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Departments of 1Physiology and Pharmacology and 2Anatomy; and 3Comparative Medicine Unit, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio
Submitted 31 March 2005 ; accepted in final form 1 September 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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cardiac fibroblasts; fibrosis; extracellular matrix; 
-smooth muscle actin
A key aspect of CF activation is differentiation into myofibroblasts, which are specialized cells that play critical roles in wound healing and repair in several tissues (9, 26). It has been suggested that these cells are responsible for the majority of ECM deposition (8, 14) and that these differentiated fibroblasts are the primary mediators of fibrosis in the heart (41). In normal wound healing, differentiation to myofibroblasts is necessary for repair and stabilization, but these cells eventually undergo apoptosis. If myofibroblasts remain in the injured area for an extended period of time, excessive ECM production occurs, resulting in fibrosis.
The specific collagen types produced by fibroblasts and myofibroblasts may, in turn, affect the activation and subsequent function of these cells. In adults, the myocardial ECM is primarily composed of types I and III collagen (1, 2, 13, 42) with types IV, V, and VI collagen comprising the remainder of the collagen network (20). During cardiac pathologies, investigators (16, 24, 40) have detected an increase in both types I and III collagen, which contribute significantly to the observed cardiac fibrosis. Although type VI collagen is considered to be a minor type in the adult heart, type VI levels have been demonstrated to significantly increase in both hypertension and diabetes (34). In addition, interstitial fibrosis and cardiac dysfunction related to hypertrophic cardiomyopathy have been positively correlated with the elevation in levels of type VI and type III collagen (23).
The fact that type VI collagen is significantly elevated in these specific cardiac pathologies prompted us to hypothesize that this collagen type plays a critical role in pathological remodeling via induction of myofibroblast differentiation. The goals of our study were to determine the effects of types I, III, and VI collagen on CF differentiation and proliferation, the effect of ANG II on type VI collagen production by CFs, and whether type VI collagen and cardiac myofibroblast levels change in vivo during post-MI cardiac remodeling.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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CFs were isolated as previously described (19, 29, 30, 32). Briefly, hearts were excised from anesthetized adult male Sprague-Dawley rats weighing 275–300 g, rinsed in cold PBS, and briefly perfused with isolation media containing (in mM) 120 NaCl, 5 KCl, 20 HEPES, 5 glucose, 0.8 MgSO4, and 1 NaH2PO4, and 0.02 mg/ml phenol red, pH 7.35. The ventricles were isolated, minced, and digested with collagenase type 2 (100 U/ml) and trypsin (0.6 mg/ml; Worthington Biochemical; Lakewood, NJ). The collagenase medium containing the CFs was centrifuged for 10 min at 180 g and resuspended in DMEM with 10% FBS. Cells were plated and allowed to attach for 30 min before the first media change, which removed weakly adherent cells, including myocytes and endothelial cells. CFs were >95% pure at passage 1 as determined by positive staining for vimentin and negative staining for von Willebrand factor and -smooth muscle actin (-SMA) as previously described (19, 29, 30). The main contaminating cell type in this preparation is endothelial cells, with minimal contamination from myocytes and vascular smooth muscle cells (VSMC). CFs were used only at early passages (<3), where their morphology, expression markers, and growth properties remain consistent.
Preparation of Substrates: Isolation of Type VI Collagen, Solubilization of Types I and III Collagen and Coating of Plates
Type VI collagen was isolated according to the methods previously outlined (11). Acid-soluble collagens were removed by treatment of bovine corneas with acetic acid. The acid-insoluble fraction was homogenized in 6 M urea and ammonium sulfate, precipitated, and was then resuspended in 5% SDS in borate buffer. The sample was chromatographed by using a Sepharose 4B column, and fractions were analyzed using SDS-PAGE to identify the presence of purified type VI collagen. Both collagen types I and III were purchased from Sigma-Aldrich (St. Louis, MO). All collagens were solubilized in 20 mM carbonate buffer (pH 9.6) at a final concentration of 20 µg/ml and added to noncoated plastic wells.
