Acute and chronic wound fluids inversely influence adipose‐derived stem cell function: molecular insights into impaired wound healing

Wound healing is a complex biological process that requires a well‐orchestrated interaction of mediators as well as resident and infiltrating cells. In this context, mesenchymal stem cells play a crucial role as they are attracted to the wound site and influence tissue regeneration by various mechanisms. In chronic wounds, these processes are disturbed. In a comparative approach, adipose‐derived stem cells (ASC) were treated with acute and chronic wound fluids (AWF and CWF, respectively). Proliferation and migration were investigated using 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) test and transwell migration assay. Gene expression changes were analysed using quantitative real time–polymerase chain reaction. AWF had a significantly stronger chemotactic impact on ASC than CWF (77·5% versus 59·8% migrated cells). While proliferation was stimulated by AWF up to 136·3%, CWF had a negative effect on proliferation over time (80·3%). Expression of b‐FGF, vascular endothelial growth factor (VEGF) and matrix metalloproteinase‐9 was strongly induced by CWF compared with a mild induction by AWF. These results give an insight into impaired ASC function in chronic wounds. The detected effect of CWF on proliferation and migration of ASC might be one reason for an insufficient healing process in chronic wounds.     

Adipose‐derived stem cells and keratinocytes in a chronic wound cell culture model: the role of hydroxyectoine

Chronic wounds represent a major socio‐economic problem in developed countries today. Wound healing is a complex biological process. It requires a well‐orchestrated interaction of mediators, resident cells and infiltrating cells. In this context, mesenchymal stem cells and keratinocytes play a crucial role in tissue regeneration. In chronic wounds these processes are disturbed and cell viability is reduced. Hydroxyectoine (HyEc) is a membrane protecting osmolyte with protein and macromolecule stabilising properties. Adipose‐derived stem cells (ASC) and keratinocytes were cultured with chronic wound fluid (CWF) and treated with HyEc. Proliferation was investigated using MTT test and migration was examined with transwell‐migration assay and scratch assay. Gene expression changes of basic fibroblast growth factor (b‐FGF), vascular endothelial growth factor (VEGF), matrix metalloproteinases‐2 (MMP‐2) and MMP‐9 were analysed by quantitative real‐time polymerase chain reaction (qRT‐PCR). CWF significantly inhibited proliferation and migration of keratinocytes. Addition of HyEc did not affect these results. Proliferation capacity of ASC was not influenced by CWF whereas migration was significantly enhanced. HyEc significantly reduced ASC migration. Expression of b‐FGF, VEGF, MMP‐2 and MMP‐9 in ASC, and b‐FGF, VEGF and MMP‐9 in keratinocytes was strongly induced by chronic wound fluid. HyEc enhanced CWF induced gene expression of VEGF in ASC and MMP‐9 in keratinocytes. CWF negatively impaired keratinocyte function, which was not influenced by HyEc. ASC migration was stimulated by CWF, whereas HyEc significantly inhibited migration of ASC. CWF induced gene expression of VEGF in ASC and MMP‐9 in keratinocytes was enhanced by HyEc, which might partly be explained by an RNA stabilising effect of HyEc.     

The role of SDF‐1 in homing of human adipose‐derived stem cells

One of the putative pathophysiological mechanisms of chronic wounds is a disturbed homing of stem cells. In this project, the stromal cell‐derived factor 1 (SDF‐1)/C‐X‐C chemokine receptor (CXCR) 4 and SDF‐1/CXCR7 pathway were focused in human adipose‐derived stem cells (ASCs). ASCs were incubated with acute (AWF) or chronic wound fluid (CWF) to analyze their effects by quantitative real‐time polymerase chain reaction (SDF‐1, CXCR4, CXCR7, TIMP3), enzyme‐linked immunosorbent assay (SDF‐1 in WFs and supernatant), and transwell migration assay with/without antagonization. Whereas SDF‐1 amounted 73.5 pg/mL in AWF, it could not be detected in CWF. Incubation with AWF led to a significant enhancement (129.7 pg/mL vs. 95.5 pg/mL), whereas CWF resulted in a significant reduction (30 pg/mL vs. 95.5 pg/mL) of SDF‐1 in ASC supernatant. The SDF‐1 receptor CXCR7 was detected on ASCs. AWF but not CWF significantly induced ASC migration, which was inhibited by CXCR4 and CXCR7 antagonists. Expressions of SDF‐1, CXCR4, and CXCR7 were significantly stimulated by AWF while TIMP3 expression was reduced. In conclusion, an uncontrolled inflammation in the chronic wound environment, indicated by a reduced SDF‐1 expression, resulted in a decreased ASC migration. A disturbed SDF‐1/CXCR4 as well as SDF‐1/CXCR7 pathway seems to play an important role in the impaired healing of chronic wounds.     

