Fibrosin: A Novel Lymphokine in Wound Healing

Experimental and Molecular Pathology 71, 247–255 (2001) doi:10.1006/exmp.2001.2402, available online at http://www.idealibrary.com on

Fibrosin: A Novel Lymphokine in Wound Healing

Sadhana Prakash1 and Phillips W. Robbins Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received July 9, 2001

Several growth factors are actively synthesized during wound repair and function to stimulate different cell types involved in the process of healing. Fibrosin is a novel fibrogenic lymphokine that stimulates several biological activities that relate to in vivo scarring. To investigate the role of fibrosin, we used “punch biopsy” and linear wounding procedures in a murine model of wound healing. Histological examination showed that recombinant fibrosin stimulated epithelialization of wounds and accelerated healing of both punch biopsy and linear wounds. Fibrosin enhanced healing of linear wounds by reducing the time for healing by approximately 30–40%. From our data we estimated the healing time of control wounds to be 22–24 days; wounds treated with fibrosin appeared to heal in 14–16 days. Our observations suggest that fibrosin enhances wound healing and may be involved in accelerating epithelialization, collagen matrix formation, and also remodeling of the extracellular matrix in vivo. Thus fibrosin may function during different phases of wound healing and act as a potent inducer of scar formation and wound healing. This finding may have direct clinical applications. 䉷 2001 Elsevier Science Key Words: fibrosin; wound healing; scarring; fibrosis; fibrogenic lymphokines; cytokines; burns.

and remodeling (Martin, 1997). While these phases are discrete, they are by no means discontinuous. In fact, the beginning of one phase overlaps with the end of another. These phases collectively include the processes of coagulation, migration of macrophages, lymphocytes, neutrophils, etc., deposition and differentiation of extracellular matrix proteins, fibroplasia, epithelialization, contraction, and remodeling (Moulin, 1995). Several studies show that growth factors are secreted at the time of the injury and during other phases of healing (Kristy and Lynch, 1993). It is believed that growth factors acting independently or in combination with others may regulate many of the processes related to the wound healing process. Several growth factors have been studied for their ability to moduate the repair process in different wound healing models. Some examples of the growth factors involved in wound healing are platelet-derived growth factor (PDGF),2 keratinocyte growth factor (KGF), insulin-like growth factor (IGF-1), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor (TGF), among others (Kristy and Lynch, 1993; Schultz et al., 1991). In this report, we investigate the effect of a novel heparin

INTRODUCTION Wound healing is a complex physiologic process that is composed of different phases : inflammation, proliferation, 1

2

To whom correspondence and reprint requests should be addressed C/O Dr. P. W. Robbins, Biology Department, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Bldg. 68, Rm. 583A, Cambridge, MA 02139. E-mail: [email protected].

0014-4800/01 $35.00 䉷 2001 Elsevier Science All rights reserved

Abbreviations used: PDGF, platelet derived growth factor; KGF, keratinocyte growth factor; IGF-1, insulin-like growth factor; FGF, fibroblast growth factor; TGF␤, transforming growth factor; EGF, epidermal growth factor; ECM, extracellular matrix.

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TABLE 1A Wound Healing of “Punch Biopsy” Wounds after 8 Days of Treatment

TABLE 1B Wound Healing of Punch Biopsy Wounds after 7 Days of Treatment

Percentage of wound length with regenerated epithelium

Percentage of wound length with regenerated epithelium

Animal

Untreated

Treated

Animal

Untreated

Treated

1 2 3 4 5 6 Average

74 48 67 68 55 70 52.3

86 85 98 100 96 90 92.8

1 2 3 4 Average

55 78 55 94 71

70 100 98 100 92

Note. Wound healing of punch biopsy wounds receiving either fibrosin (treated) or vehicle gel (untreated) alone. Wounds were biopsied after 7 days.

Note. Wound healing of punch biopsy (4 ⫻ 4 mm) wounds receiving either fibrosin (treated) or vehicle gel alone (untreated). Wounds were biopsied after 8 days.

