phenotypic and functional features of myofibroblasts in sheep fetal wounds

Differentiation (1994) 56: 173-181 Differentiation On togeny, Neoplasl a and Dlfferentlatlon Therapy

0 Springer-Verlag 1994

Phenotypic and functional features of myofibroblasts in sheep fetal wounds James M. Estes ', Jerry S. Vande Berg ', N. Scott Adzick ', Thomas E. MacGillivray ', Alexis Desmoulih-e 3, Giulio Gabbiani 'Department of Surgery and The Fetal Treatment Center, University of California, San Francisco, California, USA *Core Clinical and Research Electron Microscopy Laboratory, Veterans Administration Medical Center, La Jolla, California, USA 'Department of Pathology, University of Geneva, Switzerland

Accepted in revised form November 22, 1993

Abstract. The myofibroblast is a mesenchymal cell with functional and structural characteristics in common with fibroblasts and smooth muscle cells. These cells play a critical role in wound closure and in the pathologic sequelae of healing. It has been shown in adult humans and experimental animals that the myofibroblast expresses a -smooth muscle actin (ASMA) temporarily during wound contraction and more persistcotly during fibrocontractive diseases; however, it is unclear whether this cell makes any contribution to tissue repair in utero. Experimental work in fetal animal models has demonstrated that wound repair in fetal skin occurs by reconstitution of epidermal appendages and organized restoration of the dermal collagen network. Fetal lamb wound healing studies have shown that a transition from scarless tissue repair to healing with scar formation occurs late i n gestation.

In this study we examined the ontogeny of myofibroblasts in fetal lamb wounds at early through late gestation, using transmission electron microscopy (TEM) and AS- MA immunohistochemistry.

Dramatic differences were observed in ASMA content of early as compared to late gestation fetal wound granulation tissue: ASMA was absent in wounds made at 75 days gestation but was present in progressively greater amounts in wounds made at 100 and 120 days gestation (term=145 days). TEM studies also demonstrated progressive development and organization of microfilament bundles. Early in development microfilament bundles were sparse and disorganized, but as gestation progressed the bundles became more prevalent and formed tightly parallel arrangements. The organization of microfilament bundles was also accompanied by fibronexus formation. Thus, the ASMA expression in granulation tissue rnyofibroblasts appears in relation to healing with scar formation.

Additional in vitro experiments demonstrated that the majority of fibroblasts cultured from granulation tissue of

Correspondence to: G. Gabbiani, Department of Pathology, Univer- sity of Geneva, Centre MCdical Universitaire 1 , rue Michel-Servet, CH-1211 Geneva 4, Switzerland

75, 100 and 120 days gestation lambs expressed ASMA, suggesting that factors present in fetal calf serum can in- duce ASMA expression even in fibroblasts not expressing this protein in vivo.

Our observations demonstrate an ontogenic change in the fetal myofibroblast in response to injury in vivo and suggest that local factors may regulate the variable phenotype of rnyofibroblasts during gestation. These results have implications for the therapy of pathologic scarring.

Introduction

The myofibroblast is a mesenchymal cell sharing ultrastructural features with fibroblasts and smooth muscle (SM) cells [37]. It was first identified in granulation tissue [ 161 and was believed to play an important role in wound closure and contracture [17, 29, 32, 35, 40, 461. The myofibroblast was also observed in fibrocontractive diseases such as fibromatoses [15], hepatic cirrhosis [6,33], renal and pulmonary fibrosis [ 1, 3 I], Dupuytren's contracture [47], and neoplasia-induced desmoplastic reactions [38] (for review of all these situations see [37]). The hallmark of the myofibroblastic phenotype is expression of a- smooth muscle actin (ASMA), the actin isoform prevalent in vascular SM cells. ASMA is temporarily expressed during normal wound healing and more permanently during fibrocontractive diseases (for review see [36]).

