Nitric Oxide Accelerates Wound Healing By Promoting Epidermal Stem Cell Migration Via CGMP-Rho GTPase Signaling

Wound healing is a complex process mediated by many different factors responsible for the regeneration and reorganization of wound toward its normal architecture. The mechanisms of skin repair has not been completely elucidated, and many cellular components contribute to maintenance skin homeostasis and regeneration. Epidermal stem cells that reside in the basal layer of the skin epidermis and hair follicles bulge are essential for skin homeostasis and wound healing. Under normal circumstances, the homeostasis of epidermis and hair follicles is maintained by their stem cells. However, both stem cell populations are capable of regenerating the two structures, upon skin wounding. Through fate-mapping experiments, ESCs were shown to be rebuild to the epidermis and migrate in a linear manner toward the center of the wound. However, little is known regarding the mechanisms that stimulate ESCs migration during wound repair processing.Over the past two decades, NO has emerged as a critical molecule in wound healing and cellular homeostasis. NO regulation in the wound healing processing depends on the modulation of NO from different cell types included in this complex reconstruction process. As skin damage, NO level increase quickly, peaking one day after the initial injury during inflammatory phase of wound healing and gradually decreasing as the wound healing progresses toward proliferation and remodeling. More important, NO is released by diverse immune and skin cells, causing pleiotropic effects. Among its lot of effects, NO plays a critical role in wound healing by promoting cell migration, differentiation and proliferation. The importance of NO-mediated signaling in skin has been understood by focusing on NO-based signaling in normal skin and by contrasting this signaling with pathological conditions, and that situation has been reviewed by some authors. The direct downstream pathways of NO signaling consist primarily of interactions between NO is the most important of which is guanylate cyclase. The c GMP synthesize enzyme soluble Guanylylcyclase(s GC), which is different expressed during neuronal development, is a major receptor for NO. NO signal transduction has been shown to regulate the migration of neural progenitor cells, endothelial cells, and lung cancer cells.In addition, a small GTP-binding proteins of the Rho family, including Cdc42, Rac1, and Rho A, closely regulate actin-based structure formation, proliferation and cell migration. Rho GTPase is important regulator of cytoskeletal dynamics, and each GTPase contributes to cell motility by regulating actin cytoskeletal rearrangements. Research Report that NO causes macrophage migration via the HIF-1-stimulated small GTPases Rac1 and Cdc42; NO increases the invasion of pancreatic cancer cells via the activation of Rho A pathways after carbon ion irradiation; NO was necessary to maintain Rho A expression and Rho A-dependent functions in vascular smooth muscle cells. So that, we hypothesized that NO may stimulate ESCs migration via c GMP-Rho GTPase signal transduction.Our previous primary study showed that the NO donor sodium nitroprusside(SNP) could promote a human keratinocyte cell line(Ha Ca T) migration in vitro through c GMP signalling pathway. To further clarify the mechanism of NO promoting wound healing, the effects of NO on ESCs migration in vitro and in vivo in an animal model were detected. This study elucidated the potential effects of NO in ESCs migration and wound healing and determined the underlying mechanisms.In this study, preliminary study on the NO concentration and the expression of i NOS in the wound of burn patients firstly. Then we studied that the mechanism of NO in promoting the migration of epidermal stem cells, and to further improve the basic theory and clinical application of nitric oxide in promoting wound healing. This paper focuses on the regulation of NO promoting ESCs migration regulated via c GMP/PKG/Rho GTPase, through impelling cytoskeleton rearrangement and