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Am J Physiol Heart Circ Physiol 291: H2924-H2932, 2006. First published August 4, 2006; doi:10.1152/ajpheart.00153.2006
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Cardiac myofibroblasts differentiated in 3D culture exhibit distinct changes in collagen I production, processing, and matrix deposition

Felicitta Poobalarahi, Catalin F. Baicu, and Amy D. Bradshaw

Division of Cardiology, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina

Submitted 9 February 2006 ; accepted in final form 1 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Myofibroblasts are a differentiated fibroblast cell type characterized by increased contractile capacity and elevated production of extracellular matrix (ECM) proteins. In the heart, myofibroblast expression is implicated in fibrosis associated with pressure-overload hypertrophy, among other pathologies. Although enhanced expression of ECM proteins by myofibroblasts is established, few studies have addressed the nature of the ECM deposited by myofibroblasts. To characterize ECM production and assembly by cardiac myofibroblasts, we developed a three-dimensional (3D) culture system using primary cardiac fibroblasts seeded into a nylon mesh that allows us to reversibly interconvert between myofibroblast and fibroblast phenotypes. We report that an increase in collagen I production by myofibroblasts was accompanied by a significant increase in collagen deposition into insoluble ECM. Furthermore, myofibroblasts exhibited increased levels of procollagen {alpha}1(I) with C-propeptide attached (and N-propeptide removed) relative to procollagen {alpha}1(I) compared with fibroblast cultures. An increase in production of the myofibroblast-associated splice variant of fibronectin (EDA-Fn) was seen in myofibroblast 3D cultures. Because the regulation of procollagen I processing is known to have profound effects on ECM assembly, differences in procollagen I secretion and maturation coupled with expression of EDA-Fn are shown to contribute to the production of a distinct ECM by the cardiac myofibroblast.

extracellular matrix; fibroblast; fibronectin; fibrosis

MYOFIBROBLASTS ARE A SPECIALIZED fibroblastic cell type characterized by high levels of expression of extracellular matrix (ECM) proteins and by a potent contractile capacity (14). Changes in gene expression that occur on differentiation of fibroblasts to myofibroblasts influence the cytoskeleton as well as ECM and include nascent production of {alpha}-smooth muscle actin ({alpha}-SMA), production of a splice variant of fibronectin (EDA-Fn), and increased expression of fibrillar collagen type I (31, 32). Given the increased expression of ECM proteins by these cells, the persistence of myofibroblast expression observed in many fibrotic tissues is predicted to contribute to fibrosis and hypertrophic scarring (6, 14).

Although myofibroblasts are not associated with myocardium in normal adult ventricles, myofibroblasts are detected after myocardial infarction (MI) and pressure-overload hypertrophy (6, 21). Myofibroblasts are observed as early as 4 days after MI, and persistent myofibroblast expression is found associated with infarct scars years after MI (19). Accordingly, myofibroblasts are thought to play a critical role in production of ECM in the myocardium in response to injury. Transforming growth factor (TGF)-beta1 both promotes ECM synthesis and induces conversion of myofibroblasts in vitro (14). Kuwahara et al. (19) showed that function-blocking antibodies to TGF-beta1 administered to pressure-overloaded rats prevented myofibroblast conversion in cardiac interstitium and subsequent increases in mRNA encoding collagen I. Whereas diastolic dysfunction was observed in pressure-overloaded rats treated with control antibodies, in pressure-overloaded rats receiving neutralizing TGF-beta1 antibodies diastolic function was comparable to that of sham-operated rats (19). Hence, myofibroblast expression is implicated in the overdeposition of fibrillar collagen proposed to contribute to some forms of diastolic heart failure (38).

In vitro, cardiac fibroblasts display remarkable sensitivity to myofibroblast conversion (39). Culture conditions such as serum, density, and substrata can influence their differentiated state (6). Rat cardiac fibroblasts plated on tissue culture plates spontaneously converted to myofibroblasts within 48 h (39). Presumably, force generation by cardiac fibroblasts plated on a rigid substrate induced myofibroblast [{alpha}-SMA-positive (+)] conversion, because fibroblasts plated on a nonrigid substrate, collagen-coated agar, remained {alpha}-SMA negative (–) (38). Force applied to collagen-coated magnetite beads bound to cardiac fibroblast cell surfaces induced expression of {alpha}-SMA and coincident conversion to myofibroblasts. When similar forces were applied to myofibroblasts, reversion to {alpha}-SMA(–) fibroblasts occurred (38, 39). Thus the differentiated state of the cardiac fibroblast or myofibroblast determines subsequent responses to mechanical stimuli.

