Evidence for the Nonmuscle Nature of The" Myofibroblast" of Granulation Tissue and Hypertropic Scar. an Immunofluorescence Study

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American Journal of Pathology, Vol. 130, No. 2, February 1988 Copyright i American Association of Pathologists Evidencefor the Nonmuscle Nature ofthe "Myofibroblast" ofGranulation Tissue and Hypertropic Scar An Immunofluorescence Study

ROBERT J. EDDY, BSc, JANE A. PETRO, MD, From the Department ofAnatomy, the Department ofSurgery, and JAMES J. TOMASEK, PhD Division ofPlastic and Reconstructive Surgery, and the Department of Orthopaedic Surgery, New York Medical College, Valhalla, New York

Contraction is an important phenomenon in wound cell-matrix attachment in smooth muscle and non- repair and hypertrophic scarring. Studies indicate that muscle cells, respectively. Myofibroblasts can be iden- wound contraction involves a specialized cell known as tified by their intense staining of actin bundles with the myofibroblast, which has morphologic character- either anti-actin antibody or NBD-phallacidin. Myofi- istics of both smooth muscle and fibroblastic cells. In broblasts in all tissues stained for nonmuscle but not order to better characterize the myofibroblast, the au- smooth muscle myosin. In addition, nonmuscle myo- thors have examined its cytoskeleton and surrounding sin was localized as intracellular fibrils, which suggests extracellular matrix (ECM) in human burn granula- their similarity to stress fibers in cultured fibroblasts. tion tissue, human hypertrophic scar, and rat granula- The ECM around myofibroblasts stains intensely for tion tissue by indirect immunofluorescence. Primary fibronectin but lacks laminin, which suggests that a antibodies used in this study were directed against 1) true basal lamina is not present. The immunocytoche- smooth muscle myosin and 2) nonmuscle myosin, mical findings suggest that the myofibroblast is a spe- components ofthe cytoskeleton in smooth muscle and cialized nonmuscle type of cell, not a smooth muscle nonmuscle cells, respectively, and 3) laminin and 4) cell. (Am J Pathol 1988, 130:252-260) fibronectin, extracellular glycoproteins mediating

TISSUE CONTRACTION is an important phenom- microfilaments, which resemble the myofibrils of enon in the repairing dermis of open wounds and in smooth muscle. The surface ofthese cells was covered hypertrophic scarring. Initially, the shortening ofcol- by an interrupted amorphous layer of extracellular lagen fibers in the extracellular matrix was thought to material similar to the basal lamina surrounding be responsible; however, this was challenged by the smooth muscle cells. Because these cells showed mor- demonstration that wounds made in scorbutic ani- phologic characteristics of both fibroblasts and mals contract independent ofcollagen production.' A smooth muscle cells, Gabbiani and associates2 termed cellular mechanism for contraction has been pro- them "myofibroblasts." The presence ofthese cells in posed by Gabbiani and co-workers2'3 after their dis- covery of a morphologically distinct cell found in actively granulating wounds. Upon ultrastructural Supported by funds provided by the Department ofSur- examination, this cell type appeared to have charac- gery, Division of Plastic Surgery, New York Medical Col- lege, Valhalla, New York teristics ofboth fibroblasts and smooth muscle cells.2'3 Accepted for publication September 1, 1987. In addition to having extensive rough endoplasmic Address reprint requests to Dr. James J. Tomasek, De- reticulum and Golgi apparatus characteristic offibro- partment of Anatomy, Basic Sciences Building, New York blasts, these cells contained large bundles of actin Medical College, Valhalla, NY 10595. 252 Vol. 130 * No. 2 NONMUSCLE NATURE OF THE MYOFIBROBLAST 253 actively contracting granulation tissue and hypertro- by a matrix rich in fibronectin but lacking laminin. phic scar,4 as well as other contractile tissues such as Therefore, our immunofluorescence studies ofthe cy- the palmar fascia during Dupuytren's disease,5 and toskeleton and extracellular matrix of myofibro- their morphologic characteristics have led to the pro- blasts, in human and rat granulation tissue and hy- posal that the myofibroblast is the cellular agent re- pertrophic scar, suggest that this cell has sponsible for tissue contraction.3 characteristics similar to fibroblasts, rather than After numerous morphologic