Matrix metalloproteinase inhibition delays wound healing and blocks the latent transforming growth factor‐β1‐promoted myofibroblast formation and function

The ability to regulate wound contraction is critical for wound healing as well as for pathological contractures. Matrix metalloproteinases (MMPs) have been demonstrated to be obligatory for normal wound healing. This study examined the effect that the broad‐spectrum MMP inhibitor BB‐94 has when applied topically to full‐thickness skin excisional wounds in rats and its ability to inhibit the promotion of myofibroblast formation and function by the latent transforming‐growth factor‐β1 (TGF‐β1). BB‐94 delayed wound contraction, as well as all other associated aspects of wound healing examined, including myofibroblast formation, stromal cell proliferation, blood vessel formation, and epithelial wound coverage. Interestingly, BB‐94 dramatically increased the level of latent and active MMP‐9. The increased levels of active MMP‐9 may eventually overcome the ability of BB‐94 to inhibit this MMP and may explain why wound contraction and other associated events of wound healing were only delayed and not completely inhibited. BB‐94 was also found to inhibit the ability of latent TGF‐β1 to promote the formation and function of myofibroblasts. These results suggest that BB‐94 could delay wound closure through a twofold mechanism; by blocking keratinocyte migration and thereby blocking the necessary keratinocyte–fibroblast interactions needed for myofibroblast formation and by inhibiting the activation of latent TGF‐β1.     

Wound dressing components degrade proteins detrimental to wound healing

Excessive levels of matrix metalloproteinases (MMPs) are present in chronic wounds preventing wound closure. Reducing detrimental components may be key in healing chronic wounds. Elta Protease-containing wound dressings have been observed clinically to resolve inflammation and appear to aid healing in acute and chronic recalcitrant wounds. To investigate possible mechanisms of action, in vitro tests, zymography, collagenase assays and enzyme-linked immunosorbent assays (ELISAs), were performed to evaluate the effect of the dressing proteases on detrimental and beneficial wound healing components such as MMPs, Tissue Inhibitor of Matrix Metalloproteinases (TIMPs), cytokines and growth factors. Standards of pro- and active MMP-2, MMP-9 and chronic wound fluid (CWF) were prepared. Degradation of target proteins was enhanced by increased Elta Protease concentration, temperature and incubation time. Incubation with serial dilutions of the Elta Proteases resulted in nearly complete degradation of all MMP standards. After a 30-minute incubation, twofold diluted Elta Proteases degraded >90% of the gelatinases in CWF. ELISAs showed that Elta Proteases effectively degraded MMP-9 and tumour necrosis factor (TNF-alpha). In contrast, Platelet Derived Growth Factor (PDGF) and interleukin 1 beta were resistant to degradation by Elta Proteases. These results suggest that Elta Protease dressings appear to deactivate detrimental components in CWF, which may reduce wound bed contact with harmful proteins.     

Inhibition of bone morphogenetic protein signalling promotes wound healing in a human ex vivo model

Background

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) play roles in embryonic development and postnatal remodelling of the skin. Many indications suggest that BMP signalling regulates keratinocyte proliferation and differentiation. Chronic wounds have been shown to exhibit high levels of BMP ligands; however, the effect of BMP pathway modulation on human skin healing remains undefined.

