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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 15, 2003 as doi:10.1096/fj.03-0068fje. |
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Division of Plastic Surgery, Department of Surgery, Cedars-Sinai Research Institute and University of Southern California, Los Angeles, California, USA;
* Department of Biology, Yale University School of Medicine, New Haven, Connecticut, USA; and
Hollis-Eden Pharmaceuticals, San Diego, California, USA
3Correspondence: Cedars-Sinai Research Institute, 8700 Beverly Blvd., Davis 1091, Los Angeles, CA 90048, USA. E-mail: honigmannr{at}cshs.org
SPECIFIC AIMS
This study was performed to investigate the role of leptin in wound healing. The specific aims were to determine whether leptin is present at the wound site and whether this might reflect up-regulation of leptin expression as a consequence of active synthesis in response to the tissue insult. Leptin is a hypoxia-inducible cytokine functionally related to the IL-6 cytokine family. As a well-documented angiogenic molecule, leptin may mediate wound neovascularization and have additional effects in cells involved in the healing process, including fibroblasts, macrophages, and keratinocytes. Wound resident cells actively engage in acute synthesis of leptin within the first 4 h after injury, which is maintained throughout the various phases of the healing process. Treatment of wounds with neutralizing anti-leptin antibodies severely disrupts a variety of morphological parameters of wound healing such as wound contraction, re-epithelialization, and matrix density. The increase observed in leptin synthesis within the wound results in a transient elevation in circulating leptin, arising directly from the wound bed. This report demonstrates for the first time that leptin synthesis occurs rapidly in wound ischemic tissue and that the presence of leptin is necessary for normal healing progression to occur.
PRINCIPAL FINDINGS
1. Leptin expression is rapidly induced in experimental wounds in mice
Leptin mRNA expression was determined by in situ hybridization experiments performed on frozen sections of control and wounded skin collected 6 and 24 h postinjury. Leptin mRNA was detected using digoxigenin-labeled antisense leptin RNA oligonucleotides. In contrast to normal mouse skin (Fig. 1
a, c), numerous clusters exhibiting a strong hybridization signal were detected in sections prepared from wounds 6 (Fig. 1b, d
) and 24 h (Fig. 1e
) after skin incision. In these sections, the hybridization signal is scattered throughout the dermis and the basal keratinocyte layer of the epidermis. In the dermis, many of the cells exhibiting positive hybridization signal display elongated morphology, probably corresponding to fibroblasts (right arrows, Fig. 1d
). The control sense oligonucleotide leptin probe showed no reactivity when normal (not shown) or wounded skin sections were used (Fig. 1f
). Real-time PCR experiments were performed using RNA prepared from pooled wounds obtained by punch biopsy at various times postinjury. Consistent with the in situ hybridization results shown above, leptin mRNA expression reached a maximum as early as 6 h and was sustained throughout the healing process. On day 5, leptin mRNA levels remained elevated and were found to diminish only after 10 days, when repair is complete and scar remodeling is actively taking place (Fig. 1g
). Thus, it is apparent that leptin is actively produced in the wound environment throughout the inflammatory and proliferative phases of tissue repair. However, leptin up-regulation appears to begin immediately after inflicting the wound.
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2. Leptin protein localizes in wounds throughout the healing process
Immunohistochemical staining of wounds collected at various times after incision was performed using a polyclonal anti-leptin antibody (Fig. 2
). As expected, positive staining was observed in areas containing adipose tissue in unwounded skin. However, mild immunoreactivity was also present in non-adipose tissue cell types such as keratinocytes in the epidermal basal layer, skeletal muscle, and cellular elements of the dermis (Fig. 2a
). After injury, a robust immunohistochemical staining signal was detected throughout the wound and wound edges. The increased staining signal was observed to develop progressively, indicating continued expression of leptin in the course of the wound repair process. As early as 6 h after skin incision, the wounds already exhibited distinctive immunostaining for leptin (Fig. 2b, c
), which became markedly stronger after 24 h (Fig. 2d, e
) and were conspicuous even at 48 (Fig. 2f, g
) and 72 h (Fig. 2h
). Abundant wound resident myofibroblast-like cells were intensely immunoreactive as seen at a higher magnification (Fig. 2k
). Inflammatory cells recruited within the first 48 h after injury do not display leptin immunoreactivity (Fig. 2f
). Control sections exposed to anti-leptin antibodies in the presence of excess soluble recombinant murine leptin were developed in parallel and appeared negative for immunostaining. Thus, immunoreactivity seen in tested sections is specific for leptin (Fig. 2i
).