Immunocytochemical Staining and Fluorescent Imaging
CFs were seeded onto collagen-coated, 18-well glass "dot" slides at 
10,000 cells per dot (each with an area of 28.3 mm2) and incubated for 24 h in serum-free DMEM. Subsequently, the cells were washed and then incubated with 100 nM ANG II for an additional 24 h. The cells were washed, fixed in 3.5% paraformaldehyde, permeablized in 0.1% Triton X-100, blocked in 2% goat serum, and incubated with a mouse monoclonal anti--SMA primary antibody (Sigma-Aldrich) for 1 h in a humidified incubator. The cells were next washed and incubated with a goat anti-mouse secondary antibody conjugated to Alexa Fluor 488 (Molecular Probes; Eugene, OR) for 1 h, washed, mounted with anti-fade reagent containing 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Vector; Burlingame, CA), and visualized by an Olympus BX60 microscope. Digital images were taken using x10–x40 objectives. Additional control conditions were routinely carried out, including secondary antibody alone as well as cell-free backgrounds with primary and secondary antibodies (alone and in combination).
Assessment of Cell Proliferation
CFs were grown on 96-well plates precoated with types I, III, and VI collagen for 24 h and then treated with 100 nM ANG II or vehicle for 24 h. To assess cellular proliferation, a 5-bromo-2'-deoxyuridine (BrdU) assay was performed according to the manufacturer's protocol (Roche Applied Science; Indianapolis, IN). BrdU (10 µM) was included in the growth medium after the indicated treatments, and results were analyzed by using spectrophotometry at a wavelength of 450 nm.
Western Blot Analysis of ERK1/2 and Type VI Collagen in Isolated CF Cultures
CFs were plated for 24 h on type I, III, or VI collagen or tissue culture plates. Where indicated, cells were treated with 100 nM ANG II for either 10 min (for ERK1/2 assessment) or 24 h (for type VI collagen assessment). Whole cell lysates were collected, and Western blot analysis was performed. Cells were scraped and maintained in lysis buffer containing 62.5 mM Tris·HCl, 2 mM EDTA, 2.3% SDS, 10% glycerol (pH 6.8), and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 1 µg/ml pepstatin A, and 5 µg/ml aprotinin). Cell protein was quantified by the bicinchoninic acid (BCA) assay (Pierce). Equal amounts of protein samples (15 µg) were boiled for 5 min in x2 sample buffer (100 mM Tris base, 20% glycerol, 2% SDS, and 0.01% bromophenol blue), separated by standard SDS-PAGE, and transferred by electrolysis to nitrocellulose. Western blot analysis was performed by standard techniques with the use of 5% milk in 0.1% Tween-20-TBS as a blocking reagent. Membranes were washed in 0.1% Tween-20-TBS four to five times after incubation with each antibody, and bands were visualized with enhanced chemiluminescence. The band intensity of the indicated proteins was quantified by densitometric scanning using a Kodak 1D Digital Science Imaging System.
Surgical Procedures for Infarct Induction
Rats were administered butorphanol (10 mg/kg sc) and atropine (0.04 mg/kg sc) 10 min before being placed in an induction chamber and exposed to 100% O2-4% isoflurane. The anesthetized rat was then intubated with a 14-g Venacath and ventilated with a Harvard Rodent Ventilator at a tidal volume and frequency of 2.6 ml and 74 breaths/min, respectively, with anesthesia being maintained with 0.5–1.5% isoflurane (oxygen flow of 700 ml/min). The heart was exposed via a left thoracotomy in the fourth intercostal space. The left coronary artery was ligated 1–2 mm ventral to the left atrial margin with a 6-0 Prolene suture. Lungs were hyperinflated with 10 ml of oxygen, the chest was closed with a 2-0 catgut suture, and lidocaine (1%, 0.2 ml) was injected into the surgical site. Muscle and skin were closed in layers with a 4-0 Vicryl suture. Immediately after surgery, rats were given buprenorphine (BP, 0.03 mg/kg sc) for pain and normal saline (10 ml sc) for volume replacement. BP was administered twice more for pain at 12-h intervals. This animal protocol was approved by the Institutional Animal Care and Use Committee of the Northeastern Ohio Universities College of Medicine.
