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The Journal of Immunology, 2008, 180, 2625-2633
Copyright © 2008 by The American Association of Immunologists, Inc.
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CD44 Is Critically Involved in Infarct Healing by Regulating the Inflammatory and Fibrotic Response1

Peter Huebener, Tareq Abou-Khamis, Pawel Zymek, Marcin Bujak, Xia Ying, Khaled Chatila, Sandra Haudek, Geeta Thakker and Nikolaos G. Frangogiannis2

Section of Cardiovascular Sciences, Baylor College of Medicine, Houston, TX 77030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Infarct healing is dependent on an inflammatory reaction that results in leukocyte infiltration and clearance of the wound from dead cells and matrix debris. However, optimal infarct healing requires timely activation of "stop signals" that suppress inflammatory mediator synthesis and mediate resolution of the inflammatory infiltrate, promoting formation of a scar. A growing body of evidence suggests that interactions involving the transmembrane receptor CD44 may play an important role in resolution of inflammation and migration of fibroblasts in injured tissues. We examined the role of CD44 signaling in infarct healing and cardiac remodeling using a mouse model of reperfused infarction. CD44 expression was markedly induced in the infarcted myocardium and was localized on infiltrating leukocytes, wound myofibroblasts, and vascular cells. In comparison with wild-type mice, CD44–/– animals showed enhanced and prolonged neutrophil and macrophage infiltration and increased expression of proinflammatory cytokines following myocardial infarction. In CD44null infarcts, the enhanced inflammatory phase was followed by decreased fibroblast infiltration, reduced collagen deposition, and diminished proliferative activity. Isolated CD44null cardiac fibroblasts had reduced proliferation upon stimulation with serum and decreased collagen synthesis in response to TGF-β in comparison to wild-type fibroblasts. The healing defects in CD44–/– mice were associated with enhanced dilative remodeling of the infarcted ventricle, without affecting the size of the infarct. Our findings suggest that CD44-mediated interactions are critically involved in infarct healing. CD44 signaling is important for resolution of the postinfarction inflammatory reaction and regulates fibroblast function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Postinfarction cardiac repair is dependent on release of inflammatory mediators and subsequent infiltration of the infarcted myocardium with leukocytes that clear the wound from dead cells and matrix debris (1, 2, 3). However, optimal infarct healing requires timely activation of "stop signals" that suppress chemokine and cytokine synthesis resulting in resolution of the inflammatory infiltrate. The inflammatory phase is followed by the proliferative phase as myofibroblasts accumulate in the infarct and deposit extracellular matrix proteins forming a collagen-based scar. The dynamic changes in the extracellular matrix network play an important role in regulating the cellular events involved in wound healing. Increased turnover of extracellular matrix is a hallmark of tissue injury and results in generation of degradation products. Hyaluronan, a ubiquitously present constituent of the extracellular matrix (4), exists as a high m.w. polymer under physiologic conditions, but undergoes degradation resulting in accumulation of lower m.w. species after tissue injury. Hyaluronan fragments induce the expression of a variety of inflammatory genes by endothelial cells and macrophages (5), including chemokines and cytokines, and may play an important role in regulating the inflammatory process. Furthermore, clearance of hyaluronan fragments from the injured tissue is crucial for resolution of chronic inflammation (6). Hyaluronan participates in induction and resolution of inflammation through interactions with the transmembrane adhesion molecule CD44 (7), a ubiquitously distributed glycoprotein that mediates a wide variety of cell-cell and cell-matrix interactions. CD44-hyaluronan interactions play an important role in regulating leukocyte extravasation into inflammatory sites (8, 9) and mediate efficient phagocytosis (10). In addition, a growing body of evidence suggests that CD44 serves as a key factor in resolution of inflammation through removal of matrix breakdown products and clearance of apoptotic neutrophils (6), and mediates fibroblast migration and invasion in the wound provisional matrix (11). Although numerous studies have explored the significance of CD44-mediated interactions in a variety of inflammatory and fibrotic processes, the role of CD44 signaling in healing myocardial infarcts has not been investigated.

We hypothesized that CD44 may play an essential role in infarct healing by regulating the inflammatory and fibrotic response. We found that CD44null animals exhibit enhanced and prolonged inflammation in the infarcted heart followed by reduced myofibroblast infiltration. The healing defect in CD44–/– mice was associated with impaired fibroblast function and markedly diminished collagen deposition in the scar and resulted in enhanced adverse remodeling of the infarcted ventricle.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Murine ischemia/reperfusion protocols