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3. Neutralizing anti-leptin antibodies impair healing progression in experimental wounds
A possible functional requirement for leptin during wound healing was assessed by treating wounds with neutralizing anti-leptin antibodies (or nonimmune IgG for controls) and then preparing wound sections for histological evaluation. The degree of reepithelialization was measured by computer-assisted morphometry as the transversal distance between the wound margins at the center of the spindle-shaped incision defect. This distance was on average 
1.6-fold wider in wounds treated with the anti-leptin antibody compared with control wounds treated with nonimmune IgG. The thickness of the granulation tissue present in the wound was significantly reduced in the mice treated with anti-leptin antibodies. Further evaluation of the effects of antibody on healing progression by real-time quantitative PCR showed a significant down-regulation in the transcriptional expression level of several extracellular matrix molecules such Tenascin C and collagen III. These changes may help explain the reduced matrix density found in antibody-treated wounds. mRNA expression for endothelial markers such as CD-31, Endoglin, and VEGFR-2 (FLK-1) was also diminished. These results strongly suggest that wound-associated leptin production plays an important functional role in epidermal reepithelialization, neovascularization, and development of granulation tissue, key events in the wound repair.
4. Wound-derived leptin leads to a transient increase in circulating leptin levels after wounding
To evaluate whether the increase of leptin detected in injured tissue affected circulating leptin, we measured serum leptin after wounding. Incision wounds were created in adult Balb/c mice and serum levels of leptin were measured at various times thereafter. Circulating leptin concentration increased rapidly from 2 ng/mL to 12 ng/mL (6-fold), reaching a peak by 12 h and returning to baseline after 24 h. To determine whether the surge of circulating leptin originates from the wound site, we inflicted wounds on human skin grafted on human-SCID mouse chimeras. We found that human leptin appeared in the mouse circulation after wounding whereas mouse leptin levels did not change significantly. This observation further supports our observations that there is an early surge of leptin production in wounds that has an important functional role in the healing and repair of wounds.
CONCLUSIONS AND SIGNIFICANCE
In this report, we demonstrate that wound resident cells rapidly engage in leptin synthesis shortly after an incision injury of the skin and that leptin production appears to continue throughout the entire process of healing at the wound site. These findings are consistent with previous observations suggesting that leptin may play a fundamental role in tissue repair, namely: 1) the characteristic severe healing impairment in mice lacking leptin (ob/ob) or leptin receptors (db/db), even in the absence of metabolic alterations; 2) the reversal of this defect by topical leptin treatment in leptin-deficient (ob/ob) mice; 3) the hypoxia-inducible character of the leptin gene, suggesting a mechanism for up-regulation in hypoxic, ischemic tissues such as the wound bed; and 4) the proangiogenic activity of leptin, which is likely to be involved in the neovascularization events that typically accompany wound healing. As illustrated in Fig. 3
, our results suggest that normal wound repair requires both acute local production of leptin (probably induced in response to local hypoxia) and a functionally intact leptin signaling system at the wound site.
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Leptin has been classically described as a circulating hormone primarily produced in adipocytes and responsible for regulation of energy balance through effects on the hypothalamus. However, there is a growing list of additional biological actions, some of which may result from direct, peripheral effects. Previous reports documenting the production of leptin in a variety of non-adipose cells are consistent with our finding indicating a dramatic burst of leptin production by wound resident cells of sufficient magnitude to impact blood leptin levels. Collectively, these observations illustrate an emerging pleiotropic character for leptin, a feature shared with other multifunctional IL-6-related cytokines to which leptin is structurally related, such as OSM, CNTF, LIF, IL-11, and CT-1. Our results give strength to the concept that leptin is a functionally pleiotropic cytokine with biological effects that overlap with other IL-6 cytokine family members.
The proangiogenic activity of leptin may represent an important function similar to other soluble factors actively produced in wounds (i.e., FGF-2, TGF
and ß, VEGF), all of which stimulate angiogenesis either directly or through chemoattracted macrophages primed to secrete angiogenic factors. Thus, our observations showing increased production of leptin in wounds suggest that its healing augmentation effect may be the result of its ability to promote neovascularization during tissue repair. However, it is feasible that leptin might modulate other important functions of the wound healing process. A study of the relationship between leptin induction and other cytokines produced at the wound site should provide insight into the potential role of leptin as an upstream regulator of the cytokine activation cascade that accompanies tissue repair.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0068fje; doi: 10.1096/fj.03-0068fje 
2 These authors contributed equally.
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