studies, uncertainty smooth muscle cells. still exists as to whether the myofibroblast more closely resembles the fibroblast or the smooth muscle cell. Therefore, we examined the cytoskeleton and Materials and Methods extracellular matrix of myofibroblasts from human Tissue burn granulation tissue, rat granulation tissue, and human hypertrophic scar, with immunologic probes Granulation tissue was taken from 2 patients un- for proteins characteristic of either fibroblasts or dergoing debridement ofthe burn wound. Hypertro- smooth muscle cells. Two aspects of the myofibro- phic scar tissue was taken from 1 patient undergoing blast were studied: 1) the type of myosin in the cyto- scar contracture release. Granulation tissue was also skeleton and 2) the macromolecules in the extracellu- taken from 4 female Sprague-Dawley rats each lar matrix. The selection ofthese cellularcomponents weighing approximately 250 g. Open wounds were was based on their presumed role in the generation made on rats which were anesthetized with an intra- and transmission of contractile force. peritoneal injection ofsodium pentabarbitol in phos- Myosin is a cytoskeletal protein that interacts with phate-buffered saline. There backs were shaved and actin to generate contractile force in both muscle and depilated (Nair, Carter Wallace, Inc., Cranbury, NJ), nonmuscle cells.6 The myosin associated with smooth and an antiseptic solution (Podiodine, Larson Labo- muscle cells is distinct from that found in nonmuscle ratories, Inc., Erie, Pa) was applied.A single 2 X 2-cm cells.7 To determine which species ofmyosin is asso- full-thickness excision was made on the dorsum of ciated with myofibroblasts of human burn granula- each animal and the wound left open. Two weeks tion tissue, rat granulation tissue, and hypertrophic after wounding, the animals were sacrificed and the scar, we employed antibodies that can distinguish be- granulation tissue excised. All the tissues were cut into tween smooth muscle myosin and nonmuscle myosin 2-3-cu mm pieces and prepared for histologic and in conjunction with indirect immunofluorescence.8 immunocytochemical staining by fixation in 4% Smooth muscle cells are surrounded by a highly paraformaldehyde in 0.1 mol/1 phosphate buffer, pH organized layer of extracellular material called the 7.4, for 20 minutes at room temperature. The tissue basal lamina.9 This structure functions in maintain- was infiltrated with 30% sucrose, mounted in OCT ing intercellular connections and transmitting compound (Lab-Tek Products, Miles Laboratories, changes in cell shape, such as contraction, across a Naperville, Ill), and rapidly frozen by immersion in tissue fabric. Basal laminae contain the glycoprotein liquid nitrogen. Cryostat sections (4-6,u) were thaw- laminin,'0 which binds the cell to the surrounding mounted onto gelatin-coated glass slides. Type IV collagen layer." In contrast, fibroblasts have no basal lamina and therefore lack laminin.'0 A different glycoprotein, fibronectin, attaches fibroblasts Antibodies to their surrounding stromal (Types I and III) colla- The anti-nonmuscle myosin antibody was a gift gen.'2 Previous studies of myofibroblasts in human from Dr. Keigi Fujiwara (National Cardiovascular wound granulation tissue,'3 experimental granula- Research Institute, Suita-Shi, Osaka, Japan). It is a tion tissue,'4 and hypertrophic scar'5 have demon- rabbit antiserum against human platelet myosin pre- strated fibronectin at the surface ofthese cells. How- pared as previously described.16 Previous studies with ever, the distribution of laminin in these tissues is this antibody have shown it to react with the myosin unknown. To determine the distribution of laminin ofnonmuscle cells, including fibroblasts and vascular with respect to fibronectin at the surface of myofi- endothelial cells, but not with the myosin of smooth broblasts in granulation tissue and hypertrophic scar, muscle cells.8 we employed antibodies specific for laminin or fibro- The anti-smooth muscle myosin antibody, also a nectin in conjunction with indirect immunofluores- gift from Dr. Keigi Fujiwara, is a rabbit antiserum cence. against human uterine myosin prepared as previously In this report, we demonstrate that myofibroblasts described.8 By double immunodiffusion, this anti- contain only nonmuscle myosin and are surrounded body formed a single precipitin line with extracts of 254 EDDY ET AL AJP * February 1988 human uterus but not with