The wound repair process is a complex and well-coordinated regenerative response that involves a cross-talk among several types of cells, growth factors, cytokines, ECM, and soluble factors. Various risk factors, however, such as diabetes, hypoxia, ischemia, and infection, lead to dysfunction of various types of cells and to production of soluble mediators, resulting in wound underhealing or overhealing. Recently, several studies showed that local or systemic administration of stem or progenitor cells, such as mesenchymal stem cells (MSCs) and EPCs, enhances wound repair and angiogenesis [10, 21]. In particular, these studies revealed that the use of genetically engineered cell populations could increase the therapeutic efficacy, because the harsh pathological environment, including hypoxia, drastically affects the survival rate of the unmodified transplanted cells. In the present study, the major findings are as follows: (1) Lnk deficiency in EPCs enhances the expression of functional EPC markers and bioactivities such as proliferation, migration, and capacity for tubule-like formation; (2) a transplant of Lnk-deficient EPCs enhances wound repair via inhibition of recruitment of leukocytes in the inflammatory phase and by activation of myofibroblasts in the tissue-remodeling phase; (3) administration of Lnk-deficient EPCs improves wound healing in mice with STZ-induced diabetes.

Lnk is an adaptor protein that mediates protein–protein and protein–phospholipid interactions without an intrinsic enzymatic function [22, 23]. Lnk, as a key molecular target, augments the function of EPCs and neovascularization [12]. Our findings indicated that wound repair was significantly enhanced in Lnk-deficient mice as compared with wild-type mice. However, wound repair is a complex biological process that requires several cell types [1, 2], and the Lnk adaptor protein affects various of cell types, including hematopoietic stem cells [24], T cells [25], macrophages [26], EPCs [13, 14], and endothelial cells [27]. Therefore, to focus on one of the populations involved in the wound-healing process, we performed flow cytometry in the wound tissues to search for the recruitment of a specific cell population, and demonstrated that Lnk deficiency in mice specifically increased the recruitment of the EPC population to the injury sites. In a model of bone fracture, Lnk-deficient mice show improved osteogenesis because of enhanced angiogenesis through the recruitment of EPCs to the prefracture zone [28]. Our data also revealed that the EPC bioactivities, including proliferation, migration, and tube formation capacity were significantly higher in Lnk-deficient EPCs than in wild-type EPCs. Our previous studies showed that Lnk deficiency in mice promotes EPC kinetics and neovascularization in response to angiogenic cytokines, such as SCF, VEGF, and SDF-1 [13]. In addition, Lnk-deficient EPCs increase the clonogenic proliferation via activation of the JAK-STAT3 signal pathway [14]. These findings strongly support the notion that Lnk deficiency in mice promotes wound repair through the recruitment of EPCs and improves EPC cellular bioactivities, which are initiating steps of vascular repair, which is tightly regulated by the Lnk adaptor protein for cellular homeostasis.

EPCs, as key progenitors of endothelial cells, participate in neovascularization and tissue repair. After administration of a cutaneous wound to mice, BM-derived EPC mobilization is increased via the SDF-1α/CXCR4 axis [29]. A human cord blood-derived EPC transplant accelerates wound closure in nude mice with STZ-induced diabetes by stimulation of proliferation of keratinocytes and fibroblasts [30]. Our results show that wound closure is significantly better after an EPC transplant than after PBS injection. Moreover, the transplant of Lnk-deficient EPCs significantly enhanced wound healing through the improvement of transplanted-cell proliferation and survival as well as neovascularization, as compared with a transplant of wild-type EPCs. In a hindlimb ischemia model, our previous report clearly showed that a transplant of Lnk-deficient EPC enhances proliferation and survival as well as neovascularization through regulation of the JAK2/STAT3 signaling pathway [14]. In a mouse model of spinal cord injury, we also reported that Lnk-deficient, c-Kit-positive, Sca-1-positive, and lineage marker-negative cell populations (which are a core source of EPCs) enhance angiogenesis, astrogliosis, and functional recovery [19]. These previous reports support our present findings that a transplant of Lnk-deficient EPCs promotes wound healing through enhancement of neovascularization.