The presence of myofibroblasts in normal healing and in the pathologic sequelae of healing, such as scarring and contracture, is well established. However, it is unclear what contributions this cell makes to tissue repair in utero, which appears to occur by a different process than postna- tal tissue repair. The fetus possesses the unique ability to heal skin wounds without scar formation [2, 3, 9, 18, 25, 341. This observation was first extended to clinical situations during the human fetal surgery experience at the Fetal Treatment Center of the University of California, San Francisco, USA [19]. Experimental work in several fetal animal models has demonstrated that this process

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occurs by reconstitution of epidermal appendages and organized restoration of the dermal collagen network [26, 481.

Longaker et al. have previously shown that fetal excisional wounds in lambs at 100 days gestation contract and contain myofibroblasts, based on morphologic and immunohistochemical criteria [27]. In this study we examined the development and ultrastructural features of myofibroblasts in fetal lamb wounds made at 75 through 120 days gestation (term=145 days) using transmission electron microscopy and ASMA immunohistochemistry. Our purpose was to investigate possible differences between a wound which heals without scar formation (e.g. at 75 days gestation) and another one which heals with scarring (e.g. 100 and 120 days gestation). Our results indicate that AS- MA expression appears only in myofibroblasts of wounds healing with scarring and contracture.

Methods

Surgical procedure. The techniques of fetal lamb surgery have been described previously [20]. Briefly, six time-dated pregnant ewes of mixed Western breed (Torrel Farms, Ukiah, Calif., USA) underwent midline laparotomy at 75, 100 and 120 days gestation (two animals at each gestational age). Fetuses were exposed by hysterotomy of the gravid horn of the bicornuate uterus. Multiple full thickness, cutaneous excisional wounds were created with a 9 mm circular punch and India ink was applied to mark the wound base. The excisional wounds and normal skin were harvested 3 or 7 days later and processed as described below.

Cells and culture conditions. Sheep fibroblasts were obtained from explants of granulation tissue from 75, 100 and 120 days gestation. Cells were cultured in Eagle's Minimum Essential Medium (Gibco AG, Basel, Switzerland) supplemented with 100 units/ml penicillin, 100 Fg/ml streptomycin and 2 mM L-glutamine and containing 10% fetal calf serum (FCS, Gibco AG). Cultures were incubated at 37" C in a humidified atmosphere of 5% C02/95% air with medium changes twice a week. Cells were harvested at confluence by trypsinization and passed using a 1: 2 split ratio. Immunohistochem- ical and biochemical staining of cultured cells were performed from the 3rd to the 8th passage and yielded always similar results.

Antibodies, avidin-biotin-complex peroxidase (ABC-P) and immunofluorescence staining. We used the following antibodies: anti- aSM-1 [42], an affinity purified rabbit polyclonal anti-desmin antibody [23], an affinity purified rabbit polyclonal anti-SM myosin antibody [5], a rabbit polyclonal anti-actin antibody recognizing all actin isoforms [42], and a monoclonal antibody (HHF 35) which is also known to react with muscle actin isoforms [32, 461.

Immunoperoxidase staining for ASMA was done on ethanol- fixed and paraffin-embedded tissues. Sections were pretreated with H,O, and subsequently with 0.1 M periodic acid, 0.005 M NaBH, and normal horse serum. They were incubated for 2 h with anti-a SM-1 diluted 1: 200. This first incubation was followed by ABC-P staining using Vectastain kit anti-mouse IgG (Vector Laboratories, Burlingame, Calif., USA). The revelation of peroxidase activity was done with 3-amino-9-ethylcarbazole (Sigma Chemical, St. Louis, Mo., USA). Slides were weakly counterstained with Mayer's hema- toxylin.

Immunofluorescence was performed on fixed cells in culture dishes. For double indirect immunofluorescence, the following combinations were used: anti-aSM- Vanti-actin antibody, and anti-aSM-.l/anti-desmin antibody. Tetramethylrhodamine isothio- cyanate (TR1TC)-labeled goat anti-mouse IgG (Nordic Immunolog- ical Laboratories, Tilburg, The Netherlands) and fluorescein isoth-

iocyanate (F1TC)-conjugated goat anti-rabbit IgG (Cappel Labora- tories, Cochranville, Pa., USA) were used for the second step. The percentage of a-SM actin, desmin and SM-myosin positive cells was evaluated independently by two researchers on 300 to 500 cells.