All animal studies were approved by the Animal Protocol Review Committee at Baylor College of Medicine. CD44–/– mice (12) and wild-type (WT)3 B6129PF2/J controls (purchased from The Jackson Laboratory) were used for myocardial infarction experiments. Male and female mice, 8–12 wk of age (18.0–22.0 g body weight) were anesthetized by an i.p. injection of sodium pentobarbital (60 µg/g). A closed-chest mouse model of reperfused myocardial infarction was used to avoid the confounding effects of surgical trauma and inflammation, which may influence the baseline levels of chemokines and cytokines (2). The left anterior descending coronary artery was occluded for 1 h then reperfused for 6 h to 7 days. At the end of the experiment, the chest was opened and the heart was immediately excised, fixed in zinc-formalin, and embedded in paraffin for histological studies, or snap-frozen and stored at –80°C for RNA or protein isolation. Sham animals were prepared identically without undergoing coronary occlusion/reperfusion. Animals used for histology underwent 24-h, 72-h, and 7-day reperfusion protocols (eight animals per group). Mice used for RNA extraction underwent 6 h, 24 h, and 72 h of reperfusion (eight animals per group). To assess remodeling-associated parameters, additional mice were used for perfusion-fixation of the heart (knockout (KO), n = 10; WT, n = 8) after 7 days of reperfusion. Mice used for isolation of infarct myofibroblasts underwent 3-day-reperfusion protocols (KO, n = 6; WT, n = 6).

Quantitative PCR (qPCR)

Total RNA was treated with DNase to remove any genomic contamination as described by the manufacturer (DNA-free; Ambion) First-strand cDNA was synthesized using the iScript cDNA Synthesis kit (Bio-Rad) with random primers as described in the manufacturer’s protocol. CD44 primers were specifically designed using the National Center for Biotechnology Information genome database and manufactured by Sigma-Genosys. Real-time PCR was performed and analyzed with 1/10 diluted cDNA according to the manufacturer’s instructions on an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Target gene expression was normalized to an internal control, ribosomal protein S3 (RPS3). Both CD44 and Rps3 were measured using SYBR Green chemistry and the relative standard curve method. At the end of PCR cycle, dissociation curve analysis was performed to ascertain the amplification of a single PCR product. Sequences of the murine primers were as follows: CD44 forward (5'-ATCAGCAGATCGATTTGAATGTAA-3'), CD44 reverse (5'-CATTTCCTTCTATGAACCCATACC-3'); RPS3 forward (5'-ATCAGAGAGTTGACCGCAGTTG-3'), RPS3 reverse (5'-AATGAACCGAAGCACACCATAG-3').

Immunohistochemistry and quantitative histology

Murine hearts were fixed in zinc-formalin (Z-fix; Anatech), and embedded in paraffin. Sections were cut at 3 µm and stained immunohistochemically with the following Abs: rat anti-mouse CD44 Ab (BD Pharmingen), monoclonal anti-{alpha} smooth muscle actin ({alpha}-SMA) Ab (Sigma-Aldrich), rat anti-mouse macrophage Ab clone F4/80 (Research Diagnostics), rabbit anti-mouse proliferating cell nuclear Ag (PCNA) Ab (Abcam), and rat anti-neutrophil Ab (Serotec). Staining was performed using a peroxidase-based technique with the Vectastain ELITE rat, rabbit, or goat kit (Vector Laboratories) and developed with diaminobenzidine plus nickel (Vector Laboratories). The Mouse on Mouse (MOM) kit (Vector Laboratories) was used for {alpha}-SMA immunohistochemistry. For F4/80 and PCNA staining, unmasking with trypsin was performed. Hyaluronic acid (HA) was detected using histochemical staining with biotinylated HA-binding protein (Cape Cod/Seikagaku Biochemicals) as previously described (13). Collagen was stained with picrosirius red (14). Apoptotic cells were labeled using the CardioTacs In Situ Apoptosis Detection kit (Trevigen). Quantitative assessment of macrophage density was performed by counting the number of F4/80-positive cells in the infarcted area as previously described (15). Myofibroblasts were identified as extravascular {alpha}-SMA-positive cells and counted in the infarcted myocardium. Macrophage, neutrophil, and myofibroblast density was expressed as cells per millimeter-squared. Proliferative activity in the infarct was assessed by counting the PCNA index as the percentage of cells with PCNA-positive nuclei among all cells in the infarct. Collagen density in the infarcted area was expressed as the percentage of the infarcted area stained with picrosirius red.

Perfusion fixation and assessment of ventricular volumes

For assessment of postinfarction remodeling, infarcted hearts after 7 days of reperfusion were used for perfusion-fixation as previously described (2). The entire heart from base to apex was cross-sectioned at 250-µm intervals. Ten serial 5-µm sections were obtained at each interval. The left ventricular end-diastolic volume (LVEDV), left ventricular volume, septal volume, and scar size were assessed with ImagePro software using methods developed in our laboratory (2). Left ventricular mass and septal mass were derived by multiplying the left ventricular volume and septal volume, respectively, by the specific gravity of the myocardium (1.065 g/ml). The size of the infarct was expressed as a percentage of the left ventricular volume.