extracts of human plate- matrix antibodies were blocked with normal goat lets or cardiac or skeletal muscle. Immunofluores- serum diluted 1:10 in PBS for 30 minutes at room cence microscopy shows this antibody to stain all vis- temperature. Sections were then incubated with the ceral and vascular smooth muscle tested, but no other appropriate primary antibody for 30 minutes at room cell type, including epithelial, endothelial, and fibro- temperature, except for the anti-myosin antibodies, blastic cells as well as cardiac and skeletal muscle.8 which were incubated overnight at 4 C. Antibodies A monoclonal antibody raised against quail muscle were diluted in PBS (anti-actin antibody, 1: 500; anti- actin was a gift from Dr. Michael Payne (Department nonmuscle myosin and smooth muscle myosin anti- of Anatomy, New York Medical College, Valhalla, bodies, 1: 1000; anti-laminin antibody, 1: 50; mouse NY). This antibody has been shown by immunoblot anti-fibronectin antibody, 1:100; and rabbit anti-fi- analysis and immunocytochemistry to react with bronectin antibody, 1: 50). The sections were washed both muscle and nonmuscle actins.'7 Rabbit anti- in PBS three times for 5 minutes each and incubated serum against laminin was purchased from Collabo- for 30 minutes with the appropriate secondary anti- rative Research, Inc. (Lexington, Mass). A monoclo- body (goat anti-rabbit IgG-rhodamine or fluorescein nal antibody prepared against human fibronectin was or goat anti-mouse IgA + IgG + IgM-rhodamine a gift from Dr. Albert J. T. Millis (Department of [Cappel Laboratories, Malvern, Pa] diluted 1: 100 in Biological Sciences, State University of New York, PBS). The slides were washed with PBS three times for Albany, NY). The specificity of this antibody has 5 minutes each, mounted in a 2:8 solution of PBS been demonstrated by immunoblot analysis and im- and glycerol, and coverslipped. Cryosections of rat munofluorescence. I8 granulation tissue were washed in PBS for 5 minutes The monoclonal antibodies against actin and fibro- and permeabilized in cold acetone (-20 C) for 4 min- nectin could not be used on rat granulation tissue utes and allowed to air-dry. NBD-phallacidin (Molec- because ofthe cross-reactivity ofthe goat anti-mouse ular Probes, Inc., Junction City, Ore) was prepared secondary antibody with exogenous rat immunoglob- according to manufacturer's instructions. Each sec- ulins in the tissue sections. In order to localize fibro- tion was incubated with 10,p1 NBD-phallacidin stock nectin, we used a rabbit anti-human fibronectin anti- solution diluted to 200 ,u with PBS for 20 minutes at body. This antibody was an F(ab')2, fragment gener- room temperature. Sections were washed twice rap- ously provided by Dr. Henry P. Godfrey (Department idly in PBS, mounted in a 1: 1 solution of PBS and of Pathology, New York Medical College, Valhalla, glycerol, and coverslipped. NY). It has been demonstrated to cross-react with rat Double fluorescent staining of the same sections fibronectin by immunoblot analysis and double im- with anti-actin and anti-laminin antibodies was ac- munodiffusion (personal communication, Dr. Henry complished by separate incubations of the primary P. Godfrey). antibodies, followed by simultaneous incubation with The fluorescent probe 7-nitrobenz-2-oxa-1,3-dia- the appropriate contrasting fluorescent secondary zole (NBD) phallacidin was used to identify myofi- antibodies. Double fluorescent staining of the same broblasts in rat granulation tissue. Phallacidin is an sections with NBD-phallacidin and anti-laminin was acidic derivative of phalloidin, a toxin from the poi- accomplished by incubation with anti-laminin anti- sonous green mushroom Amanita phalloides, which body and the contrasting secondary antibody, fol- binds specifically to filamentous or F-actin ofthe cy- lowed by incubation with NBD-phallacidin. toskeleton.'9 NBD-phallacidin has been reported to Immunofluorescence staining controls for both stain the F-actin ofstress fibers in isolated living cells anti-myosin and anti-laminin polyclonal antibodies and fixed tissues,20 as well as to stain myofibroblasts in were performed by incubating tissue sections with granulation tissue.2' rabbit preimmune serum at the same concentration as the antibody, followed by the appropriate fluoro- chrome-conjugated goat anti-rabbit IgG. Immuno- Immunofluorescence fluorescence controls for anti-actin and anti-fibronec- Staining tin monoclonal