cells migration, and ultimately promoting wound healing. The main results and conclusions of the study is summarized as follows:1. NO production and i NOS expression in wound after burn injury:1) Detection of NO concentration and i NOS expression in burn wounds:In order to further research NO concentration and the changes at different time points in burn wounds, twenty cases were suffered with deep partial-thickness burn patients [age range: 2-47 years, 39.4±9.944 years; range of total body surface area of burn(TBSA): 6–32%, 15.75±13.033%) 46 blister fluid and 27 plasma samples from the patients at 4-96 h after burn injury were collected.2) Assay of NO concentrations in plasma, burn blister fluid and wound tissueThe NO concentrations in different samples were quantitatively measured using a Griess’ s method. The NO concentrations in plasma and blister fluid were 5.04-7.61 μM(mean ± SD: 6.43±0.70 μM) and 5.26-9.94 μM(mean ± SD: 7.01±1.20 μM), respectively. The NO concentration in blister fluids was significantly higher than that in plasma from the same patient. The mean value of NO concentration in wound tissues was 4.98±0.91-fold higher than that in normal skins from the same patients(P<0.000).3) Detection of i NOS expressions in normal and wounded tissues by immunohistochemistrial staining and western blot:To identify the source of the increased NO production in burn wound. i NOS protein expressions in wound and matched normal skin tissues were determined by immunofluorescence and western blot analyses. It was found that i NOS expressions in all burn wounds were increased 18.90±11.06-fold compared with these in matched normal skin tissue controls(P<0.001) by western blot. By serial section immunohistochemistrial staining, i NOS and the ESC marker Cytokeratin 19(CK19) in burn wound tissues was found expressing at the same locations, which hints ESC produces NO massively in burn wound through increasing i NOS expression after injury..2. Isolation and culture of human epidermal stem cells:Human epidermal stem cells were isolated from foreskin with informed consent by the modified method of rapid adhesion to collagen Ⅳ and cultured with K-SFM medium supplied with necessary additives. The harvested cells were identified by the expressions of epidermal stem cell markers, integrin β1 and keratin 19, through immunofluorescence staining, Western Blotting and Flow Cytometry. Flow Cytometric Analyses found 94.3±4.36% isolatated cells were a6briCD71dim-positive.3. The effect of NO on hu ESC migration in vitro and its mechanism:1) The effect of NO on hu ESC migration and mobility in vitro:In the in vitro cell scratch model, it was found that the migration of the cultured hu ESCs could be enhanced by SNAP in a concentration-dependent manner. Compared with the migration of the control group(48.8±2.7%) at 12 h post-culture, the migration of the cells stimulated with 100 μmol/L SNAP reached a peak of 82.1±15.8%(P<0.01). However, 500 μmol/L SNAP inhibited cell migration compared with control(8.2±7.2% vs. 8.9±10.2%, respectively, P<0.00). The time-lapse cell motion speed to the scratched area was checked by the viable cell station. It was found that in the presence of SNAP, the time-lapse cell motion speed changed in a dose-dependent manner. Exposing ESCs to SNAP(1–100 μM) for 24 h significantly facilitated ESC migration. However, 500 μM SNAP inhibited cell motility. At this high concentration, SNAP might affect cell viability. So that exogenous NO exerts a biphasic effect on ESCs migration and motility, low doses(1–100 μM) of NO donors(SNAP) induce migration and mobility signal, whereas high doses(300μM and 500 μM in our study) show inhibitory effects.2) The mechanism to NO promoting hu ESC migration and mobility:(1) NO stimulates hu ESC migration and motility via c GMP-Rho GTPase-mediated signal transduction: To directly test whether NO promoting cell migration and motility via potential downstream target enzymes, in migration assays 100 μM SNAP was applied after the cells treated with a c GMP inhibitor(ODQ), a PKG inhibitor(Rp-8pcpt-c GMPs), a Rho-specific inhibitor(Rhosin), a Rac1 inhibitor(Z62954982) or a Cdc42 inhibitor(ZCL278) for ten minutes and single-cell motility assays. As the results of previous migration and cell motility assay shown, SNAP significantly promoted the ESC migration and enhances ESC motility. And a significant reduction of NO donor SNAP-induced cell migration and motility to varying degrees was observed when SNAP was used in combination with ODQ, Rp-8pcpt-c GMPs, Rhosin and Z62954982(Fig 3B and C). But the Cdc42 inhibitor ZCL278 slightly blocked SNAP-induced ESCs migration(P=0.344) and SNAP-induced ESCs motility(P=0.072).