Bioactive Sphingolipids and Wound Healing

Difficult to heal wounds are of significant health concern for military personnel and their family, especially those linked to diabetes, since diabetes has reached epidemic proportions in the Western Society. Despite being linked to high mortality, low quality of life and other health complications (1), wounds that are difficult to heal are still a mystery, and most pharmacologic regimens are inefficient (2,3). Wound healing is a complex process consisting of three coordinately regulated phases, namely inflammatory, proliferative and remodeling phase. Wounds that fail to repair show signs of chronic inflammation without progression to latter stages of re-epithelialization and proliferation. We propose that an enzyme called acid sphingomyelinase (ASMase) is key for the coordinated release of pro-inflammatory and proliferative mediators at the site of the wound and that A-SMase deregulation during diabetes is responsible for delayed wound healing in diabetic patients. Acid sphingomyelinase (SMase) catalyzes the first step in a chain of reactions that lead to the hydrolysis of the major structural lipid of mammalian membranes and lipoproteins, sphingomyelin (SM), to bioactive lipids ceramide, sphingosine, and sphingosine-1-phosphate (S1P). Acid sphigomyelinase exists in two forms, secretory (S-SMase) and lysosomal (L-SMase), that have a common precursor but undergo different post-translational modification (4,5). The two forms have very distinct functions and evidence suggest that the L-SMase may be key in the wound healing process. For one thing, the L-SMase product, ceramide is intrinsic inhibitor of TNFĄ Converting Enzyme (TACE), which controls the secretion of mature/active TNFĄ from macrophages (6). Macrophages with reduced L-SMase activity exhibit higher TACE activity, and respectively secrete 2 to 5 times more TNFĄ that is main culprit for the impairment of the wound healing process during diabetes. For another, due to defects in endocytic process, macrophages with low L-SMase fail to properly clear invading bacteria, resulting in a local and systemic hyperresponse to infection (7). Last, but not least, L-SMase controls the levels of the secretory bioactive lipid, sphingosine-1-phospate (S1P), which is known to induce cell proliferation, migration, and angiogenesis (8). However, the key rational for the need to establish the role of ASMase in wound healing comes for observations that macrophages from animals with diet-induced diabetes exhibit very low L-SMase activity, as compared to control macrophages, likely caused by a shift in the post-translational processing of the ASMase precursor away form L-SMase and towards S-SMase. Inspite of these indirect indications for the role of L-SMase in coordination of the wound healing process, that possibility had never been tested directly in appropriate animal models. The objective of this application is to decipher the role of ASMase in wound healing in healthy and diabetic state and test whether targeting ASMase is appropriate approach to facilitate the repair process. In this application, C57Bl6 mice fed low and high fat diet and mice genetically prompt to obesity (db/db and ob/ob mice), will be the models used. The effects of ceramide, S1P, and other modulators of endogenous ceramide and S1P metabolism will be tested for efficacy in promoting healing of full thickness wounds. The steps of the wound-healing process affected by sphingolipids will be identified. The requirement of L-SMase for normal healing processes will be tested using a bone marrow replacement approach with smpd1-deficient mice as donors.