Tissue Procurement, Histology, Immunohistochemistry, and Immunoblotting
Animals were euthanized at the indicated time points after infarction. The atria were excised, and the ventricular heart tissue was rinsed twice in ice-cold PBS, fixed in 4% paraformaldehyde for 30 min on ice, rinsed again, incubated in 7% sucrose at 4°C for a minimum of 4 h, and frozen in Tissue-Tek tissue-freezing medium (Miles; Elkhart, IN). Transverse sections were cut, including both the infarcted and noninfarcted (taken from the anterior wall adjacent to the infarct) zones of the heart at a thickness of 10 µm on a Leica cryostat and placed on albumin-coated slides.
Histology. Trichrome staining was carried out according to Masson's trichrome method (27) and was utilized to examine tissue morphology and composition by staining cell cytoplasm red, cell nuclei purple, and collagen (nonspecific) blue.
Immunohistochemistry.
Slides for immunostaining were blocked in 2% goat serum for 1 h at room temperature, incubated in the primary antibody (type VI collagen, rabbit anti-collagen VI at a 1:100 dilution; obtained from Research Diagnostics); mouse anti--SMA at 1:400 (Sigma-Aldrich); and rabbit anti-desmin at 1:100 [a gift from Dr. Carol Moncman (31)] for 1–2 h at room temperature and then washed extensively for 1 h. Slides were then incubated with secondary antibodies, either Alexa Fluor 488 (green) or Alexa Fluor 568 (red), were incubated at a dilution of 1:200 for 1 h at room temperature, were washed four times in PBS-Tween, and were mounted by using Vectashield Mounting Medium (Vector), containing DAPI for nuclear visualization.
Western blot analysis.
Heart tissue was rinsed twice in ice-cold PBS, and the infarct region was separated from the noninfarcted region. Tissue samples were minced, placed in lysis buffer, and incubated on ice for 15 min before homogenization with a polytron. This process of homogenization was repeated two more times. Samples were centrifuged at 4,500 g for 10 min, and the supernatant was removed and frozen at –20°C. Sample protein levels were quantified by the BCA method, and equal amounts of proteins (15 µg) were loaded onto a 7.5% (for type VI collagen) or 10% (for -SMA) acrylamide gel. Western blot analysis was carried out as described above.
Data Analysis
Statistical significance across treatment groups was determined by a one-way ANOVA using GraphPad Prism (GraphPad Software; San Diego, CA) statistical analysis software.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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CFs were plated on glass slides or tissue culture plates coated with type I, III, or VI collagen, and myofibroblast differentiation (as assessed by -SMA protein expression and stress fiber formation) was examined via immunofluorescence. CFs plated on the type I collagen substrate (Fig. 1A) displayed low levels of -SMA immunostaining that was similar to the level seen in CFs plated on glass (not shown). The type III collagen substrate caused a slight induction of -SMA expression (Fig. 1B). However, the cells plated on the type VI collagen substrate exhibited a marked elevation of -SMA and highly organized stress fibers (Fig. 1C). The key characteristic of mature myofibroblasts is the organization of -SMA into stress fibers (37), which was most evident on the type VI collagen substrate. ANG II induced a slight increase in -SMA expression in CFs plated on the type I and III collagen substrates compared with the substrates alone (Fig. 1, D and E). However, ANG II treatment did not further induce -SMA expression in CFs over that seen with the type VI collagen substrate alone (Fig. 1F), indicating that type VI by itself maximally induced the in vitro differentiation of CFs to myofibroblasts. The above results were confirmed via Western blot analysis for -SMA. Treatment with ANG II increased expression of -SMA on type I collagen by 7% (over type I collagen alone) and 82% on type III collagen (over type III collagen alone); however, treatment with ANG II reduced -SMA expression on type VI collagen by 18% (vs. type VI collagen alone).