antibodies were performed by Cryosections of human granulation tissue and hy- replacing the antibody with PBS followed by incuba- pertrophic scar were washed in phosphate-buffered tion of the sections with the appropriate fluoro- saline (PBS), pH 7.4, for 5 minutes. Sections to be chrome conjugated goat anti-mouse IgG. In both stained with anti-cytoskeletal antibodies were per- controls, no fluorescent staining was detected. All meabilized with 0.2% Triton X- 100 in PBS for 3 min- slides were examined with a Leitz orthoplan micro- utes and washed with PBS three times for 5 minutes scope equipped with epifluorescence optics and both each. Sections to be stained with anti-extracellular rhodamine and fluorescein filters for viewing double- Vol. 130 * No. 2 NONMUSCLE NATURE OF THE MYOFIBROBLAST 255 stained sections. Photographs were taken with Kodak Laminin Immunofluorescence Staining TRI-X Film Pan push-processed to 1000 ASA. The extracellular matrix surrounding myofibroblasts in human granulation tissue, hypertrophic scar, and rat granulation tissue was examined for laminin by indirect immunofluorescence. The sections were Results double-stained with anti-actin antibody or NBD- Actin Staining phallacidin for colocalization of actin and laminin. No staining with anti-laminin antibody was observed Human burn granulation tissue and hypertrophic at the surface of these myofibroblasts or in the sur- scar were examined by indirect immunofluorescence rounding matrix (human granulation tissue, Figure for the presence of myofibroblasts. Previous studies 1B; hypertrophic scar, Figure 2B, rat granulation tis- have demonstrated that the cytoplasm of these cells sue, Figure 3B). Anti-laminin antibody staining was stains intensely with anti-actin antibody.22 Myofi- observed in the basal lamina ofvascular smooth mus- broblasts were found to be the dominant cell type in cle cells (Figures lB and 3B). granulation tissue (Figure IA), whereas the pattern of actin staining in hypertrophic scar showed myofibroblasts to be organized into nodular structures (Fig- Fibronectin Immunofluorescence Staining ure 2A), confirming previous ultrastructural observa- The extracellular matrix surrounding myofibro- tions.4 In contrast, normal dermal fibroblasts do not blasts in human granulation tissue, hypertrophic scar, stain with anti-actin antibody (Figure IA). The inabil- and rat granulation tissue was examined for fibronec- ity ofanti-actin antibody to stain normal fibroblasts is tin by indirect immunofluorescence. Positive staining not due to the absence of actin in these cells, but is with anti-fibronectin antibody was observed through- most likely due to their lack oflarge bundles ofactin out the matrix ofhuman granulation tissue (Figure 8) microfilaments. Vascular smooth muscle cells are the and rat granulation tissue (Figure 10). In hypertrophic only other cell type in human granulation tissue and scar, fibronectin staining was concentrated in the hypertrophic scar that stains with anti-actin antibody nodular structures (Figure 9), which confirmed pre- (Figures lA and 2A). vious observations.15 The fluorescent probe NBD-phallacidin was used to stain actin in myofibroblasts ofrat granulation tis- Discussion sue. Myofibroblasts were found to be the dominant cell type, conforming to the staining pattern observed In this study, we have shown that the cytoskeleton in human granulation tissue (Figure 3A). of myofibroblasts from human burn granulation tissue, hypertrophic scar, and rat granulation tissue contain nonmuscle myosin but not smooth muscle myosin. The extracellular matrix ofthese myofibroblasts Myosin Immunofluorescence Staining contains abundant amounts of fibronectin as pre- The type ofmyosin present in human burn granu- viously reported.'3-'5 In addition, this study has dem- lation tissue, hypertrophic scar, and rat granulation onstrated that these cells lack the basal lamina-spe- tissue was determined by indirect immunofluores- cific glycoprotein laminin, associated with smooth cence. Myofibroblasts were found to stain intensely muscle cells. Previous studiesdemonstrating the mor- with anti-nonmuscle myosin antibody but not with phologic similarity ofmyofibroblasts to smooth mus- anti-smooth muscle myosin antibody (human granu- cle suggest that they may exert and transmit contract- lation tissue, Figures 4A and B; hypertrophic scar, ile forces by a smooth muscle type mechanism. Figures 5A and B; rat granulation tissue, Figures 