Methods

A human ex vivo skin wound healing model was used to analyse the expression of BMP signalling pathway components during healing and to investigate the effects of BMPs and the BMP antagonist Noggin on skin repair. Additionally, the effects of BMP signalling on keratinocyte proliferation, apoptosis and migration were tested using in vitro flow cytometry and ‘scratch’ migration assays, respectively.

Results

BMP receptor-1B (BMPR-1B) and downstream signalling protein phosphorylated-Smad-1/5/8 were highly expressed in healing epidermis. Treatment of human skin with exogenous BMPs impaired wound closure by reducing keratinocyte proliferation and increasing apoptosis. The BMP antagonist Noggin negated the inhibitory effects of BMP ligands, and when used alone, Noggin reduced keratinocyte apoptosis in the wound bed. In vitro, BMP ligands suppressed keratinocyte proliferation whilst Noggin stimulated proliferation. Keratinocyte migration was slowed following BMP treatment; in contrast, migration was significantly accelerated due to inhibition of BMP activity by either Noggin or BMPR-1B silencing.

Conclusions

BMP signalling is inherently involved in wound healing. BMPs slow skin repair by suppressing keratinocyte proliferation and migration. Thus, modulation of BMP signalling using BMP inhibitors such as Noggin may serve as a new approach to promote cutaneous wound repair.Level of evidence: Not ratable.

     

Low concentration amino-bisphosphonates stimulate human keratinocyte proliferation and in vitro wound healing

Amino-bisphosphonates (N-BPs) are widely used to treat a great variety of clinical conditions involving altered calcium metabolism, as well as to prevent bone metastases. The use of N-BPs, however, display well-known side effects, including cellular toxicity, mainly at soft tissue and mucosal level, that arise from N-BPs ability to induce cell apoptosis when administered at clinically relevant concentrations. The aim of this study was to evaluate, in an in vitro wound healing model, the effect of N-BPs low concentration (10 nM-10 μM) stimulation on keratinocyte cellular behaviour. Human keratinocytes were treated with neridronate and zoledronate, two N-BPs with different chemical structure and clinical potency, but sharing a common pharmacological target: farnesyl pyrophosphate (FPP) synthase. Surprisingly, at the tested concentrations, both drugs stimulated keratinocytes proliferation, upregulating cytokeratin 5 while downregulating filaggrin expression, and wound healing ability, without any significant effect on matrix metalloproteinase (MMP)-9 activity. The lack of N-BPs effect on MMP-9 activity indicates that wound closure, in our experimental model, is mainly due to an increase in cell proliferation rather than to an increase in cell migration. Therefore, it can be hypothesised that the observed wound healing results could be ascribed to an N-BPs mediated reduction of FPP endogenous levels, thus suggesting new possible clinical applications for these compounds.     

The effects of caffeine on wound healing

The purine alkaloid caffeine is a major component of many beverages such as coffee and tea. Caffeine and its metabolites theobromine and xanthine have been shown to have antioxidant properties. Caffeine can also act as adenosine‐receptor antagonist. Although it has been shown that adenosine and antioxidants promote wound healing, the effect of caffeine on wound healing is currently unknown. To investigate the effects of caffeine on processes involved in epithelialisation, we used primary human keratinocytes, HaCaT cell line and ex vivo model of human skin. First, we tested the effects of caffeine on cell proliferation, differentiation, adhesion and migration, processes essential for normal wound epithelialisation and closure. We used 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) proliferation assay to test the effects of seven different caffeine doses ranging from 0·1 to 5 mM. We found that caffeine restricted cell proliferation of keratinocytes in a dose‐dependent manner. Furthermore, scratch wound assays performed on keratinocyte monolayers indicated dose‐dependent delays in cell migration. Interestingly, adhesion and differentiation remained unaffected in monolayer cultures treated with various doses of caffeine. Using a human ex vivo wound healing model, we tested topical application of caffeine and found that it impedes epithelialisation, confirming in vitro data. We conclude that caffeine, which is known to have antioxidant properties, impedes keratinocyte proliferation and migration, suggesting that it may have an inhibitory effect on wound healing and epithelialisation. Therefore, our findings are more in support of a role for caffeine as adenosine‐receptor antagonist that would negate the effect of adenosine in promoting wound healing.     