Although increased gene expression of ECM proteins by myofibroblasts is well documented, much less is known regarding changes in ECM assembly and deposition orchestrated by myofibroblasts. Procollagen I is processed to mature collagen I by removal of N- and C-propeptides in the extracellular space (29). A disintegrin and metalloprotease with thrombospondin type I repeats (ADAMTS)-2, -3, and -14 can act as N-propeptide proteases, whereas bone morphogenetic protein (BMP)-1 [and splice-variant mammalian tolloid (mTld) and related family members mTld like (mTLL) 1 and 2] cleaves the C-propeptide (10, 17, 27). Spatial and temporal regulation of procollagen processing is considered a key step in the regulation of collagen fibrillogenesis (29). For example, retention of the N-propeptide after fibril incorporation has been proposed to both limit and enhance fibril expansion (29, 41). Hence, regulation of processing of procollagen I has the capacity to influence collagen I assembly into ECM.

Characterization of interstitial ECM construction in a three-dimensional (3D) environment better represents tissuelike conditions versus those of two-dimensional (2D) cultures (11). We have found that plating of cardiac fibroblasts in a 3D scaffold resulted in conversion to an {alpha}-SMA(+) myofibroblast phenotype. Subsequent application of a single static mechanical stretch induced conversion to {alpha}-SMA(–) fibroblasts. Hence, we present the first analysis of production and incorporation of collagen I into a distinct ECM synthesized by cardiac myofibroblasts.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cardiac cell culture in 3D scaffolds. Cardiac fibroblasts were isolated from the hearts of adult cats. The procedure for isolation of cardiac fibroblasts was approved by the Medical University of South Carolina Institutional Animal Care and Use Committee. Fibroblasts were routinely collected in a preplating step of weekly cardiac myocyte isolations as described previously (37). Cells were passaged one time before plating in 3D cultures. Cardiac fibroblast cultures were >90% {alpha}-SMA(–), vimentin(+), and desmin(–) at the time of plating. Skin fibroblasts were isolated according to the procedure described in Ref. 5 and cultured under the same conditions as cardiac fibroblasts.

3DTC (Orion Biosolutions, Vista, CA) consists of a nylon weave on a silicone backing (11). Cardiac fibroblasts were seeded in 3DTC in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO-BRL, Grand Island, NY), 10% fetal calf serum (FCS; Hyclone, Logan, UT) and antibiotics-antimycotic (GIBCO-BRL) at 2 x 106 cells/well in rectangular four-well plates containing 3DTC (~2 cm x 6 cm). After 2 days in culture, the medium was changed to growth medium (DMEM, 1% FCS, antibiotics-antimycotic, and 50 µg/ml ascorbic acid to enhance collagen production). Cells were maintained in 3DTC for ~2–3 wk before stretching. Medium was changed every 3–4 days.

Twenty-four-hour conditioned medium (without ascorbate) was collected from each well 1 day before stretching. Stretching of the scaffold was achieved by removing the mesh containing the cardiac cells to a fresh plate in which two sets of three sterile magnets were used to tether the scaffold in place. One magnet of each set was placed on top of the mesh; the mesh was then wrapped around and held in place by a second magnet. The third magnet was placed underneath the plate. A static stretch was applied by moving one set of magnets the appropriate distance to achieve a 30% stretch of each scaffold. Growth medium containing ascorbic acid was then added to each culture. For each scaffold that underwent mechanical stretch, a parallel unstretched culture was treated in an identical manner with the exception that stretch was not applied. Stretch experiments were performed for designated periods of time (24 h to 1 wk). On conclusion, cells within the mesh were harvested by excising the mesh between the two sets of magnets such that only cells and ECM within the mesh subjected to stretch were included in the analysis. All experiments included at least two stretched and two unstretched cultures in parallel; the majority included four stretched and four unstretched pairs.

Immunohistochemistry. Cells and ECM within stretched and unstretched scaffolds were fixed in 4% paraformaldehyde. Meshes were incubated in 0.5% Triton X-100 and 1% normal goat serum and stained with anti-{alpha}-SMA antibodies conjugated to fluorescein (Sigma, St. Louis, MO) or anti-actin (Sigma) primary antibodies. Appropriate secondary antibodies conjugated to fluorescein (Jackson Immunoresearch, West Grove, PA) were used when needed. Cells and ECM were viewed on an Olympus IX71 microscope equipped for epifluorescence, and images were captured with a Hamamatsu digital camera with accompanying Slidebook software.

Immunoblot analysis. At appropriate time points, conditioned medium and detergent-solubilized cell layers were collected. Excised mesh from stretched scaffolds and corresponding equal areas from unstretched cultures were placed in 300 µl of 1% deoxycholate with protease inhibitors (Roche, Indianapolis, IN) and tumbled overnight at 4°C. The mesh was then removed for further extraction in SDS-PAGE buffer or for collagenase analysis (see below). Conditioned medium, soluble proteins in the deoxycholate-extracted cell layer, and in some cases proteins extracted with SDS-PAGE buffer (detergent insoluble) were separated by SDS-PAGE on 3–8% gradient gels under reducing conditions and analyzed by Western blot as described previously (5). Primary antibodies used were anti-collagen I (MD Biosciences, Zurich, Switzerland), LF-41 (a kind gift of Larry Fisher, National Institutes of Health, Bethesda, MD; Ref. 13), EDA-Fn (IST-9, Abcam; Ref. 22), and {alpha}-SMA (Sigma).