6A However, the absence ofsmooth muscle myosin and and B). In addition, normal dermal fibroblasts and the basal lamina-specific attachment protein laminin vascular endothelial cells stain positively with anti- in these cells strongly argues against this hypothesis. nonmuscle myosin antibody (Figures 4A, 5A and The presence of nonmuscle myosin in the cytoskele- 6A). Individual fibers within myofibroblasts in ton and fibronectin in the extracellular matrix ofthe human burn granulation tissue and rat granulation myofibroblast suggests that they more closely resem- tissue were observed to stain with anti-nonmuscle ble a nonmuscle type of cell. myosin antibody (Figures 7A and B). Anti-smooth Normal fibroblasts do not display large bundles of muscle myosin antibody staining was seen only in actin microfilaments in situ, such as those seen in vascular smooth muscle cells around blood vessels myofibroblasts, although they can be made to do so (Figures 4B, SB, and 6B). by culture on a planar sustratum, such as a plastic 256 EDDY ETAL AJP * Febnuary 1988

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Figure 1-3-Fluorescent micrographs of cryosections of human bum granulation tissue (Figure 1), human hypertrophic sca (Figure 2), and rat granulation tissue (Figure 3) double-stainied with anti-actin antibody (IA, 2A) or NBD-phllcidin (3A) and anti-lamninin antibody (IB, 2B, 38). Figures IA, 2A, and 3A-Myofbroblastsare identified by intensefiuorescent staining of thecytoplasmforactin. Fibroblasts inthe surrounding stromadonot stain. Vasclarsmooth muscle cells around blood vessels stain brightiy for actin. Figure 2A-An aggregation of myofibroblasts characterstic of a hypertrophic scar nodule. Figure 3A-Myofibroblasts containing stress fibers display afilamentous staining patem. Figures 18,28, and 3B-No staining for laminin is observed around myofibroblasts. Asexpected, there is no staining for laminin around stromal fibroblasts. The basal lamina around vascularsmooth musclecells of arterioles stains positively. (X375) Vol. 130 * No. 2 NONMUSCLE NATURE OF THE MYOFIBROBLAST 257

Figure 4-6-Indirect immunofluorescent micrographs of cryosections of human bum granulation tissue (FIgures 4), human hypertrophic scar (Figure 5), and rat granulation tissue (Figure 6) stained with either anti-nonmuscle myosin (4A, 5A, 6A) or anti-smooth musce myosin (48, SB, 6B)antibodies. Figures 4A, SA, and OA-Myofibroblasts stain intensely for nonmuscde myosin. Vascular smooth musce does not stain with anti-nonmusie myosin antibody, whereas vascular endothelium does stain. Figures 48, 58, and 6B-Myofibroblasts do not stain with anti-smooth musce myosin antibody. As expected, vascular smooth musce stains intensely with this antibody. (X375) 258 EDDY ET AL AJP * February 1988

W W ~~~~~~~~~~ '..;-1 i : Figure 7-Indirect immunofluorescent icrorh of crosecis of hunan bum on tiss (A) and rat granutn tiss (B) staied with aFti- musce myosin antibody. Individual stess fibers wiftin myofboblas are staned (rrow). (X925) culture dish.23 These bundles of microfilaments are lar material is biochemically distinct from basal lam- referred to as stress fibers. In addition to actin, stress ina. Rather, there is a large amount of fibronectin fibers are composed ofa-actinin, nonmuscle myosin, present at the myofibroblast's surface in granulation and tropomyosin, which are organized in a periodic tissue and hypertrophic scar, as shown here as well as manner along their length.23 Ultrastructural studies in other studies.'-'5 The fibronectin-rich extracellu- have shown that stress fibersinculturedcellsresemble lar fibrils located at the myofibroblast's surface have the bundles of actin microfilaments in myofibro- been found to be in close transmembrane association blasts.3 Our study has demonstrated that myofibro- with intracellular actin microfilaments.28 This trans- blasts contain nonmuscle myosin, not smooth muscle membrane association, termed the fibronexus, has myosin (see also thepreliminary reportby Benzonana previously been observed at the surface of cultured and co-workers24). In addition, we have found that fibroblasts.29 Thus, the extracellular matrix at the sur- anti-nonmuscle myosin antibody stains myofibro- face of myofibroblasts is distinct from basal lamina blasts in a filamentous pattern, similar to the staining and resembles that observed around cultured fibro- ofstress fibers.25 These resultssuggestthat thebundles