Epithelial Wound Closure Research Articles

Transcorneal delivery of topically applied silver nanoparticles does not delay epithelial wound healing

Silver nanoparticles (AgNPs) are a common antimicrobial additive for a variety of applications, including wound care. However, AgNPs often undergo dissolution resulting in release of silver ions, with subsequent toxicity to mammalian cells. The cornea is a primary exposure site to topically administered AgNPs in and around the eye but their impact on corneal wound healing is understudied. Thus, the purpose of this study was to determine in vitro toxicity of AgNPs on corneal epithelial cells and fibroblasts as well as their effects on corneal epithelial wound healing utilizing an in vivo rabbit model. Non-coated 20 nm sized AgNP (AgNP-20) as well as 1% and 10% silver silica NPs (AgSiO2NPs) were tested at concentrations ranging from 0.05–250 μg/mL. Immortalized human corneal epithelial (hTCEpi) cells and primary rabbit corneal fibroblasts (RCFs) were incubated for 24 h with AgNPs and cell viability was tested. Additionally, a round wound healing assay was performed to determine hTCEpi cell migration. Quantitative real-time PCR and western blot analysis was performed to determine α-smooth muscle actin (α-SMA, a myofibroblast marker) mRNA and protein expression, respectively, in RCFs treated with 50 μg/mL of AgNPs. Corneal epithelial wound healing was evaluated with 1%-AgSiO2NPs (10 and 250 μg/mL) using an in vivo rabbit model. Rabbits were subsequently euthanized, and histologic sections of the enucleated globes were used to determine corneal penetration of 1%-AgSiO2NPs with autometallography and hyperspectral darkfield microscopy. Cell viability of both the hTCEpi cells and fibroblasts was significantly decreased by the three AgNPs in a dose dependent manner. Migration of hTCEpi cells was significantly inhibited by the three AgNPs. Alpha-SMA mRNA expression was significantly inhibited with three AgNPs, but only the 1%-AgSiO2NPs inhibited protein expression of α-SMA. In vivo epithelial wound closure did not significantly differ between groups treated with 10 or 250 μg/mL of 1%-AgSiO2NPs or vehicle control. The 1%-AgSiO2NPs penetrated throughout all corneal layers and into the anterior chamber in all treated eyes with no histopathological changes observed. In conclusion, the 1%-AgSiO2NPs are safe and have potential therapeutic applications through its efficacy of the corneal penetration and reduced scar formation during corneal wound healing.

Changing the Wound: Covalent Immobilization of the Epidermal Growth Factor.

Re-epithelialization of wounds is a critical element of wound closure. Growth factors have been used in combination with conventional wound management to promote closure, but the method of delivery has been limited to the topical application of ointment formulations. Cytoactive factors delivered in this way have short resident times in wounds and have met with limited success. Here, we demonstrate that methods used to covalently immobilize proteins on synthetic materials can be extended to immobilize cytoactive factors such as the epidermal growth factor (EGF) onto the wound beds of genetically diabetic mice that exhibit impaired healing. Full-thickness splinted excisional wounds were created in diabetic (db/db) mice with a well-defined silicone splint to limit wound contracture. Wound surfaces were treated with a reducing agent to expose sulfhydryl groups and subsequently treated with EGF modified with a heterobifunctional crosslinker. This allowed for the covalent immobilization of the EGF to the wound surface. The conjugation chemistry was validated in vitro and in vivo. In a separate group of mice, wounds were topically treated twice daily with soluble EGF. The mice were evaluated over 11 days for wound closure. This covalent immobilization strategy resulted in EGF being retained on the wound surface for 2 days and significantly increased epithelial wound closure by 20% compared to wounds treated with topical EGF or topical vehicle. Covalent immobilization was not only therapeutically effective but also delivered a markedly reduced load of growth factor to the wound surface compared to topical application (when only 180 ng of EGF was immobilized onto the wound surface in comparison with 7200 ng of topically applied EGF over a period of 11 days). No adverse effects were observed in treated wounds. Results obtained provide proof of concept for the effectiveness of covalent immobilization in the treatment of dysregulated wounds. The covalent immobilization of cytoactive factors represents a potentially transformative approach to the management of difficult chronic wounds.