Metabolic labeling and collagenase digestion of insoluble ECM. [3H]thymidine incorporation of cells in stretched versus unstretched cultures was performed as described in Ref. 4. Scaffolds were incubated in growth medium containing 25 µCi/ml [3H]proline (2,3,4,5-proline, Perkin Elmer, Wellesley, MA) and 1 mM proline for 24 h. Conditioned medium and cell layers were collected as described above. For collagenase assays, detergent-extracted scaffolds were cut in half; one half was incubated for 90 min in 15 µg/ml high-quality bacterial collagenase (CLSPA, Worthington, Lakewood, NJ) and the other half was incubated in buffer only as control. Metabolically labeled collagens specifically released by collagenase from the mesh were quantified by scintillation counting. The mesh was then rinsed three times in PBS and placed in 6 N HCl at 120°C for 1 h. Collagenase-resistant material was then quantified by scintillation counting.

TGF-beta quantification. Mink lung epithelial cells (MLECs) stably transfected with a plasmid encoding luciferase driven by the plasminogen activator inhibitor-1 promoter were maintained in growth medium containing geneticin (G418; Invitrogen, Carlsbad, CA) as described previously (1). MLECs were plated at 1 x 105 cells/well in 24-well plates in DMEM containing 10% FCS + G418. The following day the medium was replaced with DMEM without FCS for 18 h. Amounts of active TGF-beta in stretched versus unstretched cultures conditioned over 24 h in serum-free medium were assessed by exposing MLECs to conditioned medium concentrated twofold in a Centricon-10. Equal amounts of concentrated serum-free medium not exposed to cells were assessed as control. Total TGF-beta was determined by heating concentrated conditioned and control media for 8 min at 80°C. MLECs were incubated with concentrated conditioned medium for 18 h, and cell layers were harvested in luciferase assay buffer (Promega, Madison, WI) according to the manufacturer’s instructions. Luciferase units were quantified with luciferase assay substrate (Promega) and an Lmax II384 luminometer (Molecular Devices, Sunnyvale, CA). Results were analyzed with the SoftMax Pro software program (Molecular Devices).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cardiac fibroblasts convert to myofibroblasts in 3D culture. Cardiac fibroblasts isolated from adult cats were cultured in 3DTC, a nylon mesh scaffold with a silicone backing. This mesh has been used extensively to culture dermal fibroblasts and has been used as a scaffold to generate bioengineered products such as those used in dermal wound healing (11). As shown in Fig. 1, feline cardiac (Fig. 1, A and C) and dermal (Fig. 1, B and D) fibroblasts populated the mesh after 3 wk of culture. In contrast to skin fibroblasts, however, cardiac fibroblasts displayed positive reactivity for {alpha}-SMA (Fig. 1, C and D), which indicated that cardiac fibroblasts converted to a myofibroblast phenotype in 3DTC.


Figure 1
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  		Fig. 1. Primary cells from heart (A and C) and skin (B and D) were grown in 3DTC for 2 wk. Whereas cells from heart (C) exhibited {alpha}-smooth muscle actin ({alpha}-SMA)-positive cytoskeletal elements indicative of myofibroblasts, skin fibroblasts (D) did not. Cardiac fibroblasts grown for 2 wk and then stretched for 1 wk (F) underwent myofibroblast to fibroblast conversion, as evidenced by the substantial loss in {alpha}-SMA-positive cytoskeleton vs. unstretched cultures (E).

 
Our intent was to establish a 3D in vitro culture system to investigate differences in mechanism(s) utilized by cardiac myofibroblasts to construct a collagenous ECM different from that of cardiac fibroblasts. We reasoned, given past reports, that a single static stretch applied to the 3DTC cultures might induce myofibroblasts to revert to fibroblasts (38, 39). As shown in Fig. 1, unstretched cultures (Fig. 1E) were populated with {alpha}-SMA(+) myofibroblasts whereas cultures subjected to a 30% static stretch (Fig. 1F) contained predominantly {alpha}-SMA(–) fibroblasts. Immunoblot analysis of detergent-soluble cell layers from 1-wk stretch experiments also revealed a reduction in {alpha}-SMA immunoreactivity associated with stretched versus unstretched cultures (data not shown).

Coincident with myofibroblast to fibroblast conversion, incorporation of [3H]thymidine was significantly increased in fibroblast cultures. Shown in Fig. 2A are representative results from cardiac cultures grown for ~3 wk in 3DTC, subjected to stretch (fibroblasts) or left unstretched (myofibroblasts) for 1 wk, and incubated for 24 h in the presence of [3H]thymidine. Interestingly, metabolic activity as measured by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (25) was not significantly different in the two culture conditions after 24 h (unstretched 0.47 ± 0.2, stretched 0.56 ± 0.2 absorbance/cm2) or after 1 wk (unstretched 0.773 ± 0.3, stretched 0.713 ± 0.2 absorbance/cm2) of stretch. We conclude that on conversion of myofibroblasts to fibroblasts cell proliferation was enhanced; however, overall metabolic activity and cell viability were not substantially altered in the two culture conditions.