In vitro and in vivo studies on matrix metalloproteinases interacting with small intestine submucosa wound matrix

Small intestine submucosa (SIS), a bioactive extracellular matrix (ECM) containing critical components of the ECM including collagens, proteoglycans, and glycosaminoglycans, has been widely used for wound healing. The purpose of this study was to investigate the interaction between SIS and matrix metalloproteinases (MMPs). MMP-1, MMP-2, and MMP-9 displayed different binding affinities, indicated by a loss in activity in solution upon incubation with SIS at 53·8%, 85·9%, and 36·9% over 24 hours, respectively. A cell migration study was conducted to evaluate the effects of MMPs and SIS on keratinocytes. The results indicated that MMPs inhibit keratinocyte migration in vitro, and that the inhibition can be significantly reduced by pre-incubating the MMP solution with SIS. To evaluate activity in vivo a diabetic mouse wound healing study was conducted. Biopsy samples were collected on different days for analysis of MMP levels by gelatin zymography. MMP activity was found to be attenuated by SIS treatment on day 3 after wounding. On day 7, the attenuation became less significant indicating that the MMP binding ability of SIS had become saturated. SIS was able to reduce MMP activity immediately, and may reduce the inhibitory effects of MMPs on keratinocyte migration.     

Effects of an aqueous extract from the leaves of Chromolaena odorata (Eupolin) on the proliferation of human keratinocytes and on their migration in an in vitro model of reepithelialization

Eupolin ointment, prepared from the leaves of Chromolaena odorata, has been shown to promote the healing of soft tissue wounds and burns in Vietnam. However, the mechanism by which this agent affects cells involved in the wound healing process is unknown. Cultured human keratinocytes were used in this study to investigate the effects of the Eupolin extract in vitro on processes involved in wound reepithelialization. Keratinocyte proliferation was monitored by a colorimetric assay and migration by the closure of a denuded area scratched in a confluent monolayer. Human keratinocyte proliferation was stimulated by low concentrations of the extract (from 0.1 to 5 microg/ml), cell differentiation by higher concentrations (50 to 300 microg/ml), and migration by intermediate concentrations (5 to 60 microg/ml). The increased proliferation and migration of human keratinocytes observed in vitro might explain, in part, the beneficial effects that have been observed in the clinic.     

The enhanced healing of a high‐risk,clean, sutured surgical incision by prophylactic negative pressure wound therapy as delivered by Prevena™ Customizable™: cosmetic and therapeutic results

According to the literature, incisional closure complications may range from postoperative surgical site infections, representing 17–22% of health care‐associated infections, surgical wound dehiscence and formation of haematomas or seromas, and can lead to delayed or impaired incision healing. These kinds of situations are more common when wounds are closed under tension or in specific patient morbidities. Obesity, in particular, is associated with an impaired blood flow to tissues, predisposing the patient to increased risk of wound complications by various mechanisms. Incisional complications can become relevant economic burdens for health care systems because of an increase in the average length of hospital stay and readmissions, and additional medical and surgical procedures. Thus, a preventive therapy may have a critical role in the management of healing. Negative pressure wound therapy (NPWT) technology as delivered by Prevena? Customizable? (Kinetic Concepts Inc., San Antonio, TX) has recently been the focus of a new investigation, as a prophylactic measure to prevent complications via immediate postoperative application in high‐risk, clean, closed surgical incisions. The authors present a 62‐year‐old class II obese female, who underwent bilateral inguinal dermolipectomy. Prophylactic NPWT as delivered by Prevena? was performed successfully over surgical incisions. Cosmetic and therapeutic results are shown.     