FIG. 1. Effect of fibrosin on “punch biopsy” (⫻4 mm) wounds; wound analysis was done after 8 days. Wounds were treated with a single dose of (A) vehicle gel alone or (B) fibrosin.

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FIG. 1—Continued

binding fibrogenic lymphokine, fibrosin. Fibrosin was initially isolated from a murine model of schistosomiasis (Prakash and Wyler, 1991). It is secreted by activated lymphocytes as in egg granulomas formed in the livers of hosts infected with Schistosoma mansoni. Among its biologic properties, native as well as recombinant fibrosin is a potent mitogen for mesenchymal cells such as fibroblasts (Prakash et al., 1995; Prakash and Robbins, 1998). It stimulates synthesis of extracellular matrix proteins like collagen, fibronectin, and hyaluronan (Wyler, 1996). It also induces chemotaxis of fibroblasts and smooth muscle cells (Wyler et al., 1989; Prakash et al., 1991). These properties are akin to other growth factors, e.g., PDGF and TGF␤ (Lynch et al., 1987). And these biologic properties of fibrosin suggest that it may play an important role in the repair of tissues. We evaluated this notion and report here that fibrosin induces healing of skin injuries caused in young Balb/c mice. At present there are no reports on the in vivo effects of fibrosin on wound healing.

MATERIALS AND METHODS

Wounding procedure. Five- to eight-week-old female Balb/c mice (average weight, 15–20 g) were obtained (Jackson Laboratories, Bar Harbor, Maine). Animals were housed five per cage in a room with a natural light–dark cycle. Mice were allowed at least 1 week of acclimatization to the laboratory condition before the start of experiments. All mice were anesthetized before surgery. The backs of the mice were shaved and the area was disinfected with 70% alcohol and betadine. A sterile template (4 ⫻ 4 or 3 ⫻ 3 mm) was placed on the shaved skin to make a deep impression. The area was then excised. Two such “punch biopsy” wounds were placed on each side of the spine and were separated from each other by 2–2.5 cm. In addition, linear incisions, 4 mm long, were surgically induced. These procedures wounded both the epidermis and the dermis layers. Each mouse received two middorsal wounds, on either side

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of the spine. One incision received the experimental and the second received the control gel. Treatment of wounds. Both types of injuries were treated at the time of the injury with 50–60 ␮l of vehicle gel containing a 2% solution of sterile biocompatible methyl cellulose. Experimental wounds received recombinant fibrosin (Prakash et al., 1995) that was applied topically in a single dose of 500–1000 ng of peptide dispersed in biocompatible methyl cellulose. Control wounds received methyl cellulose admixed with sterile 0.15 M NaCl. Histopathologic analysis of wounds. Linear wounds were analyzed on days 4–10. Circular wounds were analyzed on days 7 and 8. The wound area was trimmed and fixed in 4% buffered Formalin. The fixed tissue was processed, cut into two pieces at the center, and then embedded in paraffin. For morphometric analysis, 4- to 5-␮m paraffinembedded sections were made perpendicular to the skin surface and stained using hematoxylin and alcoholic eosin (Sigma Chemicals). The wounds were photographed and the

TABLE 2 Wound Healing of Animals after 5 Days of Treatment Length of wound (␮m)

Maximum length of inflammation (␮m)

Uncovered wound (␮m)

Untreated Treated

1427 1402

3249 2732

Nil Nil

Untreated Treated

2360 1968

3701 2800

548 Nil

Untreated Treated

2749 2138

4065 3943

438 Nil

Untreated Treated

1699 1732

2113 1825

34 Nil

Untreated Treated

2760 1940

4030 2100

575 Nil

Untreated Treated

1029 1096

3209 2063

Nil Nil

Untreated Treated Average Untreated Treated

2616 1257

3607 2054

Nil Nil

2082 1627

3424 2569

532 Nil

Animal No. 1

2

3

4

5

6

7

Note. Wounds were treated with either fibrosin or gel alone and biopsied after 5 days as described under Materials and Methods.