(2) Effect of NO on the F-actin cytoskeleton in ESCs via c GMP-Rho GTPase signaling: Cells migration is a physical process that requires dramatic change in cells shape and cytoskeleton rearrangement. For the effective movement, the processes must be space-time coordinated. Morphological changes during the migration process and main body force generated by a dynamic actin cytoskeleton filaments(F-actin). F-actin polymerization is a key step for cell migration. In the current study, it was found F-actin showed an even distribution in ESCs in the absence of NO stimulation. While SNAP could increase the F-actin polymerization significantly, meanwhile this effect could be significantly depressed by ODQ, Rp-8pcpt-c GMPs, Rhosin and Z62954982(Fig 4B). But the Rho GTPase Cdc42 inhibitor ZCL278 slightly decreased the SNAP-induced ESCs F-actin polymerization.(3) NO activation of Rho GTPases Rac1 and Rho A, but not Cdc42 of hu ESC via c GMP-mediated signal transduction: In this study, we found that NO donor SNAP significantly promoted Rho A activity, that increased 3.94-fold compared with the control, and slightly increased Rac1 activation. Nevertheless, SNAP did not clearly influence Cdc42 activity. As follow, in order to investigate the pathways of SNAP regulating Rho GTPase, ODQ(a c GMP inhibitor) and Rp-8pcpt-c GMPs(a PKG inhibitor) were used respectively. We found that the SNAP-activated activation of Rho A and Rac1 could be blocked by Rp-8pcpt-c GMPs and ODQ. The results indicate that NO primarily promotes Rho A activity. Moreover, it was found that either the PKG and c GMP inhibitor could abolish the effects of SNAP on the activity of Rho A and Rac1.4. NO promoting wound healing and ESCs migration in vivo:L-arginine(L-Arg, a NOS substrate) and NG-monomethyl-L-arginine(L-NMMA, a NOS inhibitor) were used in mouse skin defect experiment to detect the effects of NO on wound healing, normal saline(NS) and glycine(Gly) were used as controls. It was found that L-NMMA significantly delayed the wound closure compared with NS or Gly control group. While L-Arg promoted wound closure markedly during the whole healing process. The wound closed completely on the day 9 after injury in L-Arg group compared with that on day 12 in SN or Gly control group, but on the day 15 in L-NMMA group. The wound reepitheliazation on the ninth day was decreased significantly in L-NMMA group detected by H&E staining, whereas L-Arg significantly promoted wound healing and epithelization. These data indicate that NO participates in skin wound reepithelization and healing.Conclusion: In this study, an NO donor, S-nitroso-N-acetylpenicillamine(SNAP), was found to facilitate the in vitro migration of human ESCs(hu ESCs) in both live-imaging and scratch models. In addition, pull-down assays demonstrated that SNAP could activate the small GTPases Rho A and Rac1 of the Rho family, but not Cdc42. Moreover, the effects of SNAP on the migration and F-actin polymerization of ESCs could be blocked by inhibitors of c GMP, PKG, Rho A or Rac1, and by a specific si RNA of Rho A or Rac1, but not by a Cdc42 inhibitor or si RNA. Furthermore, the roles of NO in ESC migration via c GMP-Rho GTPase signalling in vivo were confirmed by tracing 5-bromo-2-deoxyuridine(Brd U)-labelled cells in a wound healing model. Thus, the present study demonstrated that the NO donor SNAP could promote ESC migration in vitro and in vivo. Furthermore, NO was found to induce ESC migration via c GMP-Rho GTPase Rho A and Rac1 signalling, but not Cdc42 signalling in vitro.