Acceleration of cutaneous wound healing by brassinosteroids

Brassinosteroids are plant growth hormones involved in cell growth, division, and differentiation. Their effects in animals are largely unknown, although recent studies showed that the anabolic properties of brassinosteroids are possibly mediated through the phosphoinositide 3‐kinase/protein kinase B signaling pathway. Here, we examined biological activity of homobrassinolide (HB) and its synthetic analogues in in vitro proliferation and migration assays in murine fibroblast and primary keratinocyte cell culture. HB stimulated fibroblast proliferation and migration and weakly induced keratinocyte proliferation in vitro. The effects of topical HB administration on progression of wound closure were further tested in the mouse model of cutaneous wound healing. C57BL/6J mice were given a full‐thickness dermal wound, and the rate of wound closure was assessed daily for 10 days, with adenosine receptor agonist CGS‐21680 as a positive control. Topical application of brassinosteroid significantly reduced wound size and accelerated wound healing in treated animals. mRNA levels of transforming growth factor beta and intercellular adhesion molecule 1 were significantly lower, while tumor necrosis factor alpha was nearly suppressed in the wounds from treated mice. Our data suggest that topical application of brassinosteroids accelerates wound healing by positively modulating inflammatory and reepithelialization phases of the wound repair process, in part by enhancing Akt signaling in the skin at the edges of the wound and enhancing migration of fibroblasts in the wounded area. Targeting this signaling pathway with brassinosteroids may represent a promising approach to the therapy of delayed wound healing.     

Diabetic wound healing in a MMP9‐/‐ mouse model

Reduced mobilization of endothelial progenitor cells (EPCs) from the bone marrow (BM) and impaired EPC recruitment into the wound represent a fundamental deficiency in the chronic ulcers. However, mechanistic understanding of the role of BM‐derived EPCs in cutaneous wound neovascularization and healing remains incomplete, which impedes development of EPC‐based wound healing therapies. The objective of this study was to determine the role of EPCs in wound neovascularization and healing both under normal conditions and using single deficiency (EPC) or double‐deficiency (EPC + diabetes) models of wound healing. MMP9 knockout (MMP9 KO) mouse model was utilized, where impaired EPC mobilization can be rescued by stem cell factor (SCF). The hypotheses were: (1) MMP9 KO mice exhibit impaired wound neovascularization and healing, which are further exacerbated with diabetes; (2) these impairments can be rescued by SCF administration. Full‐thickness excisional wounds with silicone splints to minimize contraction were created on MMP9 KO mice with/without streptozotocin‐induced diabetes in the presence or absence of tail‐vein injected SCF. Wound morphology, vascularization, inflammation, and EPC mobilization and recruitment were quantified at day 7 postwounding. Results demonstrate no difference in wound closure and granulation tissue area between any groups. MMP9 deficiency significantly impairs wound neovascularization, increases inflammation, decreases collagen deposition, and decreases peripheral blood EPC (pb‐EPC) counts when compared with wild‐type (WT). Diabetes further increases inflammation, but does not cause further impairment in vascularization, as compared with MMP9 KO group. SCF improves neovascularization and increases EPCs to WT levels (both nondiabetic and diabetic MMP9 KO groups), while exacerbating inflammation in all groups. SCF rescues EPC‐deficiency and impaired wound neovascularization in both diabetic and nondiabetic MMP9 KO mice. Overall, the results demonstrate that BM‐derived EPCs play a significant role during wound neovascularization and that the SCF‐based therapy with controlled inflammation could be a viable approach to enhance healing in chronic diabetic wounds.     

Different wound healing properties of dermis,adipose, and gingiva mesenchymal stromal cells

Oral wounds heal faster and with better scar quality than skin wounds. Deep skin wounds where adipose tissue is exposed, have a greater risk of forming hypertrophic scars. Differences in wound healing and final scar quality might be related to differences in mesenchymal stromal cells (MSC) and their ability to respond to intrinsic (autocrine) and extrinsic signals, such as human salivary histatin, epidermal growth factor, and transforming growth factor beta1. Dermis‐, adipose‐, and gingiva‐derived MSC were compared for their regenerative potential with regards to proliferation, migration, and matrix contraction. Proliferation was assessed by cell counting and migration using a scratch wound assay. Matrix contraction and alpha smooth muscle actin was assessed in MSC populated collagen gels, and also in skin and gingival full thickness tissue engineered equivalents (reconstructed epithelium on MSC populated matrix). Compared to skin‐derived MSC, gingiva MSC showed greater proliferation and migration capacity, and less matrix contraction in full thickness tissue equivalents, which may partly explain the superior oral wound healing. Epidermal keratinocytes were required for enhanced adipose MSC matrix contraction and alpha smooth muscle actin expression, and may therefore contribute to adverse scarring in deep cutaneous wounds. Histatin enhanced migration without influencing proliferation or matrix contraction in all three MSC, indicating that salivary peptides may have a beneficial effect on wound closure in general. Transforming growth factor beta1 enhanced contraction and alpha smooth muscle actin expression in all three MSC types when incorporated into collagen gels. Understanding the mechanisms responsible for the superior oral wound healing will aid us to develop advanced strategies for optimal skin regeneration, wound healing and scar formation.     