Controls were performed by using mouse or rabbit non-immune IgGs or by omitting the primary antibody. Preparations were mounted and observed on a Zeiss Axiophot microscope equipped with epiillumination and specific filters for rhodamine and fluorescein (Carl Zeiss, Oberkochen, Germany). Photographs were taken, using 10x/0.30 Plan Neofluar to 63~11.40 oil Plan Apochromat objec- tives, on Kodak Ektachrome EPY-64T film or T-Max Kodak black and white film (Kodak, England).

Electron microscopy. Wound biopsies were immediately removed at surgery and placed into cold (4" C) 0.1 M phosphate buffered (pH 7.4) Karnovsky's fixative and shipped cold by overnight express for further processing. Under a dissecting microscope biopsies were then sliced into small 1-2 mm pieces in Karnovsky's buffered fixative in order to orient the wound margin in the middle of the block face. Fixation was continued for an additional hour before washing three times in cold (4" C) 0.1 M sodium phosphate buffer (pH 7.0). Following three buffer washes, the tissues were post-fixed in 0.1 M phosphate buffered 2% osmium tetroxide (pH 7.4) for 1 h at room temperature. After three buffer washes of 5 min each the tissues were dehydrated through a graded series of ethanol and propylene oxide and embedded in plastic resin (EM-bed 812, EM Sciences, Fort Washington, Pa., USA). Thick sections of all tissue blocks were stained with 1% toluidine blue and oriented by light microscopy. From these sections, areas of the wound margin were thin sectioned (60 nm), mounted on 200 mesh unsupported mesh grids, and stained in uranyl acetate and bismuth subnitrate. Multiple sections were examined and photographed with a Zeiss EM-IOB electron microscope.

SDS-PAGE and immunoblotting. For SDS-PAGE, cultured cells were trypsinized at confluence and, after hemacytometer cell counts, were mixed in buffer containing 1% sodium dodecyl sulfate (SDS, Bio-Rad Laboratories AG, Glattbrugg, Switzerland), 1 % dithiothre- itol (Fluka Chemie AG, Buchs, Switzerland), 1 mM phenylmethyl- sulfonyl fluoride (Merck, Darmstadt, Germany), 1 mM N a -P-To- syl-L-arginine methylester (Sigma Chemical) in 0.4 M TRIS HCI pH 6.8 [13]. The samples were immediately sonicated and boiled for 3 min. Protein content was determined according to Bradford [8]. Forty micrograms protein was electrophoresed on a 5-20% gradient gel and stained with Coomassie blue. For quantification of total actin, gels were scanned with a computerized laser beam densito- meter (Genofit SA, Geneva, Switzerland) as previously described [ l l , 241.

Western blotting with anti-total actin and anti-aSM-1 was performed according to Skalli et al. [42]. Briefly, 5 to 20 pg protein were electrophoresed on a 5 2 0 % gradient gel. Separated proteins were transferred to nitrocellulose filters [45] which were incubated with a rabbit affinity purified IgG recognizing all actin isoforms or with anti-aSM-1. A second incubation was dpne either with sheep anti-rabbit or a rabbit anti-mouse IgG conjugated with horseradish peroxidase (Nordic Immunological Laboratories). Enhanced chemi- luminescence (Amersham International plc, Buckinghamshire, UK) was used for detection.

Results

The surgical procedures were well tolerated and there was no fetal or maternal morbidity. Analysis of non-wounded skin at all gestational ages showed ASMA staining only in small blood vessels (Fig. l a and b). Immunohistochemical staining for ASMA revealed marked differences between wounds made at 75 days and those at 100 and 120 days gestation. ASMA staining in granulation tissue was absent in 75 day gestation wounds except for pericytes and vascu-

