035 Leptin Plays a Key Role in the Modulation of Wound Angiogenesis

Abstract

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.