TABLE 3 Wound Healing of Animals after 7 Days of Treatment Length of wound (␮m)

Maximum length of inflammation (␮m)

Uncovered wound (␮m)

Untreated Treated

1600 260

2350 342

578 Nil

Untreated Treated

1100 996

1550 1600

159 Nil

Untreated Treated

610 561

1550 943

972 Nil

Untreated Treated

1970 648

2760 2183

587 Nil

Untreated Treated

1230 295

2240 1701

Nil Nil

Untreated Treated Experiment 2 7 Untreated Treated 8 Untreated Treated 9 Untreated Treated Average Untreated Treated

1500 1500

2300 2572

286 Nil

1739 344

2256 628

Nil Nil

875 1089

2100 1858

Nil Nil

1250 347

1900 838

Nil Nil

1338 652

2069 1428

430 Nil

Animal No. 1

2

3

4

5

6

Note. Wound healing of animals receiving fibrosin (treated) or vehicle gel alone (untreated). Wounds were analyzed after 7 days of treatment.

widths of the epidermal layer and dermal layer as well as lengths of the wound and the inflammation were measured by computer-assisted planimetry. These values were then converted into micrometers using a calibration ruler viewed under the same conditions. Stastical analysis. The mean and standard deviation were calculated for all data and Student’s t test was performed to analyze differences between control and experimental sites for each pair. RESULTS Analysis of the punch biopsy wounds. We observed that both with 3 ⫻ 3 mm wounds on day 7 and 4 ⫻ 4 mm

251

FIBROSIN AND WOUND HEALING TABLE 4 Wound Healing of Animals after 10 Days of Treatment Length of wound (␮m)

Maximum length of inflammation (␮m)

Uncovered wound (␮m)

Untreated Treated

1391 294

1883 518

983 Nil

Untreated Treated

590 190

691 190

220 Nil

Untreated Treated

942 150

1603 150

365 Nil

Untreated Treated

646 150

1921 150

48 Nil

Untreated Treated

1370 613

3115 789

1027 Nil

Untreated Treated Average Untreated Treated

1237 913

2503 1563

Nil Nil

1030 404

1952 619

701 Nil

Animal No. 1

2

3

4

5

6

Note. Wounds were treated either with fibrosin or with vehicle gel alone (untreated). Wounds were biopsied after 10 days and analyzed as described under Materials and Methods, above.

wounds by day 8, the wound areas of the treated wounds were smaller and a greater percentage of the wound length was covered. None of the untreated wounds were completely covered, while at least half of the treated wounds were completely covered by the regenerated epithelium. In general, the wounds treated with fibrosin showed a substantially larger area of regenerated epithelium than untreated wounds (mean observed difference of 20–30%, P ⬍ 0.05; Tables 1A and 1B and Figs. 1A and 1B). There was a greater proliferation and differentiation of cells in the treated versus untreated wounds. Analysis of linear incisions. At 5 days after wounding, the epithelial and dermal layers of untreated and treated wounds were fairly similar in width. The average length of the wound and the length of the inflammation were approximately 1.3-fold greater in untreated versus treated wounds (P ⬍ 0.05; Table 2). At this time, there was no apparent difference in the extent of the infiltrate or the vascularization of untreated versus the treated wound. However, the experimental wounds showed a greater amount of matrix or granulation tissue. Wounds biopsied after 7 days of treatment also showed a