Synthesis of sonicated fibrin nanoparticles that modulate fibrin clot polymerization and enhance angiogenic responses

Chronic wounds can occur when the healing process is disrupted and the wound remains in a prolonged inflammatory stage that leads to severe tissue damage and poor healing outcomes. Clinically used treatments, such as high density, FDA-approved fibrin sealants, do not provide an optimal environment for native cell proliferation and subsequent tissue regeneration. Therefore, new treatments outside the confines of these conventional fibrin bulk gel therapies are required. We have previously developed flowable, low-density fibrin nanoparticles that, when coupled to keratinocyte growth factor, promote cell migration and epithelial wound closure in vivo. Here, we report a new high throughput method for generating the fibrin nanoparticles using probe sonication, which is less time intensive than the previously reported microfluidic method, and investigate the ability of the sonicated fibrin nanoparticles (SFBN) to promote clot formation and cell migration in vitro. The SFBNs can form a fibrin gel when combined with fibrinogen in the absence of exogenous thrombin, and the polymerization rate and fiber density in these fibrin clots is tunable based on SFBN concentration. Furthermore, fibrin gels made with SFBNs support cell migration in an in vitro angiogenic sprouting assay, which is relevant for wound healing. In this report, we show that SFBNs may be a promising wound healing therapy that can be easily produced and delivered in a flowable formulation.

In-situ porcine corneal matrix hydrogel as ocular surface bandage

PurposeBioactive substrates can be used therapeutically to enhance wound healing. Here, we evaluated the effect of an in-situ thermoresponsive hydrogel from decellularized porcine cornea ECM, COMatrix (COrnea Matrix), for application as an ocular surface bandage for corneal epithelial defects. MethodsCOMatrix hydrogel was fabricated from decellularized porcine corneas. The effects of COMatrix hydrogel on attachment and proliferation of human corneal epithelial cells (HCECs) were evaluated in vitro. The effect of COMatrix on the expressions of the inflammatory genes, IL-1β, TNF-α, and IL-6 was assessed by RT-PCR. The in-situ application and also repairing effects of COMatrix hydrogel as an ocular bandage was studied in a murine model of corneal epithelial wound. The eyes were examined by optical coherence tomography (OCT) and slit-lamp microscopy in vivo and by histology and immunofluorescence post-mortem. ResultsIn vitro, COMatrix hydrogel significantly enhanced the attachment and proliferation of HCECs relative to control. HCECs exposed to COMatrix had less induced expression of TNF-α (P < 0.05). In vivo, COMatrix formed a uniform hydrogel that adhered to the murine ocular surface after in-situ curing. Corneal epithelial wound closure was significantly accelerated by COMatrix hydrogel compared to control (P < 0.01). There was significant increase in the expression of proliferation marker Ki-67 in wounded corneal epithelium by COMatrix hydrogel compared to control (P < 0.05). ConclusionsCOMatrix hydrogel is a naturally derived bioactive material with potential application as an ocular surface bandage to enhance epithelial wound healing.

Early clinical outcomes and comparison between trans-PRK and PRK, regarding refractive outcome, wound healing, pain intensity and visual recovery time in a real\u2010world setup

PurposeTo compare early clinical outcomes of single-step transepithelial photorefractive keratectomy (tPRK) and photorefractive keratectomy (PRK) regarding refractive outcome, visual acuity, wound healing, pain intensity and visual recovery time.d.MethodsIn this prospective clinical observational study 200 eyes of 100 consecutive patients with mild to moderate myopia with or without mild astigmatism were included. One hundred eyes each were either treated with StreamLight™ tPRK or PRK with the WaveLight® EX500 excimer laser. Visual acuity (Decimal) was assessed preoperatively and at day 4, 7 and 6 weeks postoperatively. Wound healing (hours between surgery and complete epithelial closure) was monitored at the slit lamp. At day 4, patients subjectively rated the maximum pain intensity within the last 4 days using a numerical pain rating scale (0–15).ResultsVisual recovery was significantly faster in the tPRK group. At days 4 and 7, the mean monocular UCDVA was significantly better in the tPRK group than in the PRK group (p < 0.001). Four days after surgery 72 % of eyes in the tPRK group but no eye in the PRK had a UCDVA of 0.7 or better. At six weeks postoperatively, a UCDVA of 1.0 or better was achieved in both groups. Complete epithelial wound closure was achieved significantly faster in the tPRK group (p < 0.0001) and maximum pain level within the first 4 days after surgery was significantly lower in the tPRK group (p < 0.0001). No patient had lost a line of BCDVA and no complications or adverse effects were observed.ConclusionsAccording to our early clinical results, both treatments options appear to be safe and effective methods for the correction of low to moderate myopia with and without astigmatism. However, in our study, StreamLight™ tPRK offered faster visual recovery and epithelial healing and was associated with less pain compared to PRK. It can therefore be considered a good treatment option for patients who refuse or are not eligible for Femto-LASIK, but at the same time demand a faster and more comfortable recovery time than PRK can offer.

