Fibromodulin Is Essential for Fetal-Type Scarless Cutaneous Wound Healing
2016; Elsevier BV; Volume: 186; Issue: 11 Linguagem: Inglês
10.1016/j.ajpath.2016.07.023
ISSN1525-2191
AutoresZhong Zheng, Xinli Zhang, Catherine Dang, Steven R. Beanes, Grace Chang, Yao Chen, Chenshuang Li, Kevin M. Lee, Kang Ting, Chia Soo,
Tópico(s)Burn Injury Management and Outcomes
ResumoIn contrast to adult and late-gestation fetal skin wounds, which heal with scar, early-gestation fetal skin wounds display a remarkable capacity to heal scarlessly. Although the underlying mechanism of this transition from fetal-type scarless healing to adult-type healing with scar has been actively investigated for decades, in utero restoration of scarless healing in late-gestation fetal wounds has not been reported. In this study, using loss- and gain-of-function rodent fetal wound models, we identified that fibromodulin (Fm) is essential for fetal-type scarless wound healing. In particular, we found that loss of Fm can eliminate the ability of early-gestation fetal rodents to heal without scar. Meanwhile, administration of fibromodulin protein (FM) alone was capable of restoring scarless healing in late-gestation rat fetal wounds, which naturally heal with scar, as characterized by dermal appendage restoration and organized collagen architectures that were virtually indistinguishable from those in age-matched unwounded skin. High Fm levels correlated with decreased transforming growth factor (TGF)-β1 expression and scarless repair, while low Fm levels correlated with increased TGF-β1 expression and scar formation. This study represents the first successful in utero attempt to induce scarless repair in late-gestation fetal wounds by using a single protein, Fm, and highlights the crucial role that the FM–TGF-β1 nexus plays in fetal-type scarless skin repair. In contrast to adult and late-gestation fetal skin wounds, which heal with scar, early-gestation fetal skin wounds display a remarkable capacity to heal scarlessly. Although the underlying mechanism of this transition from fetal-type scarless healing to adult-type healing with scar has been actively investigated for decades, in utero restoration of scarless healing in late-gestation fetal wounds has not been reported. In this study, using loss- and gain-of-function rodent fetal wound models, we identified that fibromodulin (Fm) is essential for fetal-type scarless wound healing. In particular, we found that loss of Fm can eliminate the ability of early-gestation fetal rodents to heal without scar. Meanwhile, administration of fibromodulin protein (FM) alone was capable of restoring scarless healing in late-gestation rat fetal wounds, which naturally heal with scar, as characterized by dermal appendage restoration and organized collagen architectures that were virtually indistinguishable from those in age-matched unwounded skin. High Fm levels correlated with decreased transforming growth factor (TGF)-β1 expression and scarless repair, while low Fm levels correlated with increased TGF-β1 expression and scar formation. This study represents the first successful in utero attempt to induce scarless repair in late-gestation fetal wounds by using a single protein, Fm, and highlights the crucial role that the FM–TGF-β1 nexus plays in fetal-type scarless skin repair. Cutaneous fibrosis (scarring) affects up to 100 million patients each year.1Sund B. New Developments in Wound Care. PJB Publications, London2000: 1-255Google Scholar It is characterized by disorganized extracellular matrix and lack of normal dermal appendages such as hair follicles.2Eming S.A. Martin P. Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation.Sci Transl Med. 2014; 6: 265sr6Crossref PubMed Scopus (1523) Google Scholar Interestingly, skin transplantation or grafting methodologies have shown that fetal skin itself, rather than the intrauterine environment, possesses the intrinsic cells and molecular signals to heal scarlessly, with restoration of normal dermal extracellular matrix architecture and appendages.3Larson B.J. Longaker M.T. Lorenz H.P. Scarless fetal wound healing: a basic science review.Plast Reconstr Surg. 2010; 126: 1172-1180Crossref PubMed Scopus (304) Google Scholar, 4Rolfe K.J. Grobbelaar A.O. A review of fetal scarless healing.ISRN Dermatol. 