In vivo characterization of keratinocyte growth factor-2 as a potential wound healing agent

Human keratinocyte growth factor-2 exerts a proliferative effect on epithelial cells and mediates keratinocyte migration. It has also been shown to increase both deposition of granulation tissue and collagen and maturation of collagen. Because these properties should affect the healing trajectory of wounds, this study set out to investigate the effects of keratinocyte growth factor-2 on the healing of three different types of wounds. Human meshed skin grafts explanted to athymic "nude" rats, surgical incisions in Sprague-Dawley rats, and acute excisional rat wounds inoculated with Escherichia coli were used. Two concentrations of recombinant human keratinocyte growth factor-2 were compared to a vehicle control and keratinocyte growth factor-1. Keratinocyte growth factor-2 significantly accelerated the rate of epithelialization in the meshed skin graft model and effected a modestly more rapid gain in breaking strength of surgical incisions than keratinocyte growth factor-1 or the vehicle control treatment. Neither keratinocyte growth factors accelerated wound closure by contraction of the excisional wounds. Based on these data, keratinocyte growth factor-2 may be useful in accelerating healing in wounds healing mainly by the process of epithelialization such as venous stasis ulcers, partial thickness burn wounds, and skin graft donor sites. It might also accelerate the gain in incisional wound strength in acute surgical or traumatic wounds.     

Endoplasmic reticulum stress in human chronic wound healing: Rescue by 4‐phenylbutyrate

During wound healing, cells have a high rate of protein synthesis and many proteins need to be folded post‐translationally to function, which occurs in the endoplasmic reticulum (ER). In addition to proliferation, several cellular stress conditions, such as hypoxia, in the wound micro‐environment lead to the accumulation of unfolded or misfolded proteins in the ER, causing ER stress. Eukaryotic cells have a signalling system to manage ER stress called the unfolded protein response (UPR). Mild UPR activation has a beneficial homeostatic effect; however, excessive UPR induces cell death. Herein, we examined venous leg ulcer biopsies versus normal acute incisional wounds in age‐matched elderly subjects and found a large increase in ER stress markers. To study the underlying mechanism, we established several cell cultures from amputated legs from the elderly that showed inherent ER stress. While both keratinocytes and fibroblasts migration was impaired by ER stress, migration of elderly leg skin keratinocytes was markedly improved after treatment with the chemical chaperone and clinically established drug 4‐phenylbutyrate (4‐PBA) and demonstrated a reduction in ER stress markers. In a full‐thickness human skin wound healing model, 4‐PBA improved the reepithelialisation rate, which suggests it as a promising drug repurposing candidate for wound healing.     