RNA extraction and RNase protection assay (RPA)

Inflammatory gene expression in murine hearts was assessed using RPA as previously described (2, 16). The mRNA expression level of the chemokines MIP-1{alpha}, MIP-1β, MIP-2, MCP-1, and IFN-{gamma}-inducible protein (IP)-10, the cytokines TNF-{alpha}, IL-1β, osteopontin, IL-6, and IL-10, the growth factors TGF-β1, 2, and 3, and M-CSF were determined using a RPA (RiboQuant; BD Pharmingen) according to the manufacturer’s protocol. Phosphorimaging of the gels was performed (Storm 860; Molecular Dynamics) and signals were quantified using Image QuaNT software and normalized to the ribosomal protein L32 mRNA.

Protein extraction and Western blotting

Protein isolation and Western blot analysis were conducted as previously described (17, 18). WT (n = 4) and CD44–/– (n = 5) mice undergoing reperfused infarction protocols (1 h ischemia/24 h reperfusion) were used for protein extraction. A total of 15 µg of protein were separated on SDS-polyacrylamide gels in a Tris-HCl buffer system, transferred onto nitrocellulose membranes, and blotted according to standard procedures using a polyclonal rabbit anti-Smad2 (1/1,000) or a polyclonal rabbit anti-phospho-Smad2 (Ser465/467; 1/200) Ab (both obtained from Cell Signaling). The specific bands of target proteins were visualized by chemiluminescence, and band intensities were evaluated using Image QuaNT. Membranes were then stripped and reblotted with monoclonal anti-GAPDH (1/10,000; Advanced ImmunoChemical). Target signals were normalized to GAPDH signal. The ratio of p-smad2 to total smad2 expression was used as an indicator of activation of the TGF-β-signaling pathway.

Isolation and stimulation of murine cardiac fibroblasts

Fibroblasts were isolated from normal mouse hearts by enzymatic digestion with a collagenase buffer as previously described (19). Three noninfarcted WT or CD44–/– hearts were used for each experiment. The hearts were dissected free of vessels and atria, transferred to 1 ml of collagenase buffer, and quickly minced into small pieces. Digestion with collagenase buffer continued until no visible tissue fragments were left. The isolated cell suspensions from each round were pelleted and washed. All cell suspensions were combined, plated on a T75 tissue-culture flask (Corning) in full medium supplemented with 10% of FBS (HyClone) and antibiotic-antimycotic solution. After overnight incubation, nonadherent cells were removed and adherent cells were cultivated. Upon reaching confluence, cells were detached with trypsin/EDTA, split in a 1:2 or 1:4 ratio and recultured. Characteristic fibroblast morphology was determined visually under a light microscope. Because the phenotype of fibroblasts can be influenced by growth conditions such as passage and cell density (20), only fibroblasts at passages 1–3 were used for experiments. Pure fibroblast cultures were confirmed by immunocytochemistry using Abs against vimentin (a mesenchymal cell marker), {alpha}-SMA (both obtained from Sigma-Aldrich), and collagen type I (Rockland). To study the effects of TGF-β on control WT and CD44–/– cardiac fibroblasts, cells were stimulated with rTGF-β1 (100 ng/ml; R&D Systems) for 4 h. At the end of the experiment, protein was extracted from the cell lysates and Western blotting was performed to assess collagen type I expression using a rabbit anti-collagen type I Ab (Rockland) and to quantitate Smad2 phosphorylation (18) as previously described.

Fibroblast proliferation assay

Proliferation was determined by bromodeoxyuridine incorporation using a commercially available colorimetric kit (Roche Applied Science) as previously described (21). To normalize data from different experiments, proliferation in response to 5% serum was expressed as fold increase to cells maintained in serum-free medium. The proliferative response to serum was compared between fibroblasts isolated from WT and CD44null hearts.

Isolation of myofibroblasts from healing infarcts

To examine the role of CD44 deficiency on the phenotypic characteristics of fibroblasts in the healing infarcts, infarct myofibroblasts were isolated from infarcted WT and CD44–/– hearts after 1 h of ischemia and 72 h of reperfusion (n = 6/group). This time point represents the peak of the proliferative phase, when fibroblast density in the infarcted heart is maximal. The infarct and the border zone area were excised and used for isolation of fibroblasts as described above. Isolated cells were used for immunocytochemical studies (n = 3/group) or for protein extraction (n = 3/group). Characterization of the cells was performed using immunocytochemistry for {alpha}-SMA, vimentin, CD31, and collagen type I. To compare activation of fibrogenic pathways in CD44–/– and WT infarct myofibroblasts, the protein extracts were used for Western blotting to assess collagen type I expression and p-Smad2:Smad2 ratio.