Figure 2
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  		Fig. 2. Decreased cell proliferation and increased protein secretion in myofibroblast cultures. A: cardiac fibroblasts grown for 2 wk and subjected to stretch (open bar) or left unstretched (filled bar) for 1 wk were monitored for [3H]thymidine incorporation over an 18-h culture period. Fibroblasts (stretched) displayed increased incorporation of [3H]thymidine vs. myofibroblast (unstretched) cultures. Similar results were found with 3 separate preparations of primary cell isolates. cpm, Counts per minute. Error bars indicate SE. P < 0.0005. B: nascent production of secreted proteins is substantially decreased in fibroblast vs. myofibroblast cultures. Primary fibroblasts were cultured for 2.5 wk and subjected to stretch or left unstretched for 24 h (lanes 1 and 2) or 1 wk (lanes 3 and 4). Autoradiography of [3H]proline-labeled proteins from conditioned media of fibroblast (lanes 1 and 3) and myofibroblast (lanes 2 and 4) cultures is shown. Fn, fibronectin; Pro, procollagen I; {alpha}1(I), {alpha}2(I), collagen {alpha}1(I) and {alpha}2(I). C: Western blots of detergent-soluble cell layers from 2 separate experiments for the myofibroblast-specific splice variant of fibronectin (EDA-Fn) showed that myofibroblasts (lanes 2 and 4) produced greater amounts of EDA-Fn (arrow) than fibroblasts (lanes 1 and 3). Bottom: actin levels in detergent-soluble extracts shown at top.

 
In fact, although myofibroblast cultures exhibited reduced incorporation of [3H]thymidine, robust production of collagen I by these cells was observed. As shown in Fig. 2B, incorporation of [3H]proline into bands comigrating with collagen {alpha}1(I) and {alpha}2(I), the mature subunits of collagen I, was significantly increased in myofibroblast (lane 2) versus fibroblast (lane 1) conditioned media after 24 h of stretch. Cultures subjected to static stretch for up to 1 wk displayed similar decreases in amounts of labeled collagen I in medium conditioned for 24 h (Fig. 2B; 1 wk: lane 3, fibroblasts, lane 4, myofibroblasts). Our interpretation is that a single static stretch gave rise to a stable population of fibroblasts from myofibroblasts that exhibit increases in proliferation at the expense of collagen production.

In addition to production of more mature collagen I, increased levels of nascent procollagen I (the precursor form of collagen I) were noted in medium conditioned by myofibroblast (Fig. 2B, lanes 2 and 4) versus fibroblast (lanes 1 and 3) cultures at 24 h and 1 wk of stretch. A high-molecular-weight band predicted to be fibronectin (Fig. 2B) was also notably increased in myofibroblast cultures.

EDA-Fn is a splice variant of fibronectin shown previously to be expressed by myofibroblasts (3). As shown in a representative immunoblot in Fig. 2C, myofibroblast cultures (lanes 2 and 4) displayed elevated levels of EDA-Fn associated with detergent-soluble cell layers compared with fibroblast cultures (lanes 1 and 3) at 24 h. Interestingly, very little EDA-Fn was detectable in conditioned media, which suggested preferential association of this splice variant with myofibroblast cell surfaces (data not shown). We conclude that conversion from cardiac myofibroblasts to fibroblasts leads to increased ECM production accompanied by changes in levels of specific ECM components.

Production of collagen I. Ascorbate (vitamin C) is a cofactor for enzymes in the secretory pathway that perform posttranslational modifications of collagen I. Hence, addition of ascorbate enhances collagen secretion in collagen-producing cells. In response to addition of ascorbate in the absence of stretch (Fig. 3A, lanes 4 and 8), cardiac myofibroblasts in 3DTC displayed higher levels of collagen in 24-h conditioned media versus the previous 24-h period (compare lanes 3 and 4 and lanes 7 and 8). In contrast, cultures treated with ascorbate and subjected to mechanical stretch for 24 h did not exhibit increased collagen production (compare Fig. 3A, lanes 1 and 2 and lanes 5 and 6). In addition, the conditioned medium from stretched cultures contained smaller-molecular-weight bands immunoreactive for collagen I (Fig. 3A) that suggested that increased degradation of collagen I was associated with fibroblast conversion. Western blot analysis of conditioned medium confirmed a sustained reduction in collagen I production by cultures stretched for 1 wk [Fig. 3B; fibroblasts: lanes 1 (prestretch) and 2 (poststretch + ascorbate); lanes 3 (pre-unstretch) and 4 (post-unstretch + ascorbate)].