similar trend. Both the epidermal and dermal layers of untreated wounds were 30–40% wider than those of the treated wounds. The length of the untreated wound was approximately 2-fold greater than that of the treated wounds (mean observed difference of 556 ␮m, P ⬍ 0.01; Table 3). The maximum length of inflammation of untreated wounds was approximately 1.5-fold greater when compared with wounds receiving recombinant fibrosin (mean observed difference of 754 ␮m, P ⬍ 0.01). More than half (five of nine) of the untreated wounds showed areas that were incompletely covered with regenerated epithelium. At this time, the granulation tissue of the treated wound was very dense; matrix formation could be detected in control mice also. It may be noted that the untreated wounds appeared to be covered faster in Experiment 2, day 7. We believe this may be due to the fact that the animals were younger (approx. 5 weeks) in this group compared to the other group. We believe that the untreated wounds in younger animals appeared to heal faster than the untreated wounds of mice that were slightly older. At 10 days there was a marked morphological difference between the untreated and the treated wounds. The maximum difference in the untreated and treated wounds was observed in wounds biopsied at 10 days after injury. The dermal area of the treated wounds contained dense granulation tissue and began to look very similar to the uninjured dermis. The length of the wound reflecting the wound area was much less when the wounds were treated with the growth factor (Table 4). Thus there appeared to be a greater contraction of the wound in the case of treated wounds. The average length of the untreated wound was 2.5-fold greater than the treated wound (mean observed difference of 626 ␮m, P ⬍ 0.01). The maximum length of inflammation of untreated wounds was 3.2-fold greater than that of the control (mean observed difference of 1334 ␮m, P ⬍ 0.01). All the wounds receiving the growth factor appeared to be completely covered with regenerated and well-differentiated epithelium. However, approximately two thirds of the untreated wounds still showed areas that were not completely covered. The epidermal and the dermal layers of untreated wounds were at least 30–40% and 60–70% respectively, wider than the treated wounds. Based on the rate of reduction in wound length, the estimated time of complete healing of treated (linear) wounds may be 14–16 days, as opposed to 22–24 days for untreated or control wounds. The circular defects or the punch biopsy wounds also showed a greater degree of healing when they received fibrosin. These observations suggest that the healing of wounds receiving recombinant fibrosin is accelerated by application of recombinant fibrosin.

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FIG. 2. Effect of fibrosin on linear skin wounds. Wounds (4 ⫻ 0.5 mm) received either a single dose of (A) vehicle gel alone or (B) fibrosin. Wounds were biopsied 7 days following wounding. Wounds treated with fibrosin appear to be histologically different from untreated wounds at 7 days. Arrows demarcate the length of the wound. (hematoxylin and eosin stain).

Further investigations should reveal the potential therapeutic use of fibrosin in wound healing.

DISCUSSION The aim of the present study was to investigate the effect of fibrosin, a novel fibrogenic cytokine, on wound healing. Fibrosin was applied topically to the injured skin in mice, and its effect on wound repair was noted over a period of time. Two types of incisions were made—a punch biopsy and a linear incision wound. In both cases, wounds treated with fibrosin showed a greater degree of wound healing. The area of regenerated epithelium, the extent of proliferating, differentiating cells, and the rate of appearance of granulation tissue was markedly enhanced in the experimental

wounds. Treated wounds appeared to be covered with regenerated epithelium much faster than untreated wounds (Tables 1–4; Figs. 1 and 2). In the case of the linear incisions, by day 7, the length of inflammation and the length of the wound were reduced by at least two- to threefold when wounds were treated with fibrosin. By day 10, all wounds treated with fibrosin were closed. However, not all untreated wounds were completely covered by the epidermis. The length of the wound, reflecting the wound area, was much less when wounds were treated with fibrosin. On an average, wound length was at least two- to threefold greater in untreated wounds than in treated wounds. The epidermal and the dermal layers were 30–40% and 60–70% wider (respectively) at day 10. At this time, the treated wounds resembled the uninjured skin morphologically, whereas the untreated wounds still showed considerable unresolved inflammation. Based on the observations, we estimate that the average time