2012; 2012: 698034Crossref PubMed Google Scholar, 5Lo D.D. Zimmermann A.S. Nauta A. Longaker M.T. Lorenz H.P. Scarless fetal skin wound healing update.Birth Defects Res C Embryo Today. 2012; 96: 237-247Crossref PubMed Scopus (92) Google Scholar For example, 15- to 22-week human fetal skin grafted s.c. into athymic nude mice retained the ability to heal without scar, while this ability for scarless skin repair was lost with increasing gestational age.3Larson B.J. Longaker M.T. Lorenz H.P. Scarless fetal wound healing: a basic science review.Plast Reconstr Surg. 2010; 126: 1172-1180Crossref PubMed Scopus (304) Google Scholar, 5Lo D.D. Zimmermann A.S. Nauta A. Longaker M.T. Lorenz H.P. Scarless fetal skin wound healing update.Birth Defects Res C Embryo Today. 2012; 96: 237-247Crossref PubMed Scopus (92) Google Scholar Significant research efforts have demonstrated that fetal wounds express less profibrotic factors such as transforming growth factor (TGF)-β1, and more antifibrotic factors such as TGF-β33Larson B.J. Longaker M.T. Lorenz H.P. Scarless fetal wound healing: a basic science review.Plast Reconstr Surg. 2010; 126: 1172-1180Crossref PubMed Scopus (304) Google Scholar, 6Soo C. Beanes S.R. Hu F.Y. Zhang X. Dang C. Chang G. Wang Y. Nishimura I. Freymiller E. Longaker M.T. Lorenz H.P. Ting K. Ontogenetic transition in fetal wound transforming growth factor-beta regulation correlates with collagen organization.Am J Pathol. 2003; 163: 2459-2476Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar and IL-107Liechty K.W. Kim H.B. Adzick N.S. Crombleholme T.M. Fetal wound repair results in scar formation in interleukin-10-deficient mice in a syngeneic murine model of scarless fetal wound repair.J Pediatr Surg. 2000; 35 (discussion 72–73): 866-872Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 8Gordon A. Kozin E.D. Keswani S.G. Vaikunth S.S. Katz A.B. Zoltick P.W. Favata M. Radu A.P. Soslowsky L.J. Herlyn M. Crombleholme T.M. Permissive environment in postnatal wounds induced by adenoviral-mediated overexpression of the anti-inflammatory cytokine interleukin-10 prevents scar formation.Wound Repair Regen. 2008; 16: 70-79Crossref PubMed Scopus (77) Google Scholar as well as a higher ratio of type III to type I collagen.3Larson B.J. Longaker M.T. Lorenz H.P. Scarless fetal wound healing: a basic science review.Plast Reconstr Surg. 2010; 126: 1172-1180Crossref PubMed Scopus (304) Google Scholar, 9Beanes S.R. Hu F.Y. Soo C. Dang C.M. Urata M. Ting K. Atkinson J.B. Benhaim P. Hedrick M.H. Lorenz H.P. Confocal microscopic analysis of scarless repair in the fetal rat: defining the transition.Plast Reconstr Surg. 2002; 109: 160-170Crossref PubMed Scopus (85) Google Scholar However, Phase 3 human clinical trials with recombinant human TGF-β3 (avotermin; Juvista, Renovo, Manchester, UK)10Longaker M.T. Rohrich R.J. Greenberg L. Furnas H. Wald R. Bansal V. Seify H. Tran A. Weston J. Korman J.M. Chan R. Kaufman D. Dev V.R. Mele J.A. Januszyk M. Cowley C. McLaughlin P. Beasley B. Gurtner G.C. A randomized controlled trial of the Embrace advanced scar therapy device to reduce incisional scar formation.Plast Reconstr Surg. 2014; 134: 536-546Crossref PubMed Scopus (63) Google Scholar and three Phase 2 studies with IL-10 failed to show efficacy in scar reduction.11Kieran I. Knock A. Bush J. So K. Metcalfe A. Hobson R. Mason T. O'Kane S. Ferguson M. Interleukin-10 reduces scar formation in both animal and human cutaneous wounds: results of two preclinical and phase II randomized control studies.Wound Repair Regen. 2013; 21: 428-436Crossref PubMed Scopus (85) Google Scholar, 12Kieran I. Taylor C. Bush J. Rance M. So K. Boanas A. Metcalfe A. Hobson R. Goldspink N. Hutchison J. Ferguson M. Effects of interleukin-10 on cutaneous wounds and scars in humans of African continental ancestral origin.Wound Repair Regen. 