ZINC40099027 activates human focal adhesion kinase by accelerating the enzymatic activity of the FAK kinase domain.

Focal adhesion kinase (FAK) regulates gastrointestinal epithelial restitution and healing. ZINC40099027 (Zn27) activates cellular FAK and promotes intestinal epithelial wound closure in vitro and in mice. However, whether Zn27 activates FAK directly or indirectly remains unknown. We evaluated Zn27 potential modulation of the key phosphatases, PTP‐PEST, PTP1B, and SHP2, that inactivate FAK, and performed in vitro kinase assays with purified FAK to assess direct Zn27‐FAK interaction. In human Caco‐2 cells, Zn27‐stimulated FAK‐Tyr‐397 phosphorylation despite PTP‐PEST inhibition and did not affect PTP1B‐FAK interaction or SHP2 activity. Conversely, in vitro kinase assays demonstrated that Zn27 directly activates both full‐length 125 kDa FAK and its 35 kDa kinase domain. The ATP‐competitive FAK inhibitor PF573228 reduced basal and ZN27‐stimulated FAK phosphorylation in Caco‐2 cells, but Zn27 increased FAK phosphorylation even in cells treated with PF573228. Increasing PF573228 concentrations completely prevented activation of 35 kDa FAK in vitro by a normally effective Zn27 concentration. Conversely, increasing Zn27 concentrations dose‐dependently activated kinase activity and overcame PF573228 inhibition of FAK, suggesting the direct interactions of Zn27 with FAK may be competitive. Zn27 increased the maximal activity (V max) of FAK. The apparent K m of the substrate also increased under laboratory conditions less relevant to intracellular ATP concentrations. These results suggest that Zn27 is highly potent and enhances FAK activity via allosteric interaction with the FAK kinase domain to increase the V max of FAK for ATP. Understanding Zn27 enhancement of FAK activity will be important to redesign and develop a clinical drug that can promote mucosal wound healing.

Discovery of Novel Small-Molecule FAK Activators Promoting Mucosal Healing.

Gastrointestinal mucosal wounds are common to patients injured by factors as diverse as drugs, inflammatory bowel disease, peptic ulcers, and necrotizing enterocolitis. However, although many drugs are used to ameliorate injurious factors, there is no drug available to actually stimulate mucosal wound healing. Focal adhesion kinase (FAK), a nonreceptor tyrosine kinase, induces epithelial sheet migration and wound healing, making FAK a potential pharmacological target in this regard. In our previous research, we found a lead compound with drug-like properties, ZINC40099027, which promotes FAK phosphorylation, inducing mucosal healing in murine models. Herein we describe the design and optimization of a small library of novel FAK activators based on ZINC40099027 and their applications toward human intestinal epithelial wound closure and mouse ulcer healing.

Fibroblasts Close a Void in Free Space by a Purse-String Mechanism

How stromal cells fill voids in wounded tissue remains one of the most fundamental questions in regenerative medicine. Fibroblasts are known to fill voids by depositing extracellular matrix (ECM) proteins while migrating towards the wound site; however, their ability to adopt an epithelial-like purse-string behaviour remains unexplored. Here, we fabricated an artificial wound with a deep void space to investigate fibroblasts’ behaviour during gap closure. We found that fibroblasts can form a free-standing bridge on deep microvoids and consequently close the void through the purse-string contraction, which was previously believed to be exclusively an epithelial wound closure mechanism.

VEGF TrapR1R2 Suspended in the Semifluorinated Alkane F6H8 Inhibits Inflammatory Corneal Hem- and Lymphangiogenesis.