Statistical analysis

Statistical analysis was performed using ANOVA followed by a t test corrected for multiple comparisons (Student-Newman-Keuls). Data were expressed as mean ± SEM. Statistical significance was set at 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Expression and localization of CD44 and its main ligand, hyaluronan, in healing mouse infarcts

qPCR demonstrated that CD44 mRNA was markedly induced in the infarcted mouse myocardium, peaking after 6 h of reperfusion (CD44:RPS3 ratio, 0.77 ± 0.09 in sham mice vs 4.90 ± 0.63 after 6 h of reperfusion, p < 0.01). CD44 mRNA levels remained elevated after 24 h (p < 0.05) and 72 h (p < 0.05) of reperfusion (Fig. 1A). Immunohistochemical studies showed that CD44 was predominantly localized on infiltrating cells after 24 h of reperfusion (Fig. 1B). After 72 h-7 days of reperfusion, the majority of granulation tissue cells in the infarcted myocardium stained for CD44 (Fig. 1, C and D), including macrophages, myofibroblasts (identified as {alpha}-SMA-positive spindle-shaped cells) (Fig. 1, E and F), and endothelial cells. Using affinity histochemistry, we identified a thin rim of hyaluronan in the endoperimysium of the murine myocardium. After 24 h of reperfusion, the hyaluronan network in the infarcted area exhibited extensive fragmentation (Fig. 1G). Healing of the infarct was associated with hyaluronan deposition in the wound forming an organized network after 7 days of reperfusion (Fig. 1I).


Figure 1
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  		FIGURE 1. Expression and localization of CD44 and its main ligand hyaluronan in healing mouse myocardial infarcts. A, qPCR demonstrated that CD44 mRNA levels were markedly induced in the infarcted heart, peaking after 6 h of reperfusion (*, p < 0.05). B–E, CD44 immunohistochemistry in mouse infarcts. CD44 was localized in infiltrating leukocytes after 24 h of reperfusion (B). After 72 h (C) 7days (D) of reperfusion, many granulation tissue cells infiltrating the infarct exhibited CD44 expression. After 72 h of reperfusion, CD44 was localized in infarct myofibroblasts, identified as nonvascular {alpha}-smooth muscle actin-expressing spindle-shaped cells (arrows, E and F). In contrast, vascular smooth muscle cells did not show CD44 immunoreactivity (arrowhead). G–I, Affinity histochemistry for hyaluronan in murine myocardial infarcts. A thin rim of hyaluronan was identified in the endoperimysium of the mouse heart. After 24 h of reperfusion, disruption of the hyaluronan network was observed in the infarcted area (G, arrows). Hyaluronan deposition was noted in the infarct after 72 h of reperfusion (H, arrows) forming an organized matrix network after 7 days (I). Magnification x100, counterstained with eosin.

 
CD44null mice exhibited enhanced and prolonged inflammatory leukocyte infiltration in the healing infarct

Mortality rates following reperfused infarction were comparable in WT and CD44null mice (CD44–/–: 8.3% vs WT: 6% p not significant (pNS)). CD44–/– mice exhibited increased neutrophil (862.4 ± 34.32 cells/mm2 vs 502.6 ± 47.17 cells/mm2, p < 0.05; Fig. 2, A, C, and D) and macrophage (423.4 ± 22.4 cells/mm2 vs 248.9 ± 19.4 cells/mm2, p < 0.05; Fig. 2B) density in the infarcted myocardium after 24 h of reperfusion. In addition, CD44null mice had prolonged infiltration with inflammatory leukocytes showing significantly higher neutrophil and macrophage density after 72 h and 7 days of reperfusion (Fig. 2, A, B, E–H). To examine whether prolonged neutrophil and macrophage infiltration in CD44-deficient animals was due to defective clearance of apoptotic leukocytes from the infarcted myocardium, we assessed the density of TUNEL-positive cells in the infarct. CD44 deficiency was not associated with an increased number of apoptotic cells in the infarcted myocardium. After 24 h of reperfusion, the percentage of apoptotic cells was not significantly different between CD44–/– and WT mice (CD44–/–: 11.8 ± 1.5% vs WT: 12.8 ± 1.4%, pNS). After 72 h of reperfusion, CD44null infarcts had slightly reduced percentage of apoptotic cells in comparison with WT mice (CD44–/–: 5.5 + 0.5% vs WT: 9.97 ± 0.9%; p < 0.05).


Figure 2
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  		FIGURE 2. Effects of CD44 gene disruption on the time course of neutrophil and macrophage infiltration in the infarcted myocardium. CD44null mice exhibited enhanced and prolonged neutrophil (A) and macrophage infiltration (B) in the infarct in comparison with WT animals (**, p < 0.01). However, resolution of the neutrophilic infiltrate was noted after 7 days of reperfusion in both CD44–/– and WT infarcts. C–F, Immunohistochemistry identifies neutrophils (PMN) in WT (C and E) and CD44–/– infarcts (D and F). G and H, Macrophages (MP) in WT (G) and CD44null (H) infarcts were identified with F4/80 immunohistochemistry.