2014; 22: 326-333Crossref PubMed Scopus (17) Google Scholar In an effort to better identify key factors in scarless fetal-type repair, we used a fetal rat skin model that transitions from fetal-type scarless healing to adult-type repair with scar between embryonic days 16.5 (E16; early gestation) and 18.5 (E18; late gestation) (term, 21.5 days).3Larson B.J. Longaker M.T. Lorenz H.P. Scarless fetal wound healing: a basic science review.Plast Reconstr Surg. 2010; 126: 1172-1180Crossref PubMed Scopus (304) Google Scholar, 9Beanes S.R. Hu F.Y. Soo C. Dang C.M. Urata M. Ting K. Atkinson J.B. Benhaim P. Hedrick M.H. Lorenz H.P. Confocal microscopic analysis of scarless repair in the fetal rat: defining the transition.Plast Reconstr Surg. 2002; 109: 160-170Crossref PubMed Scopus (85) Google Scholar We previously reported a significant decrease of fibromodulin (Fm) expression associated with the transition from scarless fetal-type to adult-type repair with scar.13Soo C. Hu F.Y. Zhang X. Wang Y. Beanes S.R. Lorenz H.P. Hedrick M.H. Mackool R.J. Plaas A. Kim S.J. Longaker M.T. Freymiller E. Ting K. Differential expression of fibromodulin, a transforming growth factor-beta modulator, in fetal skin development and scarless repair.Am J Pathol. 2000; 157: 423-433Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar Fibromodulin protein (FM) is a small leucine-rich proteoglycan involved in angiogenesis and fibrillogenesis,14Jian J. Zheng Z. Zhang K. Rackhon T.M. Hsu C. Levin A. Enjamuri D.R. Zhang X. Ting K. Soo C. Fibromodulin promoted in vitro and in vivo angiogenesis.Biochem Biophys Res Commun. 2013; 436: 530-535Crossref PubMed Scopus (45) Google Scholar, 15Zheng Z. Jian J. Velasco O. Hsu C.Y. Zhang K. Levin A. Murphy M. Zhang X. Ting K. Soo C. Fibromodulin enhances angiogenesis during cutaneous wound healing.Plast Reconstr Surg Glob Open. 2014; 2: e275Crossref PubMed Scopus (33) Google Scholar, 16Adini I. Ghosh K. Adini A. Chi Z.L. Yoshimura T. Benny O. Connor K.M. Rogers M.S. Bazinet L. Birsner A.E. Bielenberg D.R. D'Amato R.J. Melanocyte-secreted fibromodulin promotes an angiogenic microenvironment.J Clin Invest. 2014; 124: 425-436Crossref PubMed Scopus (50) Google Scholar, 17Khorasani H. Zheng Z. Nguyen C. Zara J. Zhang X. Wang J. Ting K. Soo C. A quantitative approach to scar analysis.Am J Pathol. 2011; 178: 621-628Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 18Iozzo R.V. Goldoni S. Berendsen A.D. Young M.F. Small leucine-rich proteoglycans.in: Mecham R.P. The Extracellular Matrix: An Overview. Springer, Berlin, Heidelberg2011: 197-231Crossref Google Scholar but its role in fetal and adult cutaneous repair is not fully understood. Our current study used Fm loss- and gain-of-function wound models to determine the necessity and sufficiency of FM in fetal scarless healing. All animal surgeries were performed under the institutionally approved protocols provided by the Chancellor's Animal Research Committee at the University of California, Los Angeles (Los Angeles, CA; protocol number 2000-058). Three-month-old male and female Sprague-Dawley rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA), and housed in a light- and temperature-controlled animal facility at UCLA. Pregnant rats carrying fetuses at E16 were anesthetized. Using aseptic technique, laparotomy was performed via a midline incision to expose the uterus. A 7-0 Nylon purse-string suture was then placed in the uterine wall. The myometrium and amniotic sac were incised at the center of the purse string to expose the fetus as previously described.13Soo C. Hu F.Y. Zhang X. Wang Y. Beanes S.R. Lorenz H.P. Hedrick M.H. Mackool R.J. Plaas A. Kim S.J. Longaker M.T. Freymiller E. Ting K. Differential expression of fibromodulin, a transforming growth factor-beta modulator, in fetal skin development and scarless repair.Am J Pathol. 2000; 157: 423-433Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar Using a microinjector (Hamilton Co., Reno, NV), 10 μL of sterile phosphate-buffered saline (PBS) solution consisting of permanent dye and 8 mg/mL rabbit anti-FM antibody19Plaas A.H. Wong-Palms S. Biosynthetic mechanisms for the addition of polylactosamine to chondrocyte fibromodulin.J Biol Chem. 1993; 268: 26634-26644PubMed Google Scholar was injected into the fetus to raise a skin wheal. A 2-mm-diameter, circular, full-thickness skin excisional wound was generated at the site of injection. The wound was then marked with additional permanent dye, and the hysterotomy was closed by tightening of the purse-string suture. Two control groups received 10 μL of 20 mg/mL rabbit immunoglobulin (Santa Cruz Biotechnology, Santa