blasts. ofmicrofilaments present in myofibroblasts are simi- The cytoskeleton and extracellular matrix ofmyo- lar to stress fibers. fibroblasts in Dupuytren's disease, a human disease Fibroblasts and smooth muscle contain different characterized by a progressive contraction ofthe pal- types ofactin.26 Recently, a monoclonal antibody has mar fascia, have recently been examined.30 This im- been prepared against a-actin,27 a type of actin spe- munocytochemical study has demonstrated that Du- cific for smooth muscle.26 Immunocytochemical puytren's myofibroblasts contain nonmuscle myosin studies ofhuman wound granulation tissue using this and fibronectin and lack smooth muscle myosin and antibody have demonstrated the lack of ca-actin in laminin, similar to that observed here forgranulation myofibroblasts.27 These results are consistentwith the tissue and hypertrophic scar. Myofibroblasts from hypothesis that the microfilament bundles present in Dupuytren's disease as well as those from infiltrating myofibroblasts in granulation tissue are similar to ductal breast carcinomas have also been shown to stress fibers present in cultured fibroblasts. contain the intermediate filament protein vimentin The extracellular matrix at the surface of myofi- but to lack desmin,31 a protein characteristic ofmost broblasts in granulation tissue and hypertrophic scar smooth muscle cells.32 Therefore, myofibroblasts that was found to lack laminin. Previous ultrastructural form in a variety ofcontractile tissues appear to be a studies have described the extracellular material atthe nonmuscle cell type. However, it should be stressed surface ofmyofibroblasts as basal lamina-like.2-5 The that although our results have demonstrated the non- absence oflaminin demonstrates that this extracellu- muscle nature ofthe myofibroblast, they do not indi- Vol. 130 * No. 2 NONMUSCLE NATURE OF THE MYOFIBROBLAST 259 cate the origin of this cell type. Recent studies have convincingly shown that smooth muscle myosin dis- appears, while nonmuscle myosin appears, in rapidly proliferating smooth muscle cells during culture in -x7 vitro.33 Therefore, the lack ofsmooth muscle myosin in myofibroblasts does not rule out the possibility that 8~~~ these cells are derived from smooth muscle. If the myofibroblast is responsible for the contrac- 40 tion in granulation tissue and hypertrophic scarring, it must be capable ofgenerating an intracellular contractile force and possess a means oftransmitting this force to the surrounding tissue. Nonmuscle cells contain a cytoplasmic actomyosin system that is capable of generating contractile forces6 and a collagen- and fibronectin-rich extracellular matrix that is capable of transmitting such a force across a tissue fabric. Stress fibers present in cultured fibroblasts have been dem- 9.. onstrated to contract under appropriate condi- tions.333m Thus, there exists in the myofibroblast a nonmuscle contractile mechanism with the potential to generate the intracellular forces necessary fortissue contraction. Fibronectin, which is present at the sur- in8ce m h of face myofibroblasts and extends into the collagen- rich matrix,'3'28 could function in the formation of cell-to-cell and cell-to-matrix connections. These connections would then serve to transmit the contractile forces generated by the myofibroblast across the tissue as a whole. Fibronectin is ideally suited for such a role, because it is divided into functional do- mains that can bind to both the surface ofcells andthe surrounding collagen.'2 haaa- In conclusion, we suggest that the myofibroblast is a nonmuscle cell that has formed stress fibers in vivo and accumulated large amounts of fibronectin at its surface. Our findings indicate that the resemblance of this cell to smooth muscle is strictly morphologic and does not extend to characteristic cytoskeletal orextra- cellular components. Future studies oftissue contraction in wound healing, its possible control in hypertrophic scarring, and the origin and fate of this interesting cell type should take into account these findings on the properties ofits cytoskeleton and extracellular matrix.

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Gabbiani G, Ryan GB, Lamelin JP, Vassalli P, Majno York Medical College, and the staffofthe Westchester Burn G, Bouvier CA, Cruchaud A, Luscher EF: Human Center for their assistance and support throughout this smooth muscle autoantibody: Its identification as an- project.