CCR2 mediates the uptake of bone marrow-derived fibroblast precursors in angiotensin II-induced cardiac fibrosis

Animals.

Animal experiments were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85–23, revised 1996). The CCR2 KO mice on a background of C57BL/6J were purchased from the Jackson Laboratory. Disruption of CCR2 gene was confirmed by Western blot with an anti-CCR2 antibody from Abcam (Fig. 1). Male wild-type (WT) or CCR2 KO mice at 8–12 wk old, weighing 20–30 g, received ANG II (1.5 μg·kg⁻¹·min⁻¹; Sigma) or vehicle (0.9% NaCl) continuously via subcutaneous osmotic minipumps (Alzet) for 7 or 28 days following uninephrectomy. Uninephrectomy was performed to augment blood pressure and organ injury (8, 20). All mice were supplied 1% saline drinking water ad libitum.

Blood pressure and heart rate.

Systolic blood pressure (SBP) and heart rate (HR) were measured in conscious mice using a tail-cuff system (Visitech Systems), as reported (14).

Cardiac morphology.

Cardiac sections were stained with hematoxylin and eosin for initial evaluation and picrosirius red or Masson trichrome to identify collagen fiber. The cross-sectional areas of LV myocytes were measured on the mid-free wall of the LV from sections. Suitable cross-sectional areas were defined as having nearly circular capillary profiles and nuclei. A total of 100 cells were counted in each section, and the average area was used for analysis. The picrosirius red-stained sections were scanned using a microscope equipped with a digital camera (Nikon, Melville, NY), and quantitative evaluation was performed using NIS-Elements Br 3.0 software. The area of interstitial fibrosis was calculated as a percentage of the total area, and the area of perivascular fibrosis was determined as the ratio of the area of fibrosis surrounding the vessel wall to the total vessel area.

Immunohistochemistry.

After fixation, cardiac sections were then incubated with rabbit anti-Col I antibody (Rockland), followed by Alexa-488 conjugated donkey anti-rabbit antibody (Invitrogen), rabbit anti-fibronectin antibody (Sigma), followed by Alexa-488 conjugated donkey anti-rabbit antibody (Invitrogen), or rat anti-CD68 antibody (AbD Serotec), followed by Alexa-598 conjugated donkey anti-rat antibody (Invitrogen). For double immunofluorescence, cells were fixed and stained with primary antibodies, followed by appropriate secondary antibodies sequentially. Slides were mounted with VECTASHIELD HardSet mounting medium with 4,6-diamidino-2-phenylindole (DAPI). Fluorescent intensity was visualized using a microscope equipped with a digital camera (Nikon, Melville, NY). For α-SMA staining, paraffin-embedded tissues were cut at 5-μm thickness, deparaffinized, and quenched with 3% hydrogen peroxide in methanol for 10 min. After heat retrieval, slides were incubated with rabbit anti-α-SMA antibody, followed by biotin-conjugated goat anti-rabbit IgG, and then incubated with Vectastain ABC reagent. Bound antibodies were detected using diaminobenzidine as a substrate and counterstained with hematoxylin. Quantitative evaluation of sections was performed using NIS-Elements Br 3.0 software. The positively stained interstitial area was calculated as a percentage of the total area, and the positively stained perivascular area was calculated as the ratio of the area surrounding the vessel to the total vessel area.

Cell isolation and flow cytometry.

Cardiac fibroblasts were isolated as described (17). Cells (5 × 10⁵) were incubated with phycoerythrin (PE)-anti-CD34, FITC-anti-CD45, (all from BD Biosciences, San Jose, CA), PE-anti-α-SMA (R&D Systems, Minneapolis, MN), or biotin-anti-Col I (Rockland, Gilbertsville, PA)/streptavidin-allophycocyanin (APC) (BD Biosciences). Peripheral blood was obtained via cardiac puncture. Samples were lysed with BD PharM Lyse for 15 min, washed in PBS containing DNase, and blocked with PACS buffer for 30 min at 4°C. Cells were stained for CD45, Col I, and CCR2. If necessary, cells were fixed and permeabilized using the Cytofix/Cytoperm kit (PE BD Biosciences), in accordance with the manufacturer's protocol. Cells incubated with irrelevant isotype-matched antibodies and unstained cells were used as controls. The cutoffs were set according to results of controls. FITC/PE/APC fluorescence intensities were measured using a LSR II flow cytometer (BD Biosciences). Data were analyzed using BD FACSDiva software.

Quantitative real-time RT-PCR.