Cruz, CA) in PBS, or 10 μL of PBS alone. Sterile normal saline was injected into the amniotic sac to replace lost amniotic fluid before hysterotomy closure. In a typical litter of 12 fetuses, 6 littermates were wounded, and the remaining fetuses were left unwounded. Operations were performed in 10 maternal rats. On completion of the fetal surgeries, the maternal rat was given an i.p. normal saline fluid bolus (30 mL/kg), and the laparotomy incision was closed. Maternal rats were monitored closely for 6 hours after surgery and given food and water ad libitum once they had recovered from anesthesia.13Soo C. Hu F.Y. Zhang X. Wang Y. Beanes S.R. Lorenz H.P. Hedrick M.H. Mackool R.J. Plaas A. Kim S.J. Longaker M.T. Freymiller E. Ting K. Differential expression of fibromodulin, a transforming growth factor-beta modulator, in fetal skin development and scarless repair.Am J Pathol. 2000; 157: 423-433Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar Using the same technique, operations were performed on pregnant rats carrying E18 fetuses. On exposure of the fetus, 10 μL of 0.1 mg/mL sterile recombinant human FM20Zheng Z. Nguyen C. Zhang X. Khorasani H. Wang J.Z. Zara J.N. Chu F. Yin W. Pang S. Le A. Ting K. Soo C. Delayed wound closure in fibromodulin-deficient mice is associated with increased TGF-beta3 signaling.J Invest Dermatol. 2011; 131: 769-778Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar in 1 mg/mL Vitrogen (type I collagen, aka PureCol; Advanced BioMatrix, San Diego, CA; to keep FM localization) was injected superficially into the dorsum of the fetus to raise a skin wheal. After the injection, a 2-mm-diameter, circular, full-thickness skin excisional wound was generated. The wounds were then marked with permanent dye, and 10 μL of a half-strength Fm solution was applied topically to the wound. Two control groups received 10 μL of 1 mg/mL Vitrogen, or 10 μL PBS alone. Operations were performed in a total of 13 maternal rats. Using the same hysterotomy technique described for fetal rat surgery, operations were performed on pregnant maternal 129/sv wild-type and Fm-null (Fmod−/−) mice21Svensson L. Aszodi A. Reinholt F. Heinegard D. Oldberg A. Fibromodulin-null mice have abnormal collagen fibrils, tissue organization, and altered lumican deposition in tendon.J Biol Chem. 1999; 274: 9636-9647Crossref PubMed Scopus (379) Google Scholar carrying E16 fetuses (term, 20 to 21 days). A 1-mm-diameter, circular, full-thickness skin excisional wound was generated on the dorsum of only one fetus of each litter, and the remaining littermates were left unwounded. The wounds were then marked with permanent dye. For rescue experiments, 10 μL of recombinant human FM (0.1 mg/mL) in Vitrogen (1 mg/mL) was applied topically to the wound. A separate group treated with Vitrogen alone was used as control. Operations were performed in 12 mice per group. Unwounded E16 and E18 fetal rat skin samples were sent to Miltenyi Biotec GmbH (Cologne, Germany) for microarray analysis. Briefly, four E16 and three E18 total RNA samples were pooled. A total of 1 μg of each RNA sample was amplified and labeled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies, Santa Clara, CA) following the manufacturer's protocol. Then, the hybridization procedure was performed according to the 60-mer oligo microarray processing protocol using the Gene Expression Hybridization Kit (Agilent Technologies). A volume of 825 ng of the corresponding cyanine 3– and cyanine 5–labeled fragmented cRNA was combined and hybridized overnight (17 hours, 65°C) to Whole Rat Genome Oligo Microarray Kit, 4 × 44K (Agilent Technologies) using the manufacturer's recommended hybridization chamber and oven. Fluorescence signals of the hybridized microarrays were detected using a DNA microarray scanner (Agilent Technologies). The Feature Extraction software package version 9.5.1.1 (Agilent Technologies) was used to read and process the microarray image files. For the determination of differential gene expression, Feature Extraction software–derived output data files were further analyzed using the Rosetta Resolver gene expression data analysis system version 6.0 (Rosetta Biosoftware, Seattle, WA). The microarray data were submitted to Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo; accession number GSE74976). Animals were sacrificed at 72 hours after injury for sample harvesting. After fixation in 4% paraformaldehyde at 4°C overnight, samples were dehydrated, paraffin-embedded, and sectioned at 5-μm increments for hematoxylin and eosin staining, or at 10-μm increments for Picrosirius red staining. To ensure that the histologic sections were confined to the wound and not the nearby skin, the samples were sectioned with the index of the permanent dye. Immunohistochemical staining was performed and analyzed as previously described.20Zheng Z. Nguyen C. Zhang X. Khorasani H. Wang J.Z. Zara J.N. Chu F. Yin W. Pang S. Le A. Ting K. Soo C. Delayed wound closure in fibromodulin-deficient mice is associated with increased TGF-beta3 signaling.J Invest Dermatol. 2011; 131: 769-778Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Primary antibody against TGF-β1 was purchased from Santa Cruz Biotechnology. Computerized immunolocalization intensity analyses were performed using Image-Pro Plus software version 6.0 (Media Cybernetics Inc., Rockville, MD). Relative dermal protein expression was quantified as the Mean optical density of staining signal × Percentage of area positively stained × 100.20Zheng Z. Nguyen C. Zhang X. Khorasani H. Wang J.Z. Zara J.N. Chu F. Yin W. Pang S. Le A. Ting K. Soo C. Delayed wound closure in fibromodulin-deficient mice is associated with increased TGF-beta3 signaling.J Invest Dermatol. 2011; 131: 769-778Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 22Allen A. Southern S. A novel technique of computer-assisted image analysis to quantify molecular stress in cetaceans.2002Google Scholar, 23Zheng Z. Lee K.S. Zhang X. Nguyen C. Hsu C. Wang J.Z. Rackohn T.M. Enjamuri D.R. Murphy M. Ting K. Soo C. Fibromodulin-deficiency alters temporospatial expression patterns of transforming growth factor-beta ligands and receptors during adult mouse skin wound healing.PLoS One. 2014; 9: e90817Crossref PubMed Scopus (25) Google Scholar After Picrosirius red staining, the dermal collagen deposition pattern of the upper dermis was evaluated by confocal microscopy on a Carl Zeiss LSM 510 META Laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) by previously published methods.17Khorasani H. Zheng Z. Nguyen C. Zara J. Zhang X. Wang J. Ting K. Soo C. A quantitative approach to scar analysis.Am J Pathol. 2011; 178: 621-628Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Since fractal dimension (FD) and lacunarity (L) analyses are more sensitive than are conventional methods such as polarized light microscopy, X-ray diffraction, laser scattering, and Fourier transform analysis, collagen organization architecture was assessed by FD and L analyses to quantify collagen organizational architecture in scar area as described previously.17Khorasani H. Zheng Z. Nguyen C. Zara J. Zhang X. Wang J. Ting K. Soo C. A quantitative approach to scar analysis.Am J Pathol. 2011; 178: 621-628Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Total RNA of unwounded E16 and E18 fetal rat skin was isolated using the RNeasy Mini Kit with DNase treatment (Qiagen, Valencia, CA), while wound tissues were collected by microdissection from tissue sections.24Erickson H.S. Gillespie J.W. Emmert-Buck M.R. Tissue microdissection.Methods Mol Biol. 2008; 424: 433-448Crossref PubMed Scopus (20) Google Scholar Total RNA was isolated using the RNeasy FFPE Kit (Qiagen). One microgram of total RNA was reverse-transcribed into cDNA in a 20-μL reaction mixture with 50 pmol of oligo(dT)20 primer and 1 μL (200 U) of SuperScript III Reverse Transcriptase (Invitrogen, Foster City, CA). Expression of mRNA was measured by real-time quantitative RT-PCR using TaqMan Gene Expression Assays on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Concurrent expression of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was also assessed in separate tubes for each RT reaction with TaqMan Rodent Gapdh control reagents (Applied Biosystems). Three separate sets of real-time quantitative RT-PCR analysis were performed using different complementary DNA templates. All statistical analyses were conducted as per consultation with the UCLA Statistical Biomathematical Consulting Clinic. Statistical