Fig. 1. Alpha-smooth muscle actin (ASMA) staining in fetal wounds. a, b normal skin at 75 and 100 days gestation respectively. Note the preqence of a similar stain around small blood vessels in both cases. Hair follicles are visible at 100 days gestation. A positive staining for ASMA is present in fibroblastic cells surrounding these follicles [22]. c, d 3-day-old wound at 75 and 100 days gestation respectively. In (c), staining is present only around small blood ves-

sels. In (a), there is a substantial staining of fibroblastic cells in the wound bed. e, f ASMA staining in a 7-day-old wound at 75 and 100 days gestation respectively. g, h higher power micrographs of ASMA staining in a 7-day-old wound at 75 and 100 days gestation respectively. In (e) and (g), staining remains evident only around small blood vessels. In (0 and (h), most fibroblastic cells in the wound bed are positive to ASMA staining. a-f, x400; g, h, ~800

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lar smooth muscle (positive control) (Fig. lc, e and g). In contrast, granulation tissue from 100 day gestation wounds revealed substantial ASMA staining in fibroblastic cells; this staining increased from 3 to 7 days post wounding. At 3 days post wounding the density of ASMA positive cells was sparse (Fig. Id), while at 7 days ASMA- positive cells comprised approximately 90% of the granulation stromal cells (Fig. If and h). ASMA staining was also strongly positive in 7-day-old wounds made at 120 days gestation (data not shown). The presence of ASMA was also confirmed in all wounds by staining with HHF 35 (data not shown), a monoclonal antibody which is known to react with SM actin [46]. Desmin and SM myosin staining were always negative in wound stromal cells.

Fig. 2. Seventy-five day gestation, 7-day-old wound. The myofibroblast contains patches of small microfilament bundles with electron dense bodies (arrowheads). Mi- crofilament bundles were found near Golgi bodies (G) and the cell membrane. Bar, 0.4 pm Fig. 3. Seventy-five day gestation, 7-day-old wound. The most well differentiated myofibroblast at 75 days displayed bundles that .were composed of loosely arranged microfilaments (MF) among electron dense bodies. Ribosomal clusters (RC) appeared near the rough endoplasmic reticulum. Polyribo- somes (arrowheads) sometimes associated with small filaments were observed between ribosomal clusters and microfilament bundles. Bar, 0.2 pm

Electron microscopic analysis was performed on the excisional wounds and normal skin. Tissue from wounds made at 75, 100 and 120 days gestation revealed consider- able variation in the cytoskeletal features characteristic of myofibroblasts. Seven-day-old wounds made at 75 days gestation presented cells which appeared to be in the early stages of microfilament development. Evidence for this interpretation was derived from the presence of loose clusters of randomly-oriented microfilaments (60 to 80 Angstroms) and electron dense bodies. The hallmark of these organizational sites was represented by clusters of polyribosomes and concentrated areas of microfilaments. In other later stage myofibroblasts, small patches of more organized bundles were observed between Golgi bodies and the cell membrane (Fig. 2). The most developed my-

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ofibroblasts presented longer microfilament bundles with electron dense bodies along the cell membrane (Fig. 3). Higher magnification showed that these microfilaments remained loosely arranged among electron dense bodies.

In wounds made at 75 days gestation, fibronexi were present but appeared to be very undeveloped (data not shown). Occasionally some cells demonstrated these transmembrane complexes between clusters of organizing intracellular microfilaments along with sparsely dis- tributed collagen fibrils in the extracellular matrix.

Seven-day-old wounds made at 100 days gestation showed increased numbers of myofibroblasts. Bundles of microfilaments with electron dense bodies were well formed along the long axis of the cell (Fig. 4). Unlike 7-day-old wounds made at 75 days gestation, microfila-

Fig. 4. One hundred day gestation, 7-day-old wound. Most observations displayed myofibroblasts with microfilament bundles (MF) composed of parallel, tightly wo- ven microfilaments among electron dense bodies. An undeveloped fibronexus (FN) was seen with at- tachments to collagen fibers (C). Clusters of glycogen (GL) parti- cles were observed in many myofibroblasts and small amounts of fibrin (F) were also noted. Bur, 0.4 l m Fig. 5. One hundred twenty day gestation, 7-day-old wound. Net- works of myofibroblasts were joined via fibronexi (FN) between microfilament bundles (MF) of neighboring cells. These syncytial arrangements of myofibroblasts appeared throughout the wound bed. Bur, 2.0 Fm

ments from cells at the same interval of wound repair at 100 days bestation were parallel and tightly packed, form- ing broad microfilament bundles (Fig. 4). Many of these latter cells also displayed noticeable clusters of glycogen throughout their cytoplasm (Fig3. 4). Like their 75 day gestation counterparts, myofibroblasts from 100 day gestation wounds contained well developed Golgi bodies and rough endoplasmic reticulum.