253

FIBROSIN AND WOUND HEALING

FIG. 2—Continued

for the resolution of the treated wound may be 14–16 days, whereas the average time for the untreated wound may be 22–24 days. We therefore estimate a reduction in the time taken for wound healing of wounds treated with fibrosin by about 30–40%. Several growth factors, such as PDGF, TGF␤, IGF, KGF etc., have been shown to be involved in different phases of wound healing (Kristy, 1993; Lynch et al., 1987). Like other growth factors which are known to play a role in wound healing, fibrosin may enhance wound formation at different points in the wound healing cascade. Native fibrosin as well as recombinant fibrosin stimulates activation, proliferation, and induction of matrix synthesis by fibroblasts (Prakash and Wyler, 1991; Prakash et al., 1995; Prakash and Robbins, 1998; Wyler, 1996). In addition, it stimulates chemotaxis of fibroblasts. All these activities of fibroblasts are necessary for wound contraction and wound healing (Skalli and Gabbiani, 1998). As an early response to injury, resident dermal fibroblasts in the neighborhood of the wound margin begin to

proliferate and then 3 to 4 days after the wound insult, they begin migration into the provisional wound clot, where they lay down their own matrix (Seemayer and Gabbiani, 1992; Desmouliere et al., 1993). The premigratory lag phase appears to be largely due to the time required for the fibroblasts to emerge from quiescence, because it does not occur a second time around if the wound is rewounded and a new provisional matrix is laid down. It is possible that fibrosin acts to shorten the lag time necessary to emerge from the state of quiescence. Our data suggest that fibrosin may also enhance reepithelialization by acting as a direct mitogen and/or a motogen for epithelial cells. Alternatively, it may exert its effects indirectly by stimulating production of the EGF family of growth factors or TGF␤ or KGF—all of which appear to regenerate epidermal repair (Kovacs, 1991; Clark, 1993). For example, TGF␤1 and some other proinflammatory cytokines appear to stimulate the expression of some of the integrin subunits that facilitate keratinocyte migration (Grinell,

254 1992). EGF, TGF␣, and HB-EGF all act as ligands for the EGF receptor—which is the key regulator for keratinocyte proliferation at the wound edge. Exogenous application of EGF or TGF␣ appears to enhance reepithelialization of burn wounds on the backs of pigs (Brown et al., 1986). KGF is also upregulated and induces both proliferation and motility of epithelial cells when applied to skin wounds (Tsuboi et al., 1993). Our data also suggest that wounds treated with fibrosin appear to show contraction of the wound at a faster rate. Like in vitro activities (Prakash and Robbins, 1998), fibrosin may activate fibroblasts to invade the wound clot and stimulate the cells to synthesize and remodel new collagen matrix. At this stage fibrosin may also be involved in inducing transformation of fibroblasts into myofibroblasts, which express a smooth muscle cell actin and may be responsible for generating strong contractile forces necessary for wound contraction. Other growth factors such as TGF␤ (Desmouliere et al., 1993) and NGF (Micera et al., 2001), have been shown to affect transformation of fibroblasts into myofibroblasts. Further experiments will reveal if fibrosin can indeed trigger differentiation of fibroblasts into myofibroblasts. Different cell types may produce fibrosin. It may be considerably upregulated in fibroblasts, keratinocytes, or other inflammatory cells at the site of injury, e.g., macrophages, lymphocytes, etc. In addition, it is possible that fibrosin is produced by other cell types involved in ECM synthesis, such as chondrocytes. And it may possibly play a role in cartilage and bone repair. These observations suggest that fibrosin can potentially enhance wound healing. Further investigations to study the role of wound healing in other animal models, including hamsters and pigs, will substantiate our current observations. The model system described here represents healing of the epidermis and the dermis and would be similar to disease states involving the skin. Its role in enhancing wound healing under conditions where wound healing is impaired, for example, in diabetes, and burn victims etc., remains to be explored.