Figure 3
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  		Fig. 3. Collagen I production by cardiac fibroblasts in 3DTC. A: representative Western blots from 2 experiments using anti-collagen {alpha}1(I) antibodies demonstrate that levels of procollagen {alpha}1(I) and processed forms of collagen {alpha}1(I) were increased in the media of myofibroblast cultures in response to ascorbate addition (lanes 3 and 7, media without ascorbate collected 24 h before experiment; lanes 4 and 8, media conditioned for 24 h after addition of ascorbate) compared with fibroblast cultures (lanes 1 and 5, media without ascorbate collected 24 h before experiment; lanes 2 and 6: media conditioned for 24 h after addition of ascorbate in cultures stretched for 24 h). B: retention of fibroblast ECM phenotype after 1 wk of stretch. Western blot analysis performed with anti-collagen I antibodies demonstrated that before stretch (lanes 1 and 3) cultures produced similar amounts of collagen I. After 1 wk of stretch, addition of ascorbate resulted in increased amounts of collagen I produced by myofibroblasts (lane 4), whereas amounts of collagen I in fibroblast conditioned media (lane 2) were not significantly enhanced by ascorbate. Detergent-soluble cell layers displayed an increase in amounts of collagen I associated with myofibroblasts (lane 6) vs. fibroblasts (lane 5). Actin levels in detergent-soluble extracts are shown. procol {alpha}1(I), procollagen {alpha}1(I); pC, intermediate of procollagen {alpha}1(I) with the C-terminal propeptide; pN, intermediate with the N-terminal propeptide retained.

 
Quantification of immunoblot experiments from three separate cell isolations is shown in Fig. 4. The percent change in amounts of collagen I present in medium (conditioned for 24 h) in response to ascorbate was consistently greater in unstretched myofibroblast cultures versus stretched fibroblasts. The percent change was similar whether the cultures were stretched 24 h or 1 wk.


Figure 4
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  		Fig. 4. Quantification of amounts of collagen {alpha}1(I) in conditioned media was determined from the density of scanned bands taken from 8 separate experiments at 24-h duration and 6 separate experiments at 1-wk duration, each performed with 3 different cell isolates. A significant decrease in the amount of collagen {alpha}1(I) produced by fibroblasts (stretched, open bars) vs. myofibroblasts (unstretched, filled bars) was observed in each case. Error bars indicate SE. P < 0.05 for 24 h and P < 0.002 for 1 wk.

 
The detergent-soluble cell layer is predicted to contain intracellular proteins and proteins associated with the cell surface but not yet incorporated into detergent-insoluble ECM. A substantial reduction in collagen I associated with detergent-soluble cell layers from stretched (Fig. 3B, lane 5) versus unstretched (Fig. 3B, lane 6) cells was apparent in 1-wk stretch experiments. Although some procollagen I in these samples was most likely from intracellular collagen, we attributed a significant contribution to cell surface-associated collagen for the following reasons: 1) fibroblast cultures continued to synthesize low levels of collagen I as evidenced by metabolically labeled collagens in the medium (Fig. 2B, lane 3), and 2) similar differences in the levels of nascent protein were present in the medium after 24 h and 1 wk of stretch (vs. respective unstretched cultures), indicating a sustained low-level production of collagen I by fibroblasts. We conclude that myofibroblasts exhibited a disproportionate amount of pericellular-associated collagen I versus fibroblasts.

Procollagen processing. Western blot analysis of detergent-soluble cell layers using antibodies against the C-propeptide of collagen I (LF-41; Ref. 13) revealed that myofibroblasts (Fig. 5, lanes 2 and 4) had a higher percentage of total collagen present in the pC intermediate form (procollagen with the C-propeptide attached and the N-propeptide removed) than fibroblast cultures (Fig. 5, lanes 1 and 3) after 24 h of stretch. Levels of actin are shown as a control for cellular contribution from each cell layer fraction (Fig. 5, bottom).


Figure 5
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  		Fig. 5. Differences in procollagen processing in myofibroblast cultures. A representative Western blot of detergent-soluble cell layers from fibroblast (lanes 1 and 3) and myofibroblast (lanes 2 and 4) cultures from 2 separate experiments probed with anti-C-propeptide of collagen {alpha}1(I) antibodies is shown. Cultures were stretched for 24 h or left unstretched. The antibody (LF-41) recognizes procollagen I and the pC intermediate (N-propeptide removed). Bottom: actin levels in detergent-soluble extracts shown at top.

 
Quantification of immunoblots using LF-41 to detect levels of pC collagen I in cell layers is shown in Fig. 6A. In experiments in which cultures were stretched for 24 h or 1 wk (or left unstretched), a consistent increase in the amount of collagen I present as pC collagen I was observed in myofibroblast versus fibroblast cultures. By 1 wk, the differences in pC collagen I in cell layers was further increased in myofibroblast cultures. Whereas cells stretched for 24 h displayed differences in procollagen intermediates in cell layers only (Fig. 6A and data not shown), after 1 wk of stretch a persistent increase in processed pC intermediate relative to procollagen I was also detectable in conditioned medium (Fig. 6B). Therefore, over time differences in procollagen maturation initiated at the cell surface become apparent in the medium fraction as well.