In areas where new collagen fibrils and fibrin appeared near the cell membrane, fibronexus junctions appeared to be more developed. Some myofibroblasts displayed these transmembrane complexes parallel to the long axis of the cell as well as near the tips of cell processes (Fig. 4).

Seven-day-old fetal wounds made at 120 days gestation exhibited microfilament bundles that appeared to be more

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numerous and more developed than in myofibroblasts from earlier gestational ages. These cells appeared to be very metabolically active with well developed Golgi and rough endoplasmic reticulum. Frequently, parallel arrangements of myofibroblasts were attached via fibronexi into a syncytium (Fig. 5 ) . The extracellular space was filled with new collagen fibrils and very little fibrin.

Immunofluorescence studies performed on cultured fibroblasts with anti-aSM-1 showed that ASMA was expressed in all fibroblasts (Fig. 6). The expression of desmin and SM myosin was always very low (less than 5%); however, in 120 day gestation granulation tissue fibroblasts, the proportion of desmin and SM myosin positive cells slightly increased compared to 75 and 100 day gestation granulation tissue fibroblasts.

Our experiments with cultured fibroblasts show that granulation tissue fibroblasts from wounds in 75, 100 and 120 day gestation lambs all possess the capacity of expressing ASMA when exposed in vitro to growth factors in fetal calf serum. Quantitative changes of total actin in cultured fibroblasts were examined by densitometric analysis of SDS-PAGE gels. The actin band was recog- nized by Western blotting with the anti-total actin antibody. The total actin content was constantly 10% of total cellular protein (the mean of all samples was 9.89+ 0.06%

of total protein). Immuaoblot with anti-aSM- 1 consistent- ly showed high levels of ASMA expression in all sadples (Fig. 7).

Discussion

It has been previously shown that myofibroblastic cells, i.e. cells containing bundles of microfilaments or stress fibers, can express varied cytoskeletal features during wound healing and in different pathologic settings [36]. For example, one can distinguish among myofibroblasts containing vimentin and p- and y-cytoplasmic actin isoforms (V cells); vimentin, cytoplasmic actin isoforms, and ASMA (VA cells); and vimentin, cytoplasmic actin isoforms, ASMA and desmin (VAD cells). Thus, myofibroblasts can present several phenotypic variants proba- bly evolving one into another [36]. Cells expressing AS- MA (VA) are present in granulation tissue in high propor- tions during uncomplicated wound healing and particu- larly during wound contraction [ 101. Our experiments ex- tend the observation concerning different myofibroblastic features to fetal wound healing [27]. Most importantly they show for the first time that in the fetus the transition from scarless tissue repair to healing with scar formation

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a b c d e f

Fig. 7. ASMA expression in 75 (a, d), 100 (b, e), and 120 (c, f) day gestation granulation tissue fibroblasts. Densitometric scanning of Coomassie blue-stained gels (a-c) shows that the percentage of actin per total protein is not modified (for value, see Results). Immuno- blotting after SDS-PAGE (d-f) shows the high level of ASMA expression (A=actin)

coincides with the expression of ASMA by myofibroblasts and thus support the possibility that ASMA is not only a marker of granulation tissue contraction but partic- ipates actively in this phenomenon.