 
CD44-deficient mice showed increased myocardial cytokine expression but comparable chemokine mRNA levels following myocardial infarction

Reperfused murine myocardial infarction triggers a robust but transient up-regulation of proinflammatory cytokines and chemokines (16). In comparison to their WT littermates, CD44–/– animals showed significantly increased peak mRNA expression of the proinflammatory cytokines, IL-1β (Fig. 3B), TNF-{alpha} (Fig. 3C), IL-6 (Fig. 3D), and M-CSF (data not shown). Expression of the matricellular protein osteopontin, a marker of monocyte to macrophage differentiation was significantly higher in CD44null infarcts (Fig. 3F). In addition, mRNA expression of the inhibitory cytokine IL-10 was significantly higher in CD44null infarcts compared with WT animals (Fig. 3E).


Figure 3
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  		FIGURE 3. Cytokine mRNA expression in infarcted CD44null and WT hearts. RPA analysis (A) demonstrated that CD44–/– mice had enhanced peak mRNA levels of the proinflammatory cytokines IL-1β (B), TNF-{alpha} (C), and IL-6 (D), the inhibitory cytokine IL-10 (E), and the matricellular protein OPN (F), a marker of monocyte to macrophage differentiation (*, p < 0.05; **, p < 0.01). However, both WT and CD44null infarcts demonstrated timely repression of the cytokine response (U, unprotected probe).

 
In contrast, peak expression of the chemokines MCP-1, MIP-1{alpha}, MIP-1β, and MIP-2 was comparable between CD44–/– and WT infarcts, whereas IP-10 mRNA levels were modestly but significantly higher in CD44null infarcts (p < 0.05) (Fig. 4).


Figure 4
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  		FIGURE 4. Effects of CD44 gene disruption on chemokine mRNA expression in the infarcted heart. A, RPA analysis was used for assessment of chemokine mRNA expression in the infarcted heart (U, unprotected probe). CD44null and WT mice had comparable peak MIP-2 (B) and MCP-1 (D) mRNA expression in the infarcted heart, whereas peak IP-10 mRNA expression was higher in CD44–/– infarcts (C). CD44 gene disruption did not affect the time course of chemokine mRNA repression in the infarcted heart (**, p < 0.01; *, p < 0.05).

 
Effects of CD44 deficiency on repression of inflammatory cytokine and chemokine synthesis

In both WT and CD44null infarcts the postinfarction inflammatory response was followed by repression of cytokine and chemokine synthesis after 24 h of reperfusion. mRNA levels of the proinflammatory cytokines TNF-{alpha}, IL-1β, M-CSF (Fig. 3), and the chemokines MIP-1{alpha}, MIP-1β, MIP-2, and IP-10 were comparable between WT and CD44null animals, whereas MCP-1 (0.0755 + 0.01 vs 0.1402 + 0.02, p < 0.05) and IL-6 (0.0113 + 0.0015 vs 0.0196 + 0.002, p < 0.05) levels were lower in CD44null infarcts indicating that the absence of CD44 did not affect the timely repression of chemokines and cytokines in the infarcted myocardium (Figs. 3 and 4).

CD44null mice exhibited decreased myofibroblast infiltration and reduced collagen deposition in the healing infarct

During the proliferative phase of healing murine myocardial infarcts exhibit intense infiltration with myofibroblasts, phenotypically modulated fibroblasts that express {alpha}-SMA and are the main collagen-producing cells in the infarct (22). CD44null mice had significantly lower myofibroblast density in the infarcted myocardium after 3 days of reperfusion (WT: 233.3 ± 16.9 cells/mm2 vs –/– 123.3 ± 13.0, p < 0.05) in comparison with WT animals (Fig. 5). PCNA immunohistochemistry demonstrated that in the absence of CD44, decreased myofibroblast density was associated with significantly reduced proliferative activity in the infarcted myocardium (PCNA index WT: 24.05 ± 2.0% vs CD44–/–: 13.5 ± 1.2%, p < 0.05). After 7 days of reperfusion, mouse infarcts show deposition of collagen and formation of a scar. CD44null mice had reduced collagen content in the infarct compared with WT mice (CD44–/–: 12.3 ± 1.1% vs WT: 20.99 ± 0.6%, p < 0.05) (Fig. 5, F–H).


Figure 5
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  		FIGURE 5. CD44 gene disruption results in decreased infiltration of the infarcted myocardium with myofibroblasts and markedly reduced collagen deposition. A, Myofibroblast density was significantly lower in CD44null infarcts after 3 days of reperfusion (**, p < 0.01). B and C, Immunohistochemistry for {alpha}-SMA identified myofibroblasts as spindle-shaped interstitial cells in WT (B) and CD44null (C) mice. Myofibroblast infiltration was significantly decreased in CD44–/– animals after 72 h of reperfusion (counterstained with eosin). D and E, Immunohistochemical staining for PCNA was used to label proliferating cells in the infarcted myocardium (arrows). The PCNA index was calculated as the percentage of cells with PCNA positive nuclei among all cells in the infarct (identified with hematoxylin counterstaining). At the peak of the proliferative phase (72 h of reperfusion), CD44null infarcts exhibited significantly lower number of proliferating cells in comparison with WT animals. F, Quantitative assessment of the collagen-stained area in the infarcted myocardium demonstrated that after 7 days of reperfusion, CD44–/– infarcts had lower collagen deposition than WT infarcts (**, p < 0.01). G and H, Sirius red-staining labels the collagen network in WT (G) and CD44null infarcts (H) after 7 days of reperfusion. Dense deposition of collagen is noted in WT, but not in CD44 –/– infarcts.