Figure 6
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  		Fig. 6. A: quantification of 7 separate experiments (using 3 different cell isolates) revealed a shift from predominantly unprocessed procollagen I in fibroblast (stretched) detergent-soluble cell layers to pC collagen I in myofibroblast (unstretched) cell layers in experiments performed with 24 h or 1 wk of stretch. Density of collagen I bands was quantified, and % contribution of each band to the total density of bands was calculated. Error bars indicate SE. P < 0.005 for respective % of procollagen and pC collagen I in fibroblasts relative to myofibroblast cultures at 24 h; P < 0.05 for 1-wk experiments. B: the differences in proportion of collagen I processing forms were magnified in extracts from conditioned media of fibroblasts (stretched) compared with myofibroblasts (unstretched) in 1-wk stretch experiments. Error bars indicate SE. P < 0.005 for % procollagen in fibroblasts relative to myofibroblasts; P < 0.001 for % pC collagen in fibroblasts relative to myofibroblasts.

 
Interestingly, whereas pC collagen I was the dominant intermediate in conditioned medium from myofibroblasts cultured for ~2 wk (Fig. 3A), pN collagen I [procollagen {alpha}1(I) with the N-propeptide retained and the C-propeptide removed] was more prominent than pC collagen I in conditioned medium of cultures ~3 wk in duration (Fig. 3B).

Collagen deposition. To assess amounts of metabolically labeled ([3H]proline) collagen deposited by cardiac cells grown in 3DTC, detergent-extracted cellular scaffolds were incubated with high-quality bacterial collagenase, an enzyme specific for triple helical collagen. This assay was adapted from a traditional assay used to quantify collagen in 2D cultures and in tissues (Ref. 24; see MATERIALS AND METHODS). Figure 7 demonstrates that myofibroblast cultures contained approximately twofold higher total counts per minute (cpm) per mesh than those of fibroblasts. Similarly, collagenase-sensitive counts were also approximately twofold higher in myofibroblast versus fibroblast cultures.


Figure 7
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  		Fig. 7. Extracellular matrix (ECM) deposition by myofibroblasts (unstretched) was more substantial than that by fibroblasts (stretched) as monitored by labeling with [3H]proline for 24 h before detergent extraction and collagenase digestion with subsequent extraction in 6 N HCl (see MATERIALS AND METHODS). After 1 wk of stretch, fibroblast (open bars) cultures continued to display reduced incorporation of total radiolabeled and collagenase-sensitive proteins produced in 3D cultures vs. that of unstretched myofibroblast (filled bars) cultures. Eight separate experiments contributed to the results. Error bars indicate SE. P < 0.04 for cpm in fibroblast relative to myofibroblast cultures at 24 h; P < 0.01 for collagenase-sensitive cpm in fibroblasts relative to myofibroblasts at 1 wk; P < 0.005 for total detergent-insoluble cpm in fibroblasts relative to myofibroblasts.

 
After 1 wk of stretch, the amount of metabolically labeled protein deposited into detergent-insoluble ECM over a 24-h period continued to be approximately twofold higher in myofibroblast cultures compared with those of fibroblast cultures (Fig. 7). The differences in the amount of cpm released by collagenase from myofibroblast versus fibroblast cultures were also comparable to that observed following 24 h of stretch and suggested that amounts of nascent collagen deposited by the two fibroblast types was similar after 1 day of differentiation versus 1 wk.

In three of four experiments in which cells were grown for 4 wk and stretched for 1 wk or left unstretched, we found a consistent ~27.6% ± 2.6 decrease in collagen content as measured by hydroxyproline analysis associated with stretched fibroblast cultures (25). We conclude that differences in collagen expression and deposition observed over a 24-h period resulted in significant disproportionate accumulation of collagen in the ECM of myofibroblast cultures after 1 wk.

Levels of TGF-beta. TGF-beta is a primary mediator of myofibroblast conversion in vitro. Hence, we sought to determine whether levels of TGF-beta were disproportionately present in myofibroblast versus fibroblast 3D cultures. Surprisingly, we found an increase in total amounts of TGF-beta in the conditioned media of fibroblast versus myofibroblast cultures (Fig. 8). No significant differences in levels of active TGF-beta were found between fibroblast and myofibroblast conditioned media, although a slight trend toward higher levels of active TGF-beta was associated with myofibroblast cultures. We interpret the higher levels of total TGF-beta in fibroblast medium as resulting from increased protease activity coincident with conversion of myofibroblasts to fibroblasts rather than nascent production of TGF-beta. Remodeling of the ECM due to expression of extracellular proteases by fibroblasts is predicted to liberate TGF-beta from ECM stores deposited by myofibroblast cultures before stretching.


