Leptin, a 16 kDa pleiotropic cytokine primarily expressed in adipose tissue, has been shown to cause multiple systemic biological actions. Recently, leptin has also been documented as an important component of the wound healing process and its receptor appears to be expressed in wound tissue. We have previously demonstrated that leptin is a potent angiogenic factor exerting direct effects on endothelial cells and that transcription of its encoding gene is regulated by hypoxia. Here, we hypothesize that leptin expression is acutely up-regulated in the ischemic tissue of experimental wounds. Using a combination of in situ hybridization and quantitative RT-PCR experiments, we show that leptin expression is rapidly and steadily up-regulated in skin tissue from incisional and excisional wounds. By immunohistochemistry, we demonstrate increased and sustained leptin protein levels in basal keratinocytes, blood vessel walls, and fibroblasts. To determine whether leptin is required for normal healing, excisional wounds were treated with neutralizing anti-leptin antibodies. This treatment markedly hampered healing progression and prevented wound closure and contraction. Finally, a transient rise in circulating blood leptin levels was detected within the first 24 h after inflicting the injury; we present evidence suggesting that this elevation is due to increased leptin production at the ischemic wound site. We conclude that leptin is acutely up-regulated in the injured skin and propose that this local production of leptin serves a critical functional role as an autocrine/paracrine regulator of normal wound healing.

Leptin is a pleiotropic cytokine constitutively expressed in adipose tissue and upregulated by hypoxia in sites of tissue injury and in the placenta. Leptin has been demonstrated to play a role in normal and pathological wound healing and it is gene is acutely expressed in experimental wounds. Angiogenesis is among the most salient and well‐documented biological actions of leptin. Therefore it was hypothesized that leptin may play in the modulation of wound neovascularization. Using a murine model of experimental wounds the modulation of wound angiogenesis was investigated by treatment of the wounds to either recombinant leptin or a neutralizing anti‐leptin antibody. The parameters measured were: a)woundblood flow using laser Doppler imaging; b)vesseldensity by counting the number of CD‐31 positive vessels in wound sections; c)angiogenic transcriptionalprofiling using quantitative real‐time RT‐PCR; and d)validation of transcriptionalprofiling by immunohistochemistry and immunoblotting. The treatment of wounds with leptin had a marked effect in wound blood flow that was most significant at 24hours post‐wounding with average increase of 55% with respect to the controls. The effect diminished significantly by 72 hours (24%) and became negligible thereafter. Leptin also accelerate wound neovascularization by 24 hours where the number of small caliber vessels averaged 30/mm2 in leptin treated vessels and 13/mm2 in the controls (n = 10; P < 0.005). However, the number of vessels measured by 72 hours not significantly different between controls and leptin‐treated wounds. In addition transcriptional profiling reveled changes in gene expression of molecules related to angiogenesis such as VEGF, PlGF, Tie‐1, Tie‐2, Flt‐1, Flk‐1, FGF‐2 and endothelial markers like CD‐31, endoglin and eNOS. Overall, time course analysis throughout the healing process showed that leptin treatment induced an early shift in the expression of many of the analyzed transcripts. The most salient changes could be observed as early as 6 hours post‐wounding and included: TSP‐1 (+8.7); FLK‐1(+9); Ang‐2 (+18); CD31(+5.3); endoglin (+7.6); eNOS (+4.2); FGF‐2 (+4.3); etc. Leptin mRNA was also significantly increased (+10) as well as the long‐form of its receptor ObR‐b (+9). Conversely, when wounds were exposed to anti‐leptin antibodies, healing progression was impaired and changes in the transcriptional profile showed diminished expression of several genes related to wound neovascularization. Some examples by 24 hours: Endoglin(−2.5); Flk‐1(−2.8); eNOS (−5.1); Ang‐2 (−5.1); CD31(−3); VEGF‐A (−3.6). In conclusion, leptin had and early and significant effect in modulating wound neovascularization. The effect of treatment of wounds with exogenous leptin did not elicit hypervascularization of the wound and appeared to only accelerate what appeared to be a self‐limited process of wound angiogenesis. Funded by a Cedars‐Sinai grant from the Skirball‐Kenis Center for Plastic and Reconstructive Surgery.