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Assessment of proliferation was carried out via BrdU incorporation in fibroblasts plated on type I, III, or VI collagen substrates. Administration of ANG II increased proliferation by 157.3 ± 8.6% of basal levels (Fig. 2A) after 24 h. The types I and III collagen substrates alone markedly increased proliferation of CFs by 240.7 ± 10.3% and 271.7 ± 21.8% of basal levels, respectively. The type VI collagen substrate induced proliferation to a level similar to that of ANG II (143.3 ± 13.6% of basal) but not to the extent stimulated by the types I and III substrates. Stimulation by ANG II enhanced CF proliferation on the types I and III substrates in an additive manner but failed to enhance mitogensis on the type VI substrate. Because ERK1/2 is a major mitogenic signal in CFs, we measured ERK1/2 activation in response to the collagen substrates. Collagen I alone significantly increased ERK1/2 activity 5.7 ± 2.4-fold over control (P < 0.05; Fig. 2, B and D), whereas types III and VI collagen caused little or no change in ERK phosphorylation. We next assessed ANG II-induced ERK activation on each collagen substrate (Fig. 2, C and E). After 10 min of treatment with ANG II, ERK activation was enhanced in CFs plated on types I and III collagen substrates by 1.3 ± 0.2- and 1.3 ± 0.3-fold, respectively, over ANG II alone. In contrast, ANG II did not induce ERK phosphorylation over control when CFs were plated on type VI collagen, indicating that type VI collagen in combination with ANG II does not induce proliferation via ERK activation.
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We next aimed to confirm that isolated CFs are an inducible source of type VI collagen in vitro. Unstimulated CFs produced type VI collagen (Fig. 3A), and ANG II treatment induced an elevation of type VI protein expression (Fig. 3B), as indicated by Alexa Fluor 488 fluorescence. Western blot analysis confirmed that type VI collagen protein was expressed by unstimulated CFs (Fig. 3C, lanes 1 and 2), and production was enhanced by stimulation with ANG II by 3.0 ± 0.4-fold (Fig. 3C, lanes 3 and 4). Western blot assessment for total ERK served to demonstrate equal loading of protein samples (Fig. 3D). Thus CFs produce significant amounts of hormonally inducible type VI collagen.
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To determine the morphology and collagen content of the myocardium after a 20-wk ligation of the left anterior descending coronary artery, the infarcted heart was separated into noninfarcted and infarcted regions, and trichrome staining was carried out on 10-µ-thick transverse cryosections. The noninfarcted region of the ligated hearts exhibited an organized staining pattern of muscle tissue with little evidence of collagen deposition; conversely, trichrome staining in the infarcted region indicated a disrupted network of cells, along with significant increases in total collagen (data not shown). The infarcted region appeared to have some dead or dying cells, an expected result due to the extent of the damage that was incurred in this area. These data confirm successful induction of fibrosis via coronary ligation and prompted us to investigate the specific collagen subtypes involved in pathological remodeling.
We next quantitated the region-specific type VI collagen expression in the infarction model. The age-matched control hearts (Fig. 4, A and D, lane 1) contained low, but observable, levels of type VI collagen. Immunohistochemisty revealed a modest increase in type VI collagen in the noninfarcted region (Fig. 4B) and a marked elevation in the infarcted region (Fig. 4C). Quantitative Western blot analysis determined these increases in type VI collagen to be 4.5-fold and 17-fold over control for the noninfarcted (Fig. 4D, lane 2) and infarcted region (Fig. 4D, lane 3) of this heart, respectively. Thus the remodeling that occurs post-MI leads to interstitial fibrosis and to an elevation of type VI collagen throughout the myocardium, with the highest levels being evident in the infarcted region.