071TGF‐ß‐Mediated Paracrine Regulation of Keratinocyte Wound Closure

The importance of stromal‐epithelial interactions in wound healing is well established. These interactions likely involve autocrine and paracrine action of multiple growth factors, including members of the TGF‐ß family. TGF‐ß1, ß2 and ß3 isoforms signal by sequentially binding to the TGF‐ß type II and type I receptors, respectively. We address the role of TGF‐ß signaling in dermal fibroblasts using a conditional fibroblastic TGF‐ß type II receptor knockout mouse model (termed FßKO). We found that the loss of TGF‐ß signaling in the dermal fibroblasts results in accelerated excision‐wound closure compared with similar wounds in wild type mice. The mechanism of the altered rate of re‐epitheliaization in the FßKO mice was examined with regard to keratiocyte motility and proliferation. The migration of keratinocytes through collagen I coated 8 μm pore filters in the presence or absence of fibroblast‐conditioned media was tested. These experiments showed increased keratinocyte migration when incubated with FßKO dermal fibroblast conditioned media compared to media conditioned in wild type fibroblasts. Immuno‐histochemical staining of paraffin embedded intact skin indicated both wild type and FßKO mice had similar low levels of keratinocyte proliferation, based on Ki67 staining. In healing wounds, only the distal wound edges of wild type mice were proliferative. In contrast, the FßKO mice exhibited elevated proliferation across the length of the wound, including the leading edge of epithelial closure. Together our results suggest TGF‐ß signaling by the dermal fibroblasts suppresses re‐epithelialization of excision wounds by regulating keratinocyte motility and proliferation through paracrine mechanisms.
Funding: DOD BC99184 and NIH CA85492.     

KCNQ1OT1 mediates keratinocyte migration to promote skin wound healing through the miR-200b-3p/SERP1 axis

BackgroundThe basic functions of keratinocyte are crucial steps during skin wound healing. KCNQ1OT1 long noncoding RNA was found to accelerate the migration and proliferation of keratinocyte in psoriasis. Here, we elucidated the action and mechanism of KCNQ1OT1 in skin wound healing.MethodsExpression levels of genes and proteins were evaluated by quantitative real-time PCR (qRT-PCR) and western blotting. Cell migration was assessed by using scratch and transwell assays. The interaction between miR-200b-3p and KCNQ1OT1 or SERP1 (Stress Associated Endoplasmic Reticulum Protein 1) was confirmed by bioinformatics analysis, dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and pull-down assay.ResultsKCNQ1OT1 had increased significantly in wound edge 1 day and 7 day after injury. Functionally, overexpression of KCNQ1OT1 promoted keratinocyte migration. Mechanistically, KCNQ1OT1/miR-200b-3p/SERP1 constituted a competing endogenous RNA (ceRNA) network in keratinocytes. A series of rescue experiments showed that miR-200b-3p up-regulation in keratinocytes attenuated the pro-migration action of KCNQ1OT1 in cells. Moreover, knockdown of miR-200b-3p could promote keratinocyte migration, which was abolished by SERP1 silencing. KCNQ1OT1 competitively sponged for miR-200b-3p to elevate the expression of its target SERP1.ConclusionKCNQ1OT1 could promote keratinocyte migration by miR-200b-3p/SERP1 axis, suggesting that KCNQ1OT1 might play a crucial role in skin wound healing.     

Functional characterisation of bioactive peptide derived from terrestrial snail Cryptozona bistrialis and its wound‐healing property in normal and diabetic‐induced Wistar albino rats

A peptide might be an exciting biomaterial or template for the development of novel wound‐healing agents. In this report, it was isolated from the terrestrial snail Cryptozona bistrialis by enzymatic digestion and was evaluated for its in vitro wound‐healing activity in NIH/3T3 mouse fibroblasts cell line and in vivo wound‐healing activity in normal and diabetic‐induced Wistar albino rats. The C. bistrialis protein was digested by the papain enzyme, and 21.79 kDa peptide (Cb‐peptide) was purified by reversed‐phase high‐performance liquid chromatography and identified by MALDI (matrix‐assisted laser desorption/ionization)‐TOF analysis. The isolated Cb‐peptide was characterised by various analytical methods. The peptide demonstrated a capacity to prevent the development of pathogenic bacterial and fungal cultures and proved that it promotes significant wound‐healing activity in the wound scratch assay method by rapid cell migration and closure of wound. Isolated Cb‐peptide was lyophilised and formulated to ointment and analysed for in vivo wound‐healing activity in normal and diabetic (alloxan monohydrate)‐induced Wistar albino rats. Cb‐peptide ointment‐treated groups showed a greater degree of wound healing and early and complete period of epithelialisation in normal and diabetic‐induced Wistar albino rats. Cb‐peptide ointment‐treated groups showed significant excision and incision wound‐healing activity. A conclusion was reached that the peptide isolated from C. bistrialis showed greater wound‐healing activity compared with vehicle control and standard control.