 
Expression of phosphorylated Smad2 in infarcted CD44null hearts

Because TGF-β is a key mediator of fibrosis, involved in myofibroblast differentiation and extracellular matrix deposition, we examined expression of TGF-β isoforms and activation of the smad2/3-signaling pathway in the infarcted hearts. Infarcted WT and CD44null hearts had comparable TGF-β1, β2, and β3 mRNA expression after 72 h of reperfusion (data not shown). Western blotting experiments demonstrated that CD44null infarcts had significantly higher Smad2 expression levels, but markedly reduced p-Smad2:Smad2 ratio (CD44null: 0.14 ± 0.03 vs WT: 0.41 ± 0.11, p < 0.05) (Fig. 6).


Figure 6
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  		FIGURE 6. CD44–/– mice exhibit impaired Smad2 phosphorylation in the infarcted heart. CD44null infarcts had higher Smad2 levels, lower p-Smad2 expression, and a markedly reduced p-Smad2:Smad2 ratio in comparison with WT infarcts.

 
The absence of CD44 enhanced dilative, but not hypertrophic, postinfarction remodeling without a significant effect on infarct size

After 7 days of reperfusion, CD44null mice exhibited increased LVEDV compared with WT animals (WT: 33.62 ± 1.66 mm3 vs CD44 –/–: 43.81 ± 4.06 mm3, p < 0.05) (Fig. 7), although scar size was comparable between the two groups (WT: 9.7 ± 1.1% vs CD44–/–: 12.5 ± 1.4% of LV volume; pNS). Although infarcted CD44–/– animals had enhanced ventricular dilation, they showed a trend toward lower left ventricular mass in comparison with WT animals (Table I), suggesting that CD44 gene disruption enhances dilative but not hypertrophic remodeling following myocardial infarction.


Figure 7
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  		FIGURE 7. CD44 gene disruption resulted in enhanced dilative remodeling following myocardial infarction. LVEDV was significantly higher in infarcted CD44null hearts compared with WT animals (*, p < 0.05); infarct size was comparable between groups. Although CD44 absence enhanced dilative remodeling, postinfarction hypertrophy was comparable between CD44null and WT animals.

 

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  		Table I. Remodeling-associated parameters in infarcted CD44null and WT animals

 
TGF-β-induced collagen type I up-regulation is attenuated in CD44null cardiac fibroblasts

Fibroblasts isolated from CD44null and WT hearts exhibited similar morphology. Upon stimulation with 5% serum, CD44null fibroblasts had an attenuated proliferative response in comparison to WT cardiac fibroblasts (fold increase of cell proliferation in comparison to serum free-cells: WT, 4.14 + 0.59, vs CD44–/– 2.17 + 0.39, p < 0.05, n = 4). Furthermore, fibroblasts isolated from WT, but not from CD44null, mouse hearts exhibited significant up-regulation of collagen type I synthesis upon stimulation with TGF-β (Fig. 8A). The blunted response to TGF-β stimulation in the absence of CD44 was not due to impaired activation of the Smad2 pathway. Both WT and CD44null fibroblasts showed marked increase of the p-Smad2:Smad2 ratio, when stimulated with TGF-β (Fig. 8B).


Figure 8
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  		FIGURE 8. Impaired fibrogenic response in CD44null cardiac fibroblasts. A, Cardiac fibroblasts were isolated from uninjured CD44 and WT mouse hearts. WT but not CD44–/– cardiac fibroblasts showed up-regulation of collagen type I (Col I) protein synthesis upon stimulation with TGF-β (100 ng/ml, 4 h) (*, p < 0.05 vs control unstimulated fibroblasts, n = 4). B, TGF-β stimulation induced Smad2 phosphorylation in both WT and CD44–/– cardiac fibroblasts (*, p < 0.05 vs corresponding control fibroblasts, n = 4). C–F, Phenotypic characteristics of infarct myofibroblasts isolated from WT and CD44null hearts. Myofibroblasts were isolated from WT (C) and CD44null infarcts (D) after 72 h of reperfusion (at the peak of the proliferative phase) and were characterized using immunocytochemistry. Cells isolated from WT infarcts (C) had more intense staining for collagen I in comparison to CD44–/– cells (D). E and F, In additional experiments, myofibroblasts isolated from WT and CD44–/– infarcts were used for protein extraction (n = 3/group). Col I expression levels (E) and Smad2 phosphorylation (F) were quantitatively assessed. E, A representative experiment (one of three experiments with similar findings) demonstrates that CD44–/– infarct myofibroblasts had lower Col I expression in comparison to WT cells. Statistical analysis showed a trend toward lower Col I expression in cells isolated from CD44–/– infarcts (mean Col I levels, WT: 1.15 + 0.31 vs CD44 –/–: 0.66 + 0.35 p = 0.14). F, In contrast, WT and CD44–/– infarct myofibroblasts had comparable p-Smad2:Smad2 ratios.

