**1. Introduction**

Wound healing is a dynamic and complex process that consists of sequential, albeit somewhat overlapping, inflammatory, proliferative, and remodeling phases [1–4]. In the inflammatory phase, immune cells (particularly macrophages) are recruited into the wound [3,5]. Inflammatory cells not only sterilize the wound; they also generate a finely balanced assortment of factors that promotes the rapid healing in the proliferative phase [3,5], which includes angiogenesis [6]. The topical wound treatments that are currently available target some of these factors (e.g., prostaglandin E1 and basic

fibroblast growth factor). Recent studies have shown that fatty acids and their G protein-coupled receptors may also be important targets of novel wound healing treatments: several studies showed that the fatty acid receptors GPR40 and GPR120 play important roles in wound healing processes such as cell migration [7,8]. In addition, natural products such as honey, alkaloids, flavonoids, tannins, saponins, and polyphenols have been shown to promote wound healing [9,10]. We speculate here that additional emerging therapeutic targets in skin wound healing may be the sphingolipids and their biogenic enzymes.

Sphingosine-1-phosphate (S1P) is generated by sphingosine kinase-1 (SphK1) and -2 (SphK2), which are located in the cytosol and nucleus, respectively. Only SphK1-generated S1P is transported out of the cell [11]. It then binds in a paracrine or autocrine manner to S1P-specific G protein-coupled receptors (S1PR), of which there are five forms.

This binding event regulates various physiological processes in the S1P-binding cell [12], as follows. First, the binding of S1P to S1PR regulates lymphocyte tra fficking, including the recruitment of inflammatory cells into inflamed tissues [13–15]. This e ffect is mediated by the S1P concentration gradient between various tissues: this gradient shapes the egress of S1PR1-expressing lymphocytes from secondary lymphoid organs into the blood or lymphatic vessels [16,17]. This mechanism has been targeted for the treatment of multiple sclerosis: Fingolimod (FTY720), which is a functional agonist of S1PR, induces lymphocytes to sequester in lymph nodes, thereby preventing them from contributing to the autoimmune reaction that causes the disease [12]. S1P-S1PR binding also acts to retain inflammatory cells in inflamed tissues, which produce high levels of S1P [13,18–20].

Second, S1P-S1PR binding plays key regulatory roles in vasculogenesis, angiogenesis, and blood vessel permeability [13,18–20]. Specifically, S1P regulates angiogenesis by binding to S1PR1 and S1PR3 on vascular endothelial cells, thereby inducing them to form capillary-like networks [18]. Moreover, S1P (and its functional analog FTY720) increases adherens junction assembly in endothelial cells: as a result, S1P treatment potently inhibits VEGF-induced endothelial cell transmonolayer permeability in vitro and vascular permeability in mice [21]. Since there are high levels of S1P in the blood, this vascular permeability-related activity of S1P also helps maintain the endothelial barrier integrity of specific vascular beds. This function of S1P is mediated by endothelial cell S1PR1 [22]. By contrast, S1P binding to S1PR2 disrupts endothelial barrier permeability [23]. These disparate e ffects of the S1PRs are due to the fact that S1PR1 couples solely with Gi/o whereas S1PR2 couples with Gq, G12, and G13 as well as Gi/o. The activation of G12 and G13 stimulates the small GTPase Rho, which induces cortical actin destabilization, stress-fiber formation, and endothelial barrier disruption [22,24].

Given that S1P promotes lymphocyte recruitment to and retention in inflamed tissues along with vasculogenesis and angiogenesis, we hypothesized that S1P is involved in the skin wound healing process by enhancing the local recruitment of the inflammatory cells that produce various wound healing-related factors in the wound. The aim of this study was to clarify the roles of the SphK1/S1P axis in the wound healing process.
