Affiliations

Increased macrophage-derived SPARC precedes collagen deposition in myocardial fibrosis

Lindsay T McDonald et al. Am J Physiol Heart Circ Physiol. .

Abstract

Myocardial fibrosis and the resultant increases in left ventricular stiffness represent pivotal consequences of chronic pressure overload (PO) that impact both functional capacity and the rates of morbid and mortal events. However, the time course and cellular mechanisms that underlie PO-induced fibrosis have not been completely defined. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that has been shown to be required for insoluble collagen deposition and increased myocardial stiffness in response to PO in mice. As macrophages are associated with increases in fibrillar collagen, the hypothesis that macrophages represent a source of increased SPARC production in the PO myocardium was tested. The time course of changes in the myocardial macrophage population was compared with changes in procollagen type I mRNA, production of SPARC, fibrillar collagen accumulation, and diastolic stiffness. In PO hearts, mRNA encoding collagen type I was increased at 3 days, whereas increases in levels of total collagen protein did not occur until 1 wk and were followed by increases in insoluble collagen at 2 wk. Increases in muscle stiffness were not detected before increases in insoluble collagen content (>1 wk). Significant increases in myocardial macrophages that coincided with increased SPARC were found but did not coincide with increases in mRNA encoding collagen type I. Furthermore, immunohistochemistry and flow cytometry identified macrophages as a cellular source of SPARC. We conclude that myocardial macrophages play an important role in the time-dependent increases in SPARC that enhance postsynthetic collagen processing, insoluble collagen content, and myocardial stiffness and contribute to the development of fibrosis. NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in left ventricular and myocardial stiffness represent pivotal consequences of chronic pressure overload. In this study a murine model of cardiac fibrosis induced by pressure overload was used to establish a time course of collagen expression, collagen deposition, and cardiac macrophage expansion.

Keywords: collagen; extracellular matrix; matricellular proteins; postsynthetic collagen processing; secreted protein acidic and rich in cysteine.

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Figures

Fig. 1.
Fig. 1.
Consort diagram summarizing measured end points and sample sizes. Echo, echocardiography; LV, left ventricular; PO, pressure overload; Pap Ms, papillary muscle testing; rtPCR, reverse transcription polymerase chain reaction; CVF, collagen volume fraction; FACS, fluorescence-activated cell sorting.
Fig. 2.
Fig. 2.
Time course of procollagen synthesis and processing from soluble to insoluble collagen in response to left ventricular (LV) pressure overload (PO) induced by transverse aortic constriction. A: collagen transcriptional activation measured by mRNA expression of the α1-subunit of collagen type I (Col1a1) increased rapidly at 3 days of LV PO. Sustained elevations in Col1a1 expression were detected at 1 wk; levels decreased by 4 wk and remained greater than baseline. B: total (soluble + insoluble) myocardial collagen protein production measured by hydroxyproline assay increased at 1 wk of LV PO and reached a plateau that was maintained throughout 4 wk of LV PO. C: insoluble fibrillar collagen accumulation and deposition measured as collagen volume fraction did not increase until 2 wk of LV PO and increased further at 4 wk of LV PO. These data show an increase in procollagen synthesis immediately (as early as 3 days) after the imposition of PO; however, deposition of fibrillar collagen was not synchronous with increased transcription but was delayed until 2 wk of LV PO, when procollagen processing was increased. Values are means ± SE. *P < 0.05 vs. control (no LV PO); #P < 0.05 vs. 3 days of LV PO.
Fig. 3.
Fig. 3.
Time course of changes in myocardial stiffness in response to the imposition of left ventricular (LV) pressure overload (PO). A: myocardial stress versus strain curves determined by passive stretch of LV papillary muscle from control hearts compared with hearts subjected to 3 days, 1 wk, 2 wk, and 4 wk of LV PO. B: myocardial stiffness measured by the passive stiffness constant β did not increase until 2 wk of LV PO. Values are means ± SE. *P < 0.05 vs. control (no LV PO).
Fig. 4.
Fig. 4.
Time course of changes in secreted protein acidic and rich in cysteine (SPARC) expression in response to the imposition of left ventricular (LV) pressure overload (PO). Immunoblot analysis of SPARC showed that levels were significantly increased at 1 wk of LV PO and remained increased at 4 wk of LV PO. Values are means ± SE. *P < 0.05 vs. control (no LV PO).
Fig. 5.
Fig. 5.
Quantitative assessment of total number of macrophages and number of secreted protein acidic and rich in cysteine (SPARC)-producing macrophages in left ventricle (LV) after the induction of pressure overload (PO). FACS analysis at 1 wk of LV PO (red) versus control (blue) used a sequential gating strategy. A: Live/Dead fixable near-IR (APC-Cy7), depicted as side scatter (SSC) versus APC-Cy7; sized, single, live cells gated. B: SSC versus CD45-PE; sized, single, live, CD45+ (hematopoietic) cells gated. C: CD11b-FITC (myeloid) versus F4/80-BV421 (macrophage); sized, single, live, CD45+/CD11b+/F4/80+ cells gated. D: cell count versus SPARC-APC, indicated by the gate; sized, single, live, CD45+/CD11b+/F4/80+/SPARC+ cells. Note the significant increase in percentage of live CD45+/CD11b+/F4/80+ cells, i.e., an increase in the number of macrophages in LV PO versus control and an increase in the number of macrophages expressing SPARC [red (LV PO sample) versus blue (baseline control sample) counts]. Gates were set based on fluorescence − 1 (FMO) controls (gray peak in D).
Fig. 6.
Fig. 6.
Control left ventricular (LV) tissue (A) and LV tissue after 1 wk of pressure overload (PO; B) stained with anti-SPARC (red) and anti-F4/80 macrophage (green) antibodies. Arrows indicate cells that costained with anti-SPARC and anti-F4/80 antibodies (yellow). Scale bar = 20 μm.
Fig. 7.
Fig. 7.
Increases in total macrophages (F4/80+) occur with time in the left ventricle (LV) after LV pressure overload (PO) induced by transverse aortic constriction (TAC). A: control (no LV PO) LV tissue. B: LV tissue at 3 days of LV PO. C and D: increase in total macrophages (F4/80+) at 1 and 4 wk of LV PO. E: myocardium stained with secondary antibodies only. Arrows indicate F4/80+ cells. Scale bar = 50 μm.
Fig. 8.
Fig. 8.
Flow cytometric analysis of cell markers and secreted protein acidic and rich in cysteine (SPARC) over time after left ventricular (LV) pressure overload (PO). Cells positive for CD45 and CD11b, denoting hematopoietic origin and monocyte lineage, respectively, demonstrated increases in myocardial number at 3 days of LV PO (A and B). Cells positive for mature macrophages (F4/80+) showed greatest levels at 1 wk in LV PO myocardium (C and D). SPARC expression by each individual population peaked at 1 wk of LV PO. Sequential gating of cells positive for CD45, CD11b, and F4/80 cells (C and D) showed sustained expression of SPARC at 4 wk of LV PO. Box-and-whisker graphs show medians and interquartile ranges. Temporal changes of cell markers were compared between control and LV PO groups by one-way repeated-measures ANOVA followed by all pair-wise multiple-comparison procedures by Fisher’s least-significant-difference method (SigmaStat 3.5). *P < 0.05 vs. baseline; #P < 0.05 vs. 3-day transverse aortic constriction (TAC); +P < 0.05 vs. 1-wk TAC.
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