Our in vitro data, showing that fibroblasts cultured from 75,100 and 120 day granulation tissues express similar amounts of ASMA, suggest that the difference in AS- MA expression by granulation tissue fibroblasts observed in vivo between 75 and 100 or 120 day gestation lambs is due to different microenvironmental conditions in vitro and in vivo rather than to intrinsic fibroblastic properties. Further studies will be necessary to know whether in wounds made at 75 days gestation factors inhibiting AS- MA expression after cloning are produced and/or whether cultured fibroblasts from 75 day gestation animals are stimulated to produce ASMA by serum factors. It has been shown previously that different fibroblastic popula- tions and clones in vitro contain a variable proportion of ASMA-expressing cells [12]. However, it is unclear what microenvironmental factors are implicated in ASMA expression. Recently we have shown that heparin [13] and transforming growth factor-Pl (TGF-P1) [ 141, albeit with different mechanisms, stimulate the appearance of AS- MA-rich fibroblasts both in vivo and in vitro. Other inves- tigators have demonstrated that TGF-P increases the expression of ASMA in cultured smooth muscle cells [7] and a short communication has suggested that this cytokine exerts the same action on cultured fibroblasts [30]. TGF-P has been shown to play a role in the production of wound contraction and scars in vivo [39]. Moreover the role of TGF-P in the regulation of collagen synthesis during wound healing is well established [44]. All these data

point to TGF-P as a regulator of myofibroblast differentiation [14]. This cytokine appears to act during wound healing in a coordinated fashion on the metabolism of extracellular matrix components (e.g. collagen) and on the regulation of cellular contractility (e.g. ASMA expression). Work is in progress in our laboratories in order to evaluate TGF-P expression during wound healing at different gestation times. Several other cytokines and extracellular matrix components have also been proposed to modulate ASMA expression in myofibroblasts (for review see [36]).

Other lines of investigation have demonstrated the in- fluence of cell adhesion on the expression of actin in gen- eral as well as ASMA. Yamada et al. [50] and Ali et al. [4] showed that addition of a matrix glycoprotein could re- store cell adhesion and actin synthesis in transformed chick embryo fibroblasts. Willingham et al. [49] also showed that normal fibroblasts produce well organized actin bundles when cultured on an adherent substratum and that this property was lost after dissociation. Later, Singer et al. [40] demonstrated that fibronectin is one of the adhesion molecules that induces actin synthesis in fibroblasts.

In our study, fully formed microfilament bundles, typi- cally found in normal adult granulating wounds, were not found in wounds made at 75 days gestation. This may be due to an insufficient level of growth factor stimulation in the microenvironment or decreased levels of cell adhesion factors. Further studies addressing these questions are in progress.

The appearance of ASMA at 100 days gestation re- flects a progression of the myofibroblast phenotype from fibroblastic to smooth muscle-like. This transition may have important implications for wound healing as the per- sistence in wounds of ASMA-positive cells is associated with hypertrophic scarring and fibromatoses [41, 431. Progressive cellular differentiation may occpr throughout gestation in wound myofibroblasts, thus influencing tissue repair by causing more wound contraction and eventu- ally scar formation. Experimental evidence has demonstrated such a wound healing transition in fetal lambs at approximately 100 days gestation [27]. A similar transition has been described in fetal rat wounds [21], in fetal rhesus monkey wounds [28], and in mesenchymal cells of human embryonic and adult colonic muscularis mucosae W I .

Our study characterizes an ontogenic transition of the myofibroblast phenotype in fetal lamb wounds. The ac- quisition and organization of ASMA rich microfilaments in wound myofibroblasts later in gestation suggests an increasingly important role for wound contraction during fetal development. This phenomenon may be related to the development of scar in the third trimester. A better under- standing of factors modulating the fibroblastic phenotype during wound healing could lead to therapeutic strategies directed against myofibroblast proliferation or differentiation, and these may be useful in the prevention of pathologic scarring and fibrosis.

Acknowledgements. Supported by NIH grants HD 25505 and GM 27348 (N.S.A.), an NIH National Research Service Award

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HD 07462 (J.M.E.), Veterans Administration Medical Research De- partment (J.S.V.), and the Swiss National Science Foundation Grant No. 31-30796.91 (G.G.). We thank Mrs. G. Chakroun for excellent technical assistance.

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