Quantitative analysis of mRNA expression was performed with real-time RT-PCR. Total RNA was extracted from snap-frozen heart tissues with TRIzol Reagent (Invitrogen). Aliquots (1 μg) of total RNA were reverse-transcribed and amplified using IQ SYBR green supermix reagent (Bio-Rad, Hercules, CA) with Opticon real-time PCR machine (MJ Research, Waltham, MA), according to the manufacturer's instructions. The specificity of real-time PCR was confirmed via routine agarose gel electrophoresis and melting-curve analysis. The expression level of the target genes was normalized by the GAPDH level in each sample. The following are the primer sequences: Col I, forward 5′-TGCCGCGACCTCAAGATGTG-3′ and reverse 5′-CACAAGGGTGCTGTAGGTGA-3′; fibronectin, forward 5′-CTTCTCCGTGGAGTTTTACCG-3′ and reverse 5′-GCTGTCAAATTGAATGGTGGTG-3′; α-SMA, forward 5′-ACTGGGACGACATGGAAAAG-3′ and reverse 5′-CATCTCCAGAGTCCAGCACA-3′; brain natriuretic peptide (BNP), forward 5′-AAGTCCTAGCCAGTCTCCAGA-3′ and reverse 5′-GAGCTGTCTCTGGGCCATTTC-3′; and GAPDH, forward 5′-TGCTGAGTATGTCGTGGAGTCTA-3′ and reverse 5′-AGTGGGAGTTGCTGTTGAAATC-3′.

Western blot analysis.

Protein was extracted using the RIPA buffer containing cocktail proteinase inhibitors and quantified with Bio-Rad protein assay. Equal amount of protein was separated on SDS-polyacrylamide gels in a Tris·HCl buffer system, transferred onto nitrocellulose membranes, and blotted with anti-α-SMA antibody, anti-ED-A fibronectin antibody, or anti-CCR2 antibody. The specific bands of target proteins were visualized by chemiluminescence, and band intensities were quantified using National Institutes of Health Image/J. Membranes were then stripped and reblotted with anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA). Target signals were normalized to GAPDH signal.

Cardiovascular parameters.

Cardiac function was obtained by two-dimensional-directed M-mode echocardiography (Vevo770; Visual Sonics) and Doppler Ultrasound (model DSPW, Indus Instruments), as previous described (9).

Statistical analysis.

All data were expressed as means ± SE. Multiple-group comparisons were performed by one-way ANOVA, followed by the Bonferroni procedure for comparison of means. P < 0.05 was considered statistically significant.

Blood pressure and HR.

The effects of ANG II treatment on SBP and HR are summarized in Table 1. There were no significant differences among the four groups before ANG II treatment. SBP was similarly elevated in both WT and CCR2-KO mice after ANG II infusion. There was no significant difference in HR among the groups.

Cardiac fibrosis.

To test whether CCR2 deficiency influenced cardiac fibrosis, WT and CCR2-KO mice were treated with ANG II or vehicle for 4 wk. The staining for both picrosirius red and Masson trichrome was performed on cardiac sections, and the results of the two stainings were similar. Representative photomicrographs are shown in Fig. 2, A and B, and the results of morphometric analysis are shown in Fig. 2, C and D. WT mice treated with ANG II developed marked interstitial and perivascular collagen deposition in the heart. In contrast, CCR2-KO mice exhibited much less increase in collagen accumulation, despite ANG II treatment. These data indicate that CCR2 plays a critical role in the pathogenesis of cardiac fibrosis.

We next investigated the effect of CCR2 deficiency on expression and accumulation of Col I and fibronectin, two major components of ECM. As shown in Figs. 3A and 4A, there was a marked increase in the mRNA levels of Col I and fibronectin in the WT mice treated with ANG II, whereas CCR2-KO mice exhibited much less increase in the mRNA expression of Col I and fibronectin in the heart after ANG II treatment. We then examined the levels of protein expression in the heart in response to ANG II. There was minimal Col I and fibronectin protein accumulation in the normal hearts of both WT and CCR2-KO mice. After ANG II treatment for 4 wk, WT mice developed severe interstitial and perivascular fibrosis, as demonstrated by an accumulation of Col I and fibronectin deposition that was significantly lower in the heart of ANG II-treated CCR2-KO mice (Fig. 3, B–D, and Fig. 4, B–D). These data indicate that targeted deletion of CCR2 attenuates cardiac fibrosis by inhibiting ECM protein production and deposition.

Bone marrow-derived fibroblast precursors.