 
Infarct myofibroblasts isolated from CD44null animals exhibit similar p-Smad2 expression, but reduced collagen type I synthesis in comparison to WT mice

Infarct myofibroblasts were isolated from WT and CD44null infarcts and were characterized as vimentin and {alpha}-SMA-positive, CD31-negative (nonendothelial) spindle-shaped cells. A trend toward lower collagen type I expression levels was noted in cells isolated from CD44–/– infarcted hearts when compared with WT animals (mean collagen type I levels, WT: 1.15 + 0.31 vs CD44–/–: 0.66 + 0.35 p = 0.14, n = 3) (Fig. 8, C–E). In contrast, p-Smad2 expression levels were comparable between groups (p-Smad2:Smad2 ratio WT: 1.25 ± 0.03 vs CD44–/–: 1.22 ± 0.02; pNS) (Fig. 8F).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Diverse cellular functions have been attributed to CD44, including involvement in leukocyte chemotaxis and activation (23), clearance of apoptotic cells and matrix fragments (6, 24), resolution of chronic inflammation (6), regulation of fibrous tissue deposition (11), and angiogenesis (25). The present study reports the first evidence that CD44 plays an essential role in infarct healing and regulates postinfarction cardiac remodeling. CD44 expression was markedly induced in the infarcted myocardium (Fig. 1) and was localized in leukocytes, fibroblasts, and endothelial cells infiltrating the wound. CD44 deficiency resulted in enhancement of the postinfarction inflammatory response. However, despite showing an accentuated inflammatory phase, infarcted CD44–/– mice exhibited markedly attenuated myofibroblast infiltration and diminished collagen deposition in the wound, associated with decreased proliferative activity and evidence of impaired TGF-β signaling. These healing defects resulted in enhanced dilative remodeling of the infarcted ventricle.

The role of CD44 in regulation of the postinfarction inflammatory response

Numerous studies have explored the role of CD44 in models of acute inflammation generating disparate and often contradictory results that underline the complex and multifunctional role of CD44 in the inflammatory response. Administration of anti-CD44 Abs inhibited leukocyte extravasation in a model of cutaneous delayed-type hypersensitivity (26) and abrogated tissue edema and leukocyte infiltration in murine arthritis (8). Anti-CD44 treatment induced the rapid loss of CD44 from the surface of leukocytes presumably preventing cell-extracellular matrix interactions critical for leukocyte infiltration (8). Moreover, CD44null animals bred into an atherosclerosis-prone ApoEnull background had markedly reduced macrophage infiltration in aortic lesions (27). In contrast, other investigations did not support an essential role for CD44 in mediating leukocyte infiltration in inflamed tissues, but suggested that CD44-hyaluronan interactions are important for suppression of inflammation and resolution of the inflammatory infiltrate. CD44 KO animals exhibited accentuated inflammation in a model of collagen-induced arthritis (28), were more susceptible to endotoxin-induced shock (29), and showed enhanced neutrophil migration and lung injury in murine bacterial pneumonia (30). In vitro experiments demonstrated that CD44–/– neutrophils migrate faster through matrigel than WT neutrophils, suggesting that CD44 may slow neutrophil migration through extracellular matrix (30). These actions may be due to CD44-induced intracellular signaling events in leukocytes bound to hyaluronan or other ligands (30). In addition, CD44 may prevent exaggerated inflammatory responses by promoting the expression of negative regulators of TLR-4 signaling (29). Furthermore, CD44null animals succumb to unremitting inflammation following noninfectious lung injury (6) demonstrating impaired clearance of apoptotic neutrophils and hyaluronan fragments. It appears that in the absence of CD44 the highly potent surface receptor for hyaluronan-mediated motility compensates for the loss of CD44 supporting inflammatory leukocyte migration and inducing an intense inflammatory response (28). Our findings suggest that CD44 does not mediate recruitment of leukocytes in the infarcted myocardium but plays an important role in suppression of the postinfarction inflammatory response. CD44 absence resulted in an enhanced inflammatory response, associated with increased neutrophil (Fig. 2) and macrophage infiltration (Fig. 2) and increased cytokine mRNA expression (Fig. 3) in the infarcted myocardium. Enhanced neutrophil density was not due to the persistent presence of apoptotic neutrophils in the infarct. Defective clearance of hyaluronan fragments from the infarcted myocardium may have resulted in enhanced inflammation in CD44–/– animals. However, resolution of postinfarction inflammation ultimately occurred in CD44null mice, suggesting that in the absence of CD44 other inhibitory pathways mediate repression of inflammatory mediators and clearance of the leukocyte infiltrate.