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Our next goal was to determine whether myofibroblast content increased concurrently with type VI collagen in the infarcted rat myocardium. -SMA staining was observed in the endomysium of the noninfarcted tissue region (Fig. 5A). Both the staining intensity and pattern were altered in the infarcted region (Fig. 5B); a more punctate cellular staining pattern, which was of the approximate size of fibroblasts and myofibroblasts, was evident throughout the infarcted tissue. To rule out the possibility that the increase -SMA expression in the infarcted myocardium was due to the presence of VSMCs, we measured levels of the intermediate filament desmin (Fig. 5C), which is expressed in VSMCs but not myofibroblasts (39). Figure 5, B and C, contains the same field of tissue and demonstrates an elevation of -SMA in the infarcted region (Fig. 6B), whereas only a minimal amount of desmin staining was evident (Fig. 5C). Coexpression of -SMA and desmin in VSMCs in a blood vessel is presented in Fig. 5, D and E (arrows), confirming the reliability of the antibodies. Western blot analysis validated the immunostaining results: the age-matched control heart (Fig. 5H, lanes 1 and 2) exhibited low levels of -SMA expression, whereas both the noninfarcted (Fig. 5H, lanes 3 and 4) and infarcted regions (Fig. 5H, lanes 5 and 6) contained elevated -SMA levels (1.4 ± 0.2- and 2.7 ± 0.2-fold over control, respectively). In addition, Fig. 5I demonstrates that expression of desmin did not vary across the indicated conditions. Overall, myofibroblast content was highest in the infarcted myocardium, and our data demonstrate that these hypersecretory cells remain in the injured myocardium for at least 20-wk post-infarction.
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DISCUSSION |
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Our studies demonstrated that the type VI collagen substrate was a potent inducer of myofibroblast differentiation in vitro, whereas the types I and III collagen substrates alone caused significant CF mitogenesis. Treatment of cells plated on each collagen substrate in combination with ANG II caused a modest additive effect (over substrate alone) in the case of types I and III collagen, an effect that was not apparent in the type VI collagen substrate group. The differential effects that these collagen types have on CF proliferation and differentiation are supported by the idea that at any given time most cells are capable of either proliferation or differentiation (but not both), a fate that depends on the regulatory factors present (18). Thus it is important to note that in our study, types I and III collagen, the most abundant types in the heart, appear to drive proliferation of undifferentiated CFs and are poor inducers of myofibroblast differentiation. On the other hand, type VI collagen augments differentiation to a greater extent than cell division. To gain a better understanding of the signaling mechanism responsible for proliferation, we examined ERK1/2 activation, which has been shown to be a critical signal underlying hormone-stimulated CF proliferation (22, 33, 35). The type VI collagen substrate both alone and in combination with ANG II was a poor activator of ERK1/2, which is consistent with our observations, indicating that type VI collagen preferentially induces myofibroblast differentiation over proliferation.
The various factors that control transformation of CFs to myofibroblasts are not fully understood. However, specific hormones have been shown to induce fibroblast differentiation, and inhibiting the actions of these hormones has proven to be effective in limiting fibrosis. In pressure-overloaded rats, inhibition of transforming growth factor (TGF)-
with neutralizing antibodies resulted in decreased types I and III collagen mRNA transcription, myofibroblast number, and myocardial fibrosis (25). Their study demonstrated that anti-TGF- antibodies inhibited both proliferation and differentiation of CFs and abolished the increase in left ventricular end-diastolic pressure and the ratio of early-to-late filling velocity, both of which are direct measures of diastolic cardiac function. In addition to hormonal factors, our data demonstrate that specific ECM proteins may play critical roles in promoting myofibroblast differentiation. Therefore, targeting ECM-induced signal transduction may be an alternative approach to effectively reduce cardiac fibrosis by preventing excessive myofibroblast activity and/or differentiation.