Figure 8
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  		Fig. 8. Levels of total transforming growth factor (TGF)-beta were increased in conditioned medium of fibroblast (stretched, open bars) cultures stretched for 24 h vs. that of myofibroblasts (unstretched, filled bars). A slight increase in active TGF-beta was associated with myofibroblast cultures but did not reach significance. Error bars indicate SE.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We report here the first 3D culture system developed to analyze ECM deposition by cardiac myofibroblasts versus fibroblasts. As myofibroblast differentiation is a key element associated with fibrotic deposition of collagen in the heart, evaluation of ECM assembly by this cell type is essential to our understanding of cardiac remodeling. Adult feline cardiac fibroblasts grown in 3DTC scaffold were shown to exhibit a myofibroblastic phenotype based on three criteria: 1) expression of {alpha}-SMA, 2) increased expression of EDA-Fn, a splice variant of fibronectin shown to be expressed by myofibroblasts, and 3) significantly enhanced production of fibrillar collagen I. A single mechanical stretch converted myofibroblast cultures to cultures predominantly populated by {alpha}-SMA(–) fibroblasts that produced substantially less EDA-Fn and significantly less collagen I. The 3D nature of the culture environment and the ease of conversion from myofibroblasts to fibroblasts provided an excellent cellular milieu to characterize the disparate mechanisms used by myofibroblasts to assemble an ECM and to investigate differences in ECM structure and stability. In contrast to collagen gels 3DTC is biologically inert, and hence exogenous collagen does not interfere with ECM analysis.

Myofibroblast expression is not detectable in normal adult hearts (21, 40). On induction of hemodynamic stress by pressure overload-induced hypertrophy, myofibroblasts increase in number (19, 21). Because excessive accumulation of collagen in the cardiac interstitium is associated with pressure-overload hypertrophy, we speculate that myofibroblast conversion and the accompanying increase in collagenous ECM deposition by myofibroblasts contributes to increased amounts of collagen (24, 42). Overdeposition of collagen in the heart has been associated with cardiac dysfunction, particularly in respect to relaxation of the ventricles during diastole (33, 44). Potentially, a distinct ECM synthesized by myofibroblasts could induce changes in integrin-mediated cell signaling, display different susceptibility to collagenase digestion by matrix metalloproteinases, and/or exhibit disparate structural properties. Therefore, characterization of the ECM deposited by cardiac myofibroblasts is relevant to the development of strategies to control collagen in some forms of heart disease.

Previously, the conversion of cardiac fibroblasts to myofibroblasts was shown to occur in cells plated on rigid tissue culture plastic (38, 39). Presumably, internal tension generated by the cells on the substrate led to myofibroblast differentiation (38). Cardiac fibroblasts grown in 3DTC also converted to myofibroblasts, most likely because of internal tension generated by the cells adhering to the nylon substrate. Hence, cardiac fibroblasts in 3DTC would be classified as being in a "restrained matrix-high mechanical load" environment as described by Grinnell (15). This type of environment is characterized by collagen accumulation and fibronectin organization. However, an increase in cell proliferation was not associated with myofibroblasts in our 3D system (15).

A function of TGF-beta as a regulator of myofibroblast differentiation has been established (32). In rat cardiac myofibroblasts cultured in 2D, myofibroblasts secreted elevated levels of soluble TGF-beta versus those in fibroblasts (8). We anticipated that 3D myofibroblast cultures would exhibit increased TGF-beta activity. However, we were not able to detect significant increases in active TGF-beta in 3D myofibroblast cultures. Indeed, we found an increase in total amounts of TGF-beta in fibroblast-conditioned medium (Fig. 8). Increases in latent TGF-beta are not predicted to influence cell behavior (2). In fact, we attribute the increase in total TGF-beta to liberation of stored TGF-beta in the ECM deposited before fibroblast conversion. Koli et al. (18) recently reported that TGF-beta deposition into ECM occurs over weeks in cell culture, with discrete ECM components providing unique spatial and temporal sites of extracellular stores of TGF-beta. On conversion of myofibroblasts to fibroblasts, evidence of increased remodeling of deposited ECM was associated with decreases in collagen production (Figs. 3 and 7). Thus we hypothesize that the increase in total (primarily latent) TGF-beta in fibroblast-conditioned medium arises from release of extracellular stores.

Because active TGF-beta rapidly binds to cell surface receptors and is internalized, we cannot rule out differences in TGF-beta activity in the pericellular milieu of myofibroblasts versus fibroblasts (2). Nonetheless, our results argue against increased levels of soluble, active TGF-beta as the primary inducer of a myofibroblast phenotype in 3DTC and suggest that mechanical stimuli are a principal factor in conversion and maintenance of myofibroblasts in this system (23). Cardiac fibroblasts in monolayers demonstrate an increase in collagen synthesis in response to mechanical stimuli (7). A synergistic effect on collagen synthesis by profibrotic cytokines was observed in this system. Other factors implicated in myofibroblast conversion such as tumor necrosis factor-{alpha} and activation of adenylyl cyclase might contribute to phenotypic differences in 3D cultures (35, 43).

In 2D cultures, EDA-Fn expression has been shown to precede {alpha}-SMA expression and is thought to play a positive role in myofibroblast differentiation (31). We also observed production of EDA-Fn in cardiac myofibroblasts grown in 3D (Fig. 2). Of future interest is whether function-blocking antibodies against EDA-Fn might promote fibroblast conversion in 3D cultures.