Bone marrow-derived fibroblast precursors have been shown to play a critical role in the pathogenesis of cardiac fibrosis, and chemokines, through interaction with their cognate receptors, mediate the recruitment and differentiation of bone marrow-derived fibroblast precursors (17, 19). To verify if CCR2, the receptor for MCP-1, is expressed in circulating fibroblast precursors, peripheral blood nucleated cells were stained for CD45, CCR2, and Col I and examined with a deconvolution microscope. Our results showed CD45, CCR2, and Col I triple-positive cells are present in the peripheral circulation (Fig. 5A). These data confirm that circulating fibroblast precursors express CCR2. To determine the percentage of bone marrow-derived fibroblast precursors that express CCR2, peripheral blood nucleated cells were stained for CD45, CCR2, and Col I using flow cytometry. Our results showed that >80% of bone marrow-derived fibroblast precursors express CCR2 (Fig. 5B). To examine the role of CCR2 in the recruitment of bone marrow-derived fibroblast precursors into the heart, WT and CCR2-KO mice were treated with ANG II or vehicle for 7 days. Cardiac fibroblasts were isolated and cultured. These cells were stained for CD45 and Col I, or CD34 and Col I, and examined using a fluorescent microscope. Our results showed that CD45⁺ and Col I⁺ cells or CD34⁺ and Col I⁺ cells were present in the heart of ANG II-treated WT mice. In contrast, these cells were rarely found in the heart of ANG II-treated CCR2-KO mice (Fig. 6A). To quantify the number of bone marrow-derived fibroblast precursors in the heart, freshly dispersed cardiac cells were stained for CD45, CD34, and Col I and analyzed by flow cytometry. The number of bone marrow-derived fibroblast precursors identified as CD45⁺ and Col I⁺ or CD34⁺ and Col I⁺ cells was markedly increased in the heart of ANG II-treated WT mice, whereas the number of bone marrow-derived fibroblast precursors was significantly reduced in the heart of ANG II-treated CCR2-KO mice (Fig. 6, B and C). These data indicate that CCR2 mediates the trafficking of bone marrow-derived fibroblast precursors into the heart.

We then determined if CCR2 influences the number of bone marrow-derived myofibroblasts in the heart. WT and CCR2-KO mice were treated with ANG II or vehicle for 1 wk. Freshly dispersed cardiac cells were stained for CD45, a marker of hematopoietic cell, and α-SMA, a marker of myofibroblasts, and then analyzed by flow cytometry. Targeted deletion of CCR2 resulted in a significant reduction of the number of bone marrow-derived myofibroblasts in the heart treated with ANG II (Fig. 6D). Consistent with these findings, real-time RT-PCR showed that the mRNA level of α-SMA was significantly reduced in the heart of ANG II-treated CCR2-KO mice compared with that of WT mice treated with ANG II (Fig. 7A). These findings were confirmed at the protein levels, as demonstrated by immunostaining and Western blot analysis (Fig. 7, B–E). We also examined if CCR2 deficiency affects the expression level of ED-A fibronectin, another marker of myofibroblasts (36, 40). The level of ED-A fibronectin was significantly induced in the heart of ANG II-treated WT mice compared with vehicle-treated WT mice. In contrast, the level of ED-A fibronectin was dramatically reduced in the heart of ANG II-treated CCR2 KO mice compared with ANG II-treated WT mice (Fig. 8). These data indicate that CCR2 plays an important role in the activation and differentiation of bone marrow-derived fibroblasts.

Cardiac hypertrophy.

Hypertrophic responses to sustained hypertension induced by ANG II infusion were assessed in WT and CCR2-KO mice. Before ANG II infusion, WT and CCR2-KO mice did not exhibit significant differences in heart weight. In response to chronic ANG II infusion, both WT and CCR2-KO mice developed comparable cardiac hypertrophy, as indicated by increased heart-to-body weight ratio, heart weight-to-tibia length ratio, and myocyte cross-sectional area (Fig. 9, A–C). Next, we examined the expression levels of BNP, a marker for cardiac hypertrophy, using real-time RT-PCR. Our result showed that mRNA levels of BNP were upregulated in both WT and CCR2-KO mice in response to ANG II, which was not statistically different between the two groups (Fig. 9D). We also evaluated LV hypertrophy (LVH) using echocardiography. After ANG II treatment, both WT and CCR2-KO mice developed comparable cardiac hypertrophy, as judged by anterior and posterior wall thickness (Table 1). These data indicate that CCR2 deficiency does not affect the development of ANG II-induced hypertrophy.

Cardiac function and dimension.

To investigate the effect of CCR2 deficiency on cardiac function, we performed echocardiographic examination of WT and CCR2-KO mice after 4 wk of ANG II infusion. Both WT and CCR2-KO mice treated with ANG II developed impaired LV diastolic function, as indicated by increased isovolumic relaxation time corrected by the time per heartbeat that was not statistically different between the two groups (Table 1). These data indicate that CCR2 deficiency does not affect ANG II-induced diastolic dysfunction. LV systolic function, as indicated by ejection fraction and fractional shortening, was not statistically different in WT mice treated with ANG II or vehicle. In contrast, the ejection fraction and fractional shortening were decreased in ANG II-treated CCR2-KO mice compared with vehicle-treated CCR2-KO mice, indicating impaired LV systolic function (Table 1). There was no statistical difference in end-diastolic LV diameter between ANG II-treated and vehicle-treated WT mice. However, end-diastolic LV diameter was significantly increased in ANG II-treated CCR2-KO mice compared with vehicle-treated CCR2-KO mice (Table 1).