PRAKASH AND ROBBINS

REFERENCES

Brown, G. L., Curtsinger, L. IIIrd., Brightwell, J. R., Ackerman, D. M., Tobin, G. R., Polk, H. C. Jr., George-Nascimento, C., Valenzuela, P., and Schultz, G. S. (1986). Enhancement of epidermal regeneration by biosynthetic epidermal growth factor. J. Exp. Med. 163, 1319–1324. Clark, R. A. F. (1993). Biology of wound repair. Dermatol. Clin. 11, 647–666. Desmouliere, A., Geinoz, A., Gabbiani, F., and Gabbiani G. (1993). Transforming growth factor-␤1 induces a-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J. Cell Biol, 122, 103–111. Grinnell, F. (1992). Wound repair, keratinocyte activation and integrin modulation. J. Cell Sci. 101, 1–5. Kovacs, E. J. (1991). Fibrogenic cytokines. The role of immune mediators in the development of scar tissue. Immunol. Today, 12, 17–23. Kiristy, C. P., and Lynch, S. E. (1993). Role of growth factiors in cutaneous wound healing: A review. Crit. Rev. Oral Biol. Med. 4, 729–760. Lynch, S. E., Nixon, J. C., Colvin, R. B., and Antoniades, H. N. (1987). Role of platelet derived growth factor in wound healing: Synergistic effects with other growth factors. Proc. Natl. Acad. Sci. USA 84, 7696–7770. Martin, P. (1997). Wound healing—Aiming for perfect skin regeneration. Science 276, 75–81. Micera, A., Vigneti, E., Pickholtz, D., Reich, R., Papp, O., Bonini, S., Maquart, F. X., Aloe, L., and Levi-Shaffer, F. (2001). Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair. Proc. Natl. Acad. Sci. USA 98, 6162–6167. Moulin, V. (1995). Growth factors in skin wound healing. Eur. J. Cell Biol. 68, 1–7. Prakash, S., Postlethwaite, A. E., and Wyler, D. J. (1991). Alterations in the influence of granuloma derived cytokines in the course of murine Schistosoma mansoni infection. Hepatology 13, 970–976. Prakash, S., and Wyler, D. J. (1991). Fibroblast stimulation in Schistosomiasis XI: Purification to apparent homogeneity of fibroblast stimulating factor (FsF-1), an acidic heparin-binding growth factor produced by schistosomal egg granulomas. J. Immunol. 146, 1679–1684. Prakash, S., Robbins, P. W., and Wyler D. J. (1995). Cloning and analysis of murine cDNA that encodes a fibrogenic lymphokine, fibrosin. Proc. Natl. Acad. Sci. USA 92, 2154–2158. Prakash, S., and Robbins, P. W. (1998). Cloning and analysis of the cDNA for human fibrosin, a novel fibrogenic lymphokine. DNA Cell Biol. 10, 879–884.

ACKNOWLEDGMENT

The authors thank Dr. Robert Marini (Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA) for assisting in surgical procedures while animals were used.

Schurch, W., Seemayer, T. A. and Gabbiani, G. (1992). Myofibroblast. In “Histology for Pathologists” (S. S. Sternberger, Ed.), pp. 109–144. Raven Press, New York. Skalli, O., and Gabbiani, G. (1988). The biology of myofibroblast. Relationship to wound contraction and fibroconnective diseases. In “The Molecular and Cellular Biology of Wound Repair” (R. A. F. Clark and P. M. Henson, Eds. ), pp. 373–401. Plenum, New York.

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Schultz, G., Ritatori, D. S., and Clark, W. (1991). EGF and TGFa in wound healing and repair. J. Cell Biochem. 45, 346–352. Tsuboi, R., Sato, C., Kurita, Y., Ron, D., Rubin, J. S., and Ogawa, H. (1993). KGF (FGF-7) stimulates migration and plasminogen activator activity of normal human keratinocytes. J. Invest. Dermatol. 101, 49–53.

255 Wyler, D. J., Prakash, S., and Postlethwaite, A. E. (1989). Fibrosis as a complication of granulomatous inflammation: Lessons learned from schistosomiasis. In “Basic Mechanisms of Granulomatous Inflammation” (T. Yoshida and M. Torisu, Eds. ), pp. 241–252, Elsevier, Amsterdam. Wyler, D. J. (1996). Fibrosin, a novel fibrogenic protein. Int. Arch. Allergy Immun. 111, 326–329.