Role of CD44 in fibrous tissue deposition in the healing infarct

In the absence of CD44, enhanced peak expression of inflammatory cytokines and accentuated leukocyte infiltration in the infarcted myocardium was followed by decreased myofibroblast accumulation and markedly diminished collagen deposition in the healing wound (Fig. 5). Several mechanisms may be responsible for decreased fibroblast infiltration in the healing infarct. First, CD44-mediated interactions may play a direct role in fibroblast proliferation (31). CD44null mice exhibited reduced proliferative activity in the infarcted myocardium in comparison with WT animals (Fig. 5). In addition, isolated CD44null cardiac fibroblasts had decreased proliferative activity when stimulated with serum. Second, CD44 deficiency may result in impaired fibroblast migration and invasion of the provisional matrix in the infarct. Svee et al. (11) demonstrated that anti-CD44 Ab blocked fibroblast migration on the provisional matrix proteins fibronectin, fibrinogen, and HA, all important components of the healing wound (13). Third, CD44 may promote fibroblast survival by decreasing apoptosis (32). Although previous investigations demonstrated that anti-CD44 treatment induced apoptotic death in cultured fibroblasts, our experiments showed no significant difference in the density of apoptotic cells between CD44null and WT infarcts. Fourth, CD44 gene disruption may impair fibrogenic responses in stimulated fibroblasts. CD44-HA interactions stimulate TGF-βRI serine/threonine kinase activity inducing Smad2/3 phosphorylation in metastatic breast cancer cells (33). Because the TGF-β/Smad2/3 signaling cascade plays a crucial role in fibrous tissue deposition (34, 35, 36) in the infarcted heart, we examined whether CD44 deficiency directly impairs TGF-β-mediated fibrogenic responses. CD44null infarcts had significantly higher Smad2 expression levels, but markedly reduced the p-Smad2:Smad2 ratio (Fig. 6) raising the possibility that, in the absence of CD44, infarct fibroblasts may exhibit defective activation of the Smad2/3 pathway. Although CD44null fibroblasts had attenuated collagen up-regulation upon stimulation with TGF-β (Fig. 8A), this defect was not associated with impairment in Smad2 phosphorylation (Fig. 8B). Thus, the mechanism responsible for the defective response of CD44null cardiac fibroblasts to TGF-β remains unknown. The absence of CD44 appears to have profound effects on fibroblast proliferation and matrix protein synthesis; these defects may result in diminished extracellular matrix deposition in the infarcted myocardium.

The role of CD44 in cardiac remodeling

In comparison with WT animals, CD44null mice exhibited enhanced ventricular dilation following myocardial infarction (Fig. 7, Table I). However, infarct size was comparable in CD44–/– and WT hearts suggesting that accentuated dilative remodeling in infarcted CD44–/– hearts was not a consequence of increased cardiomyocyte injury due to the enhanced inflammatory reaction. Augmented dilation of the infarcted heart may be due to the healing defects associated with CD44 deficiency resulting in marked decrease in collagen deposition in the scar. Formation of a defective matrix network reduces the tensile strength of the ventricle (37) promoting chamber enlargement. In contrast, CD44 absence did not affect hypertrophic remodeling suggesting that CD44 signaling does not play a role in the development of cardiomyocyte hypertrophy following myocardial infarction.

Conclusions

CD44-mediated interactions are critical for cardiac repair and appear to regulate both inflammatory and fibrotic responses. CD44 signaling does not mediate leukocyte infiltration, but is an important inhibitory signal responsible for suppression of postinfarction inflammation. In addition, CD44 plays a key role in the formation of scar-mediating myofibroblast infiltration and collagen deposition. Defects in the CD44-signaling cascade may be important in the pathogenesis of adverse remodeling following myocardial infarction. Dissecting the pathways involved in CD44-mediated actions may facilitate design of novel therapeutic interventions to optimize infarct healing and prevent adverse remodeling.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health R01 HL-76246, R01 HL-85440, and the American Heart Association. Back

2 Address correspondence and reprint requests to Dr. Nikolaos G. Frangogiannis, Section of Cardiovascular Sciences, Baylor College of Medicine, One Baylor Plaza BCM620, Houston, TX 77030. E-mail address: ngf{at}bcm.tmc.edu Back

3 Abbreviations used in this paper: WT, wild type; KO, knockout; qPCR, quantitative PCR; RPS3, ribosomal protein S3; {alpha}-SMA, {alpha}-smooth muscle actin; PCNA, proliferating cell nuclear Ag; HA, hyaluronic acid; LVEDV, left ventricular end-diastolic volume; RPA, RNA protection assay; IP, IFN-{gamma}-inducible protein; RPA, ribonuclease protection assay; pNS, p not significant. Back

Received for publication March 8, 2007. Accepted for publication December 3, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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