Several studies have been performed in noncardiac cells that provided insight into how myofibroblast differentiation occurs. Differentiation of corneal fibroblasts to myofibroblasts is induced by the type VI collagen substrate, which appears to be dependent on the 1-integrin receptor (K. J. Doane, unpublished observations). In addition, the type VI collagen substrate also reduces apoptosis in these cells (21). Faouzi et al. (14) presented evidence of significant elevations of type VI and IV collagen in both hepatic carcinoma tissue and the cells isolated from these tumors. In their study, the rise in both collagen isotypes was accompanied by an increase of myofibroblasts rather than of endothelial cells, although there was no discussion or speculation as to a potential causative link between type VI collagen and myofibroblast differentiation. A coexistence of type VI collagen and myofibroblasts was also reported after renal injury. Expression of type VI collagen was demonstrated to be significantly elevated in diabetic glomeruli, as well as in areas of renal fibrotic injury (17). Increased amounts of -SMA-expressing cells were evident in renal fibrotic interstitium, along with elevated type VI collagen. Given the above studies, the coexistence of type VI collagen and myofibroblasts suggests a potential link between this specific collagen type and the process of myofibroblast differentiation.
The mechanism by which type VI collagen induces myofibroblast differentiation has not been currently established; however, there are several possibilities that require further investigation. Integrin receptors, which are heterodimers composed of one - and one -subunit, are responsible for mediating intracellular signaling in response to many types of collagen, as well as other ECM proteins. Specifically, 31 and the integral membrane proteoglycan NG2 on corneal fibroblasts have been identified as receptors that associate with type VI collagen during development (10). More recently, a study by Bouzeghrane et al. (3) demonstrated upregulation of 81 in myofibroblasts present in the fibrotic myocardium. In addition, evidence exists showing that the v-subunit (more specifically v3 and v5) is critically involved in myofibroblast differentiation in fibroblast cell lines (28). Thus several avenues exist to explore the mechanism by which type VI collagen induces cardiac myofibroblast differentiation and requires further study.
Evidence from two clinical studies indicates that type VI collagen is elevated during hypertension, heart failure, and diabetes, conditions in which cardiac fibrosis is prevalent and accelerated (23, 34). However, the possibility that type VI collagen might actually drive the fibrotic process was not considered. We postulated that diseases that increase type VI collagen deposition create conditions favorable for myofibroblast differentiation and that these differentiated cells are the major players in both normal and pathological remodeling. In the current study, we demonstrate that type VI collagen expression is elevated during post-MI remodeling, which is accompanied by an increase in myofibroblast content in the damaged myocardium. Our data support a novel role for type VI collagen in post-MI remodeling by augmentation of myofibroblast differentiation. Understanding the mechanisms by which type VI collagen enhances myofibroblast differentiation may provide a basis to limit excessive myofibroblast activity and pathological fibrosis following MI.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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P < 0.01 vs. type I collagen substrate alone. B and C: CFs were plated on either type I, III, or VI collagen substrates or tissue culture (TC) plates. Cells were seeded in 10% serum for 24 h and then incubated without serum for another 24 h. Where indicated, CFs were stimulated with 100 nM ANG II for 10 min, and whole cell protein lysates were collected and subjected to SDS-PAGE and Western blot analysis. Blots were probed for phospho (P)-ERK1/2 and stripped and reprobed for total ERK1/2 as protein-loading control. Blots are representative of either 3 (B) or 2 (C) similar experiments. D: densitometric analysis was performed on blot from B and normalized to total ERK. Data are expressed as mean fold change ± SE over controls. *P < 0.05 vs. control by 1-way ANOVA. E: densitometric analysis was carried out on blot from C and normalized to total ERK with data expressed as mean fold change ± SE relative to ANGII.