In addition to an increase in collagen I synthesis, myofibroblasts in 3DTC also displayed differences in the level of procollagen I intermediates. Specifically, an increase in the percentage of collagen I present as pC collagen {alpha}1(I) was detected in myofibroblasts coupled with a reduction in the percentage present as procollagen I. The difference between myofibroblasts and fibroblasts was particularly pronounced in cell layers taken from cultures stretched 1 wk. Thus, in addition to elevated levels of collagen I, processing of procollagen to mature collagen was also increased in myofibroblast cultures. ADAMTS-2, -3, and -14 have been identified as enzymes that cleave the N-propeptide of collagen I, whereas BMP-1 is primarily responsible for C-propeptide removal (10, 17).

The increase in the proportion of pC collagen {alpha}1(I) in myofibroblast cultures suggested that C-propeptide processing might be a rate-limiting step in procollagen I maturation by myofibroblasts, particularly during the first 2 wk of matrix deposition (Figs. 3–6). pC collagen {alpha}1(I) exhibits solubility at concentrations similar to procollagen I, whereas pN collagen {alpha}1(I) is significantly less soluble (28). Hence cleavage of C-propeptides from pC collagen should lead directly to collagen fibril deposition. C-propeptide processing of procollagen I is predicted to be influenced by levels of BMP-1 as well as procollagen C-proteinase enhancer protein, which specifically enhances cleavage of procollagen I by BMP-1 (36).

pN collagen {alpha}1(I) was not detectable in cells cultured for ~2 wk and subjected to stretch for 24 h and was most likely assembled into detergent-insoluble ECM. However, cultures grown for ~2 wk and subjected to 1 wk of additional culture (either unstretched or stretched) demonstrated increased amounts of pN versus pC collagen {alpha}1(I) (Fig. 3). We conclude that processing of procollagen I was most likely influenced by cell density and perhaps collagen concentrations in the mesh. Because intracellular processing of procollagen I was shown to take place in embryonic tendon, processing of the N-propeptide might be primarily an intracellular event during the first 2 wk of ECM deposition (9). Perhaps a mechanism to exclude pN collagen {alpha}1(I) from growing fibrils takes place at later times in collagen deposition, because incorporation of pN collagen {alpha}1(I) into collagen fibrils has been proposed to limit fibril aggregation.

Seemingly the cell surface of myofibroblasts binds procollagen more efficiently and perhaps serves as a better organizer of procollagen I processing enzymes than that of fibroblasts, because we observed an increase in levels of procollagen I and pC, pN, and mature collagen I associated with myofibroblasts (Fig. 3). Although collagen I fibril assembly can take place in a cell-autonomous manner, an appreciation of the receptors and structures present on the cell surface as vital components of collagen deposition and fibril formation is well established (29). Increased amounts of EDA-Fn, which has been implicated in ECM assembly and deposition, were also found to be associated with detergent-soluble cell layers of myofibroblasts (Fig. 2). In the event that EDA-Fn is actively involved in enhancing collagenous ECM assembly, the cell surface localization would facilitate its interaction with newly processed mature collagen I.

A number of studies have analyzed the effects of different forms of stretch on cardiac fibroblast synthesis of collagen in an effort to recapitulate physiological forces that might resemble those that occur in vivo in response to hypertrophy (23). These studies have used cardiac fibroblasts on 2D substrata and have shown an increase in collagen production in response to both static and cyclic stretch (3, 20, 26). We have found that cardiac fibroblasts in 3DTC convert to myofibroblasts through interaction with the scaffold and that stretch induces phenotypic conversion to fibroblasts with a concomitant reduction in collagen production. Our system is designed to characterize differential ECM assembly by myofibroblasts and not necessarily to simulate cardiac injury. However, the plasticity of cardiac fibroblasts to interconvert between myofibroblasts and fibroblasts with subsequent changes in ECM remodeling revealed in our 3D system is notable. Apoptosis of myofibroblasts has been implicated as a mechanism by which this cell type is removed from healed wounds (12). However, myofibroblasts have been found to persist in cardiac scar tissue for years after injury (34). Perhaps tissue-specific properties of myofibroblasts dictate distinct responses to extracellular cues.

Production of EDA-Fn and changes in procollagen processing intermediates confirmed that the nature of ECM deposited by myofibroblasts is distinct from that of fibroblasts. To be included in future studies is the investigation of whether changes in collagen cross-linking occur in myofibroblast cultures and whether differences in expression of other collagen-binding proteins and proteoglycans implicated in cardiac collagen deposition such as SPARC, decorin, and osteopontin, among others, might be differentially represented in myofibroblast ECM (16, 30). A better understanding of the disparate mechanisms used by myofibroblasts to construct ECM will lead to improved strategies to alleviate fibrotic deposition of collagen in the heart and in other tissues.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-02220 (A. D. Bradshaw).


   ACKNOWLEDGMENTS
 
We thank the members of the Gazes Cardiac Research Institute for helpful discussion, Mary Rackele and Mary Barnes for the isolation of adult cardiac fibroblasts, and Emmett Pinney for consultation on the culture of cells in 3DTC.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. D. Bradshaw, Div. of Cardiology, Dept. of Medicine, Medical Univ. of South Carolina, Gazes Cardiac Research Inst., 114 Doughty St., Charleston, SC 29425 (e-mail: bradshad{at}musc.edu)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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