PurposeSemifluorinated alkanes (SFAs) are used at the ocular surface as lubricants or vehicles for drugs. The purpose of this study was to test the effect of vascular endothelial growth factor (VEGF) TrapR1R2 suspended in the SFA perfluorohexyloctane (Trap/F6H8) on corneal neovascularization.MethodsSuture placement was used to induce inflammatory corneal neovascularization in mice. Treatment groups were: Trap/F6H8, VEGF TrapR1R2 as aqueous formulation dissolved in phosphate buffer (Trap), F6H8, and phosphate buffer (controls). Eye drops were applied 3×/daily for 2 weeks. Afterward, corneas were stained with CD31 and LYVE-1 to analyze corneal hem- and lymphangiogenesis. To investigate the effect of on inflammatory cell recruitment, corneal CD45+ cells were quantified. In addition, epithelial wound closure after debridement was assessed by corneal fluorescein staining.ResultsTrap/F6H8 was as effective as Trap in inhibiting corneal hemangiogenesis and lymphangiogenesis after 2 weeks of treatment. After 3 days of treatment, Trap/F6H8 was even more effective than Trap in inhibiting corneal hemangiogenesis. Both treatment groups (Trap/F6H8 and Trap) significantly reduced corneal CD45+ cell recruitment. Epithelial closure after debridement was unaffected by Trap/F6H8 or Trap.ConclusionsIn this study, we demonstrate that F6H8 is a potential carrier for VEGF TrapR1R2 to topically treat corneal neovascularization. Our findings might open new treatment avenues for local anti-angiogenic therapy at the cornea, as F6H8 is already approved for the usage at the ocular surface.Translational RelevanceWith this study we show for the first time that SFAs can serve as carriers for anti-angiogenic drugs at the ocular surface.

High CO2 Levels Impair Lung Wound Healing.

Delayed lung repair leads to alveolopleural fistulae, which are a major cause of morbidity after lung resections. We have reported that intrapleural hypercapnia is associated with delayed lung repair after lung resection. Here, we provide new evidence that hypercapnia delays wound closure of both large airway and alveolar epithelial cell monolayers because of inhibition of epithelial cell migration. Cell migration and airway epithelial wound closure were dependent on Rac1-GTPase activation, which was suppressed by hypercapnia directly through the upregulation of AMP kinase and indirectly through inhibition of injury-induced NF-κB-mediated CXCL12 (pleural CXC motif chemokine 12) release, respectively. Both these pathways were independently suppressed, because dominant negative AMP kinase rescued the effects of hypercapnia on Rac1-GTPase in uninjured resting cells, whereas proteasomal inhibition reversed the NF-κB-mediated CXCL12 release during injury. Constitutive overexpression of Rac1-GTPase rescued the effects of hypercapnia on both pathways as well as on wound healing. Similarly, exogenous recombinant CXCL12 reversed the effects of hypercapnia through Rac1-GTPase activation by its receptor, CXCR4. Moreover, CXCL12 transgenic murine recipients of orthotopic tracheal transplantation were protected from hypercapnia-induced inhibition of tracheal epithelial cell migration and wound repair. In patients undergoing lobectomy, we found inverse correlation between intrapleural carbon dioxide and pleural CXCL12 levels as well as between CXCL12 levels and alveolopleural leak. Accordingly, we provide first evidence that high carbon dioxide levels impair lung repair by inhibiting epithelial cell migration through two distinct pathways, which can be restored by recombinant CXCL12.

Dynamic heterogeneity influences the leader-follower dynamics during epithelial wound closure.