analysis was performed using OriginPro software version 8 (Originlab Corp., Northampton, MA). Data are generally presented as means ± SD. P < 0.05 was considered statistically significant. Two-sample t-tests were used to compare results between two groups. Global gene profiling revealed that the expression of Fm was significantly decreased during the transition from fetal-type scarless healing to adult-type repair with scar between E16 to E18 in a fetal rat cutaneous wound model, while the expression of other small leucine-rich proteoglycans was increased or unchanged (Supplemental Figure S1A and Supplemental Table S1) and was further confirmed by real-time quantitative RT-PCR (Supplemental Figure S1B). Furthermore, in a loss-of function experiment, injection of anti-FM antibody was sufficient to induce scar formation in normally scarless E16 rat wounds, which was not seen in PBS- and IgG-treated wounds (Figure 1A). The scarring in anti-FM antibody–treated E16 wounds resembled scarring in E18 rat wounds: Both were characterized by absent dermal appendages (eg, hair follicles) (Figure 1B) and a denser, more disorderly collagen deposition pattern (Figure 1A). To quantitate collagen organizational architecture, we performed FD and L analyses. FD provides a measure of how completely an object fills space, and it quantifies an object in terms of shape, regularity, lack of smoothness, size, and number of self-similarities (ie, invariance regardless of scale).25Smith Jr., T.G. Lange G.D. Marks W.B. Fractal methods and results in cellular morphology–dimensions, lacunarity and multifractals.J Neurosci Methods. 1996; 69: 123-136Crossref PubMed Scopus (464) Google Scholar In general, a higher FD value indicates uniform distribution.25Smith Jr., T.G. Lange G.D. Marks W.B. Fractal methods and results in cellular morphology–dimensions, lacunarity and multifractals.J Neurosci Methods. 1996; 69: 123-136Crossref PubMed Scopus (464) Google Scholar In contrast, L permits an analysis of density, packing, or dispersion through scales. In other words, L is a measure of the heterogeneity of a structure or the degree of structural variance within an object, which is related to the distribution of empty spaces (lacunas) of an image.25Smith Jr., T.G. Lange G.D. Marks W.B. Fractal methods and results in cellular morphology–dimensions, lacunarity and multifractals.J Neurosci Methods. 1996; 69: 123-136Crossref PubMed Scopus (464) Google Scholar Objects with lower L values are of a finer texture, while higher L values indicate that objects are more spatially unorganized.26Ling E.J. Servio P. Kietzig A.M. Fractal and lacunarity analyses: quantitative characterization of hierarchical surface topographies.Microsc Microanal. 2016; 22: 168-177Crossref PubMed Scopus (11) Google Scholar In this study, we showed that anti-Fm antibody treatment decreased the mean FD value but increased the mean L value of the E16 rat wounds in comparison with those in other groups (Figure 1C), indicating that the collagen fibers in those wounds were disorganized or less uniform. On the other hand, 72 hours after injury, PBS- and IgG-treated E16 rat wounds had FD and L values similar to those of age-matched unwounded skin (Figure 1C). Additionally, while unwounded Fmod−/− mice21Svensson L. Aszodi A. Reinholt F. Heinegard D. Oldberg A. Fibromodulin-null mice have abnormal collagen fibrils, tissue organization, and altered lumican deposition in tendon.J Biol Chem. 1999; 274: 9636-9647Crossref PubMed Scopus (379) Google Scholar showed normal dermal histologic architecture, E16 Fmod−/− mouse wounds healed with scarring characterized by a hypertrophic epidermis and absence of dermal appendages compared with E16 wild-type control wounds displaying normal scarless healing, which were partially rescued by application of FM protein in a Vitrogen vehicle but not by Vitrogen alone (Figure 2). These findings demonstrated that loss of a single extracellular matrix proteoglycan, Fm, caused normally scarless E16 fetal rodent skin wounds to heal with scar.Figure 2Fibromodulin (Fm) deficiency leads to scar formation in embryonic day 16.5 (E16) fetal mouse wounds. A: Unwounded embryonic day 19.5 (E19) wild-type (WT) and Fmod−/− mouse skin shows no significant histologic difference. B: At 72 hours after injury, E16 fetal mouse wounds shows scarless repair, while E16 Fmod−/− fetal mouse wounds present scar formation characterized by the absence of hair follicles and epidermal hypertrophy. C: Administration of FM with Vitrogen partially restores scarless wound healing in E16 Fmod−/− wounds, while Vitrogen control alone fails to do so. Hair follicle densities are analyzed based on hematoxylin and eosin (H&E) staining (D), while fractal dimension (Fd) and lacunarity (L) are analyzed based on Picrosirius red–confocal laser scanning microscopy (PSR-CLSM) imagery (E). Blue arrowhead, hair follicle; black arrowhead, surgical dye. Data are expressed as means ± SD. n = 6. ∗P < 0.05 (two-sample t-test). Scale bars: 50 μm (black); 25 μm (white).View Large Image Figure ViewerDownload Hi-res image Download (PPT) On the other hand, in a gain-of-function experiment, we demonstrated that the administration of FM protein in a Vitrogen collagen vehicle prevented scar formation in E18 rats; surprisingly, the wounds healed scarlessly, with hair follicles and a more organized collagen architecture that were virtually indistinguishable from those in age-matched unwounded skin (Figure 1, D and E). However, PBS- and Vitrogen-treated E18 wounds healed with scar as expected, accompanied by a lower mean FD value and a higher mean L value at 72 hours after injury in comparison with unwounded E21 skin (Figure 1, D–F). Moreover, FM treatment increased the mean FD value and decreased the mean L value of E18 wounds at 72 hours after injury to the same levels as these values in unwounded E21 skin (Figure 1F), representing more organized collagen fibers. Therefore, the addition of FM alone was sufficient to regenerate scarless fetal-type wound healing in late-gestation animals, which normally exhibit adult-type repair with scar. Taken together, these findings demonstrate that FM is both necessary and sufficient for fetal scarless repair in rodents. Importantly, FM application can reverse scarring in late-gestation fetal rodent wounds. Because previous studies revealed that fetal-type scarless wounds express less Tgf-β1 compared with adult-type scarring wounds,3Larson B.J. Longaker M.T. Lorenz H.P. Scarless fetal wound healing: a basic science review.Plast Reconstr Surg. 2010; 126: 1172-1180Crossref PubMed Scopus (304) Google Scholar, 6Soo C. Beanes S.R. Hu F.Y. Zhang X. Dang C. Chang G. Wang Y. Nishimura I. Freymiller E. Longaker M.T. Lorenz H.P. Ting K. Ontogenetic transition in fetal wound transforming growth factor-beta regulation correlates with collagen organization.Am J Pathol. 2003; 163: 2459-2476Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar we next evaluated Tgf-β1 expression in Fm gain- and loss-of-function fetal wound models to assess for a possible correlation. The scars formed in E18 rats treated with Vitrogen control presented strong Tgf-β1 immunohistochemical analysis staining throughout the epidermis and dermis (Figure 3A). Conversely, scarless wounds in FM-treated E18 rats exhibited markedly less Tgf-β1 staining in the epidermis and limited Tgf-β1 staining in the dermis (Figure 3, A and B). Accordingly, real-time quantitative RT-PCR revealed increased Tgf-β1 mRNA levels in collagen control–treated E18 rat wounds at 72 hours after injury compared with the Fm-treated wounds, the Tgf-β1 expression of which was reduced to the level in unwounded skin (Figure 3C). Similarly, transcription of Tgf-β1 downstream target Col1α1 (encoding α1 chain of type I collagen) was also elevated in E18 rat wounds treated with Vitrogen control, but Col3α1 (encoding α1 chain of type III collagen) did not show an increase (Figure 3D). Thus, it resulted in a decreased ratio of type III to type I collagen. As expected, FM application eliminated the injury-induced increase of Tgf-β1, and Col1α1 increased in E18 rat wounds (Figure 3, C and D). Meanwhile, E16 Fmod−/− mouse wounds that healed with scarring showed significantly increased Tgf-β1 expression compared with wild-type wounds accompanied by higher Col1α1 transcription. However, FM administration significantly reduced Tgf-β1 expression in E16 Fmod−/− wounds and promoted scarless healing (Figure 4). These data
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