In the inflammatory phase, a healthy inflammatory reaction is involved in wound healing through the removal of necrotic tissues, debris, and pathogen contaminants, as well as via recruiting and activating fibroblasts. Leukocytes, including macrophages and neutrophils, appear in the wound at 1–3 days after injury and continue the process of phagocytosis [31]. Nonetheless, inflammation under pathological conditions such as in a chronic disease leads to delayed healing and promotes inflammation. In the proliferation phase (4–14 days after injury), overactivated immune cells induce scar formation and fibrosis [31, 32]. The possible reason for the continued presence of inflammatory cells is their persistent recruitment and activation due to tissue injury from enhanced mechanical pressure, pathogens, leukocyte trapping, and ischemic injury [20]. Cell death and tissue necrosis also cause inflammation [33]. In addition, inflammation and oxidative stress affect EPC mobilization. Our data show that neither wild-type nor Lnk-deficient EPCs affected immune cell recruitment in the inflammatory phase (postoperative day 3), whereas transplantation of Lnk-deficient EPCs significantly decreased the recruitment of macrophages and neutrophils after postoperative day 7 as compared with transplantation of wild-type EPCs. Human gingiva-derived MSCs accelerate wound healing by eliciting M2 polarization in macrophages [34]. In corneal injury, MSCs promote corneal wound healing by their anti-inflammatory action, including secretion of IL-10, IL-6, and transforming growth factor beta 1 (TGF-β1) [35]. IL-10-deficient EPCs show decreased survival and function at ischemic sites [36]. These results suggest that Lnk-deficient EPCs have an anti-inflammatory effect after a transplant in an excisional wound. Additional studies will obviously be necessary to further elucidate the complex role of Lnk-deficient EPCs in secretion of anti-inflammatory paracrine factors and their functions during dermal wound healing.

In the remodeling phase, activated fibroblasts, which have a myofibroblast phenotype, perform the ECM remodeling. Fibroblast-to-myofibroblast differentiation represents a pivotal process during wound healing and tissue repair, because the high contractile force generated by myofibroblasts is effective for physiological tissue remodeling [32, 37]. Although the mechanism of skin contraction is different between mice and humans, reduced fibroblast proliferation leads to a strong delay in wound closure [38]. To evaluate the potential preclinical and clinical application of Lnk-deficient EPCs as a cell-based therapeutic, we focused on the bioactivity of fibroblasts through transplantation of EPCs [31, 37, 39]. The results of this study indicate that Lnk-deficient EPCs activate fibroblasts in vitro and induce the differentiation of fibroblasts into myofibroblasts in vivo. In particular, the fibrotic area was significantly decreased in mice transplanted with Lnk-deficient EPCs compared with that of mice transplanted with wild-type EPCs. Myofibroblasts perform a key function in wound healing and in contractile forces [39]. Trophic activity of MSCs increases skin wound closure by activation of dermal fibroblasts [40]. Coculture of fibroblasts with EPCs improves functional recovery after a myocardial infarction [41]. Engraftment of EPCs into an excisional wound model in diabetic mice augments wound repair via fibroblast proliferation [30]. These findings indicate that Lnk-deficient EPCs may be engaged in a cross-talk with fibroblasts for wound healing, but the precise mechanism of action of Lnk-mediated signaling cascades and the difference in skin contraction between mice and humans should be further investigated to support their preclinical and clinical application.

Finally, we assessed the effect of a transplant of Lnk-deficient EPCs on wound repair in mice with STZ-induced diabetes to confirm the beneficial effects of Lnk-deficient EPCs in a chronic disease. Our results revealed that wound repair is significantly better after a transplant of Lnk-deficient EPCs as compared with that in other groups. This study has some limitations to comprehensively determine the effect of Lnk-deficient EPCs in chronic diseases in general. In particular, we established a type 1 diabetes mouse model for wound healing; however, a type 2 diabetes model might be a more appropriate chronic disease model. To reveal the availability and possibility of EPCs for cell-based therapy in diabetes, we first confirmed the effect of Lnk-deficient EPCs in a type 1 diabetes model. In addition, the wound-healing mechanism might be different between mice and humans, since skin contraction plays a greater role in the rodent wound-healing mechanism than in that of humans. Therefore, in future studies, the effect of Lnk gene silencing will be investigated in type 1 and 2 diabetic models, and the precise role of Lnk in EPC-mediated wound healing will be evaluated in the chronic disease condition to support preclinical and clinical application.