Cells of epithelial tissue proliferate and pack together to attain an eventual density homeostasis. As the cell density increases, spatial distribution of velocity and force show striking similarity to the dynamic heterogeneity observed elsewhere in dense granular matter. While the physical nature of this heterogeneity is somewhat known in the epithelial cell monolayer, its biological relevance and precise connection to cell density remain elusive. Relevantly, we had demonstrated how large-scale dynamic heterogeneity in the monolayer stress field in the bulk could critically influence the emergence of leader cells at the wound margin during wound closure, but did not connect the observation to the corresponding cell density. In fact, numerous previous reports had essentially associated long-range force and velocity correlation with either cell density or dynamic heterogeneity, without any generalization. Here, we attempted to unify these two parameters under a single framework and explored their consequence on the dynamics of leader cells, which eventually affected the efficacy of collective migration and wound closure. To this end, we first quantified the dynamic heterogeneity by the peak height of four-point susceptibility. Remarkably, this quantity showed a linear relationship with cell density over many experimental samples. We then varied the heterogeneity, by changing cell density, and found this change altered the number of leader cells at the wound margin. At low heterogeneity, wound closure was slower, with decreased persistence, reduced coordination and disruptive leader-follower interactions. Finally, microscopic characterization of cell-substrate adhesions illustrated how heterogeneity influenced orientations of focal adhesions, affecting coordinated cell movements. Together, these results demonstrate the importance of dynamic heterogeneity in epithelial wound healing. This article is part of the theme issue 'Multi-scale analysis and modelling of collective migration in biological systems'.

Xylose-Inducible Promoter Tools for Pseudomonas Species and Their Use in Implicating a Role for the Type II Secretion System Protein XcpQ in the Inhibition of Corneal Epithelial Wound Closure.

Tunable control of gene expression is an invaluable tool for biological experiments. In this study, we describe a new xylose-inducible promoter system and evaluate it in both Pseudomonas aeruginosa and Pseudomonas fluorescens The Pxut promoter, derived from the P. fluorescensxut operon, was incorporated into a broad-host-range pBBR1-based plasmid and was compared to the Escherichia coli-derived PBAD promoter using gfp as a reporter. Green fluorescent protein (GFP) fluorescence from the Pxut promoter was inducible in both Pseudomonas species, but not in E. coli, which may facilitate the cloning of genes toxic to E. coli to generate plasmids. The Pxut promoter was activated at a lower inducer concentration than PBAD in P. fluorescens, and higher gfp levels were achieved using Pxut Flow cytometry analysis indicated that Pxut was leakier than PBAD in the Pseudomonas species tested but was expressed in a higher proportion of cells when induced. d-Xylose as a sole carbon source did not support the growth of P. aeruginosa or P. fluorescens and is less expensive than many other commonly used inducers, which could facilitate large-scale applications. The efficacy of this system was demonstrated by its use to reveal a role for the P. aeruginosa type II secretion system gene xcpQ in bacterial inhibition of corneal epithelial cell wound closure. This study introduces a new inducible promoter system for gene expression for use in Pseudomonas species.IMPORTANCEPseudomonas species are enormously important in human infections, in biotechnology, and as model systems for investigating basic science questions. In this study, we have developed a xylose-inducible promoter system, evaluated it in P. aeruginosa and P. fluorescens, and found it to be suitable for the strong induction of gene expression. Furthermore, we have demonstrated its efficacy in controlled gene expression to show that a type II secretion system protein from P. aeruginosa, XcpQ, is important for host-pathogen interactions in a corneal wound closure model.

Role of VIP and Sonic Hedgehog Signaling Pathways in Mediating Epithelial Wound Healing, Sensory Nerve Regeneration, and Their Defects in Diabetic Corneas.

Diabetic keratopathy, a sight-threatening corneal disease, comprises several symptomatic conditions including delayed epithelial wound healing, recurrent erosions, and sensory nerve (SN) neuropathy. We investigated the role of neuropeptides in mediating corneal wound healing, including epithelial wound closure and SN regeneration. Denervation by resiniferatoxin severely impaired corneal wound healing and markedly upregulated proinflammatory gene expression. Exogenous neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP) partially reversed resiniferatoxin's effects, with VIP specifically inducing interleukin-10 expression. Hence, we focused on VIP and observed that wounding induced VIP and VIP type 1 receptor (VIPR1) expression in normal (NL) corneas, but not corneas from mice with diabetes mellitus (DM). Targeting VIPR1 in NL corneas attenuated corneal wound healing, dampened wound-induced expression of neurotrophic factors, and exacerbated inflammatory responses, while exogenous VIP had the opposite effects in DM corneas. Remarkably, wounding and diabetes also affected the expression of Sonic Hedgehog (Shh) in a VIP-dependent manner. Downregulating Shh expression in NL corneas decreased while exogenous Shh in DM corneas increased the rates of corneal wound healing. Furthermore, inhibition of Shh signaling dampened VIP-promoted corneal wound healing. We conclude that VIP regