Myofibroblast induction with transforming growth factor-β1 and -β3 in cutaneous fetal excisional wounds

doi:10.1016/S0022-3468(00)90007-1

Journal of Pediatric Surgery

Volume 35, Issue 2, February 2000, Pages 183-188

Presented at the 30th Annual Meeting of the American Pediatric Surgical Association, Rancho Mirage, California, May 16-19, 1999.

https://doi.org/10.1016/S0022-3468(00)90007-1 Get rights and content

Abstract

Background/Purpose: In a noncontractile fetal rabbit model, the authors recently have shown the induction of excisional wound contraction with sustained-release cellulose implants formulated with transforming growth factor (TGF)-β. The purpose of this study was to test the hypothesis that the excisional wound contraction in this model is associated with the induction of myofibroblasts in the surrounding dermis, demonstrated by the presence of α-smooth muscle actin. Methods: Cellulose discs were formulated with either 1.0 μg of TGF-β₁ (n = 6); 1.0 μg of TGF-β₃ (n = 9); 10 μg of TGF-β₃ (n = 6); or their carrier protein, bovine serum albumin (BSA; n = 9), for sustained-release over 5 days. Each disc was implanted into a subcutaneous pocket on the back of a fetal New Zealand White rabbit in utero on day 24 of gestation (term, 31 days). A full-thickness, 3-mm excisional wound (7.4 mm²) was then made next to the implanted cellulose disc. All fetuses were harvested at 3 days. The amount of α-smooth muscle (SM) actin in the dermis around the implants and wounds was determined using immunohistochemical techniques. Results: Excisional wounds exposed to 1.0 μg of TGF-β₁ (5.6 ± 2.0 mm²), 1.0 μg of TGF-β₃ (6.9 ± 1.0 mm²), and 10 μg of TGF-β₃ (2.7 ± 1.0 mm²) were significantly smaller when compared with the BSA control group (12.8 ± 1.1 mm²; P <.05). Furthermore, there was a significant increase in staining for α-SM actin in the TGF-β₁ (1.8 ± 0.5) and 10 μg TGF-β₃ (2.8 ± 0.2) groups in comparison with the scant staining in the BSA control group (0.5 ± 0.2; P <.05). Conclusions: TGF-β₁ and -β₃ induce α-SM actin and contraction of cutaneous excisional wounds in a fetal noncontractile model. This model of inducible cutaneous excisional wound contraction may be useful in further determining the role of the myofibroblast in wound contraction and the physiology underlying this poorly understood aspect of wound healing. J Pediatr Surg 35:183-188. Copyright © 2000 by W.B. Saunders Company.

Section snippets

Animals

Time-dated pregnant New Zealand White rabbits (Myrtle's Rabbitry, Thompson Station, TN) were maintained in the animal facilities at the Medical College of Virginia and allowed to acclimate to the environment for 9 to 10 days preoperatively. Rabbits were provided chow and water ad libitum and evaluated on a daily basis by a staff veterinarian. Approval for this study was obtained from the Institutional Animal Care and Use Committee.

Implants

Discs composed of cellulose, stearates, lactose, phosphates, and

Results

Excisional wounds exposed to 1.0 μg of TGF-β₁ (5.6 ± 2.0 mm²), 1.0 μg of TGF-β₃ (6.9 ± 1.0 mm²), and 10 μg TGF-β₃ (2.7 ± 1.0 mm²) were significantly smaller when compared with the bovine serum albumin (12.8 ± 1.1 mm²), blank implant (13.3 ± 2.8 mm²), and no-implant (16.9 ± 1.5 mm²) control groups (P <.05; Figs 1A, B and 2).

. Representative excisional wounds from fetuses from the BSA control (A) and 10 μg TGF-β₃ (B) groups at the time of harvest, 3 days after wounding. The wound in the TGF-β₃

Discussion

Since its initial description and characterization over 20 years ago, the myofibroblast is thought by many to be the key effector cell in wound contraction.1, 2 This specialized fibroblast contains α-SM actin microfilaments that may generate contractile forces.¹³ Collagen lattice gels have been shown to contract significantly more when populated with myofibroblasts as opposed to normal fibroblasts.¹⁴ Also, in adult porcine excisional wounds, the number of myofibroblasts was found to correlate

Acknowledgements

The authors thank Anita Roberts, PhD, from the National Cancer Institute, and David A. Cox, PhD, from Ciba-Geigy, for donating the TGF-β₁ and TGF-β₃ used in this study. The authors also thank Luke G. Wolfe, PhD for his assistance with the statistical analysis and Sue Dennison for her technical assistance.

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TGF-β₃, a subtype of transforming growth factor-β (TGF-β), is essential to various biological processes, including endoderm development, organogenesis, epithelial hyperplasia, synthesis of extracellular matrix, and immune response. Essentially, TGF-β₃ engages the TGF-β₁/Smad signaling pathway to stimulate mesenchymal lineage cells, inhibit epithelial or neuroectodermal lineage cells, and regulate repair, remodeling, and potential scarring after cutaneous wounding. We have now expressed recombinant human TGF-β₃ in Escherichia coli Origami B (DE3), with yield 300 ± 17 mg/L monomeric protein at pilot scale. Identity was confirmed by western blot and HPLC-based peptide mapping. After purification and refolding, dimeric proteins were found to induce chondro-related genes in adipose-derived stem cells, and to suppress scarring in injured rabbit ears. Thus, the recombinant protein has excellent potential for medical applications.
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For example, fetal sheep wounds that heal scar free have no myofibroblasts within the wound, but as wounds are made on progressively older fetuses, myofibroblasts become present and this is associated with scarring (Estes et al., 1994). In fact, because TGFβ1 application to fetal wounds induces myofibroblast transformation, it is likely that the control of TGFβ isoforms plays a role in controlling this event (Lanning et al., 2000). Furthermore, scarring and nonscarring fibroblasts from different aged fetuses respond differently to the same environment.
Regeneration biology has experienced a renaissance as clinicians, scientists, and engineers have combined forces to drive the field of regenerative medicine. Studies investigating the mechanisms that regulate wound healing in adult mammals have led to a good understanding of the stereotypical processes that lead to scarring. Despite comparative studies of fetal wound healing in which no scar is produced, the fact remains that insights from this work have failed to produce therapies that can regenerate adult human skin. In this review, we analyze past and contemporary accounts of wound healing in a variety of vertebrates, namely, fish, amphibians, and mammals, in order to demonstrate how examples of skin regeneration in adult organisms can impact traditional wound-healing research. When considered together, these studies suggest that inflammation and reepithelialization are necessary events preceding both scarring and regeneration. However, the extent to which these processes may direct one outcome over another is likely weaker than currently accepted. In contrast, the extent to which newly deposited extracellular matrix in the wound bed can be remodeled into new skin, and the intrinsic ability of new epidermis to regenerate appendages, appears to underlie the divergence between scar-free healing and the persistence of a scar. We discuss several ideas that may offer areas of overlap between researchers using these different model organisms and which may be of benefit to the ultimate goal of scar-free human wound healing.
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Mature TGF-β1 and TGF-β3 peptides show a high degree of sequence identity (77% in human, 72% in chicken) and activate the same receptors and subsequent downstream signaling. Other reports in fact demonstrate very similar activities of both isoforms in the induction of αSMA expression and myofibroblastic differentiation both in vitro and in vivo (Jobling et al., 2009; Lanning et al., 2000). We chose to compare the responsiveness of selected genes to MPPN treatment in CEDM cells and to high sustained TGF-β3 secretion in DF1 cells to rule out possible objection of TGF-β signaling unrelated effects of the chemical inhibitor.
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Partial regeneration of adult organs has been accomplished by induction. This is increasingly an alternative to organ transplantation. Skin was the first adult organ to be induced to regenerate partly in animals and in humans. Skin regeneration was induced following grafting of a highly porous scaffold based on a graft copolymer of type I collagen and chondroitin 6-sulfate (CG scaffold). Peripheral nerves and the conjunctiva have been similarly induced to regenerate. The structural determinants of active CG scaffolds have been determined. The data support a theory of induced regeneration in which contraction of a severe wound in an organ is blocked by the CG scaffold. Contraction blocking appears to require reduction of MFB (myofibroblasts) density in the wound as well as integrin-specific binding of contractile fibroblasts (MFB) on the extensive surface of the CG scaffold. When present in such reduced numbers and oriented quasi-randomly in the plane of the wound, MFB appear to lose their ability to contract wounds. Active CG scaffolds are powerful insoluble regulators of cell function. They appear to modify the adult wound healing to a process that resembles early fetal healing.

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^☆: Supported by NIH grants GM 47566 and 08695-01, a Jeffress Memorial Trust Research Grant, and a Plastic Surgery Educational Foundation Grant.

^☆☆: Address reprint requests to Jeffrey H. Haynes, MD, Medical College of Virginia, Virginia Commonwealth University, Box 980015, Richmond, VA 23298.

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Myofibroblast induction with transforming growth factor-β1 and -β3 in cutaneous fetal excisional wounds☆,☆☆

Abstract

Section snippets

Animals

Implants

Results

Discussion

Acknowledgements

Pathology

J Pediatr Surg

J Surg Res

Am J Surg

Differentiation

Dev Biol

J Pediatr Surg

J Pediatr Surg

Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction

Experientia

Granulation tissue as a contractile organ: A study of structure and function

J Exp Med

The spatial and temporal quantification of myofibroblasts

Plast Reconstr Surg

Cytoskeletal protein modulation in pulmonary alveolar myofibroblasts during idiopathic pulmonary fibrosis. Possible role of transforming growth factor beta and tumor necrosis factor alpha

Am J Respir Crit Care Med

Relationship between alpha-smooth muscle actin expression and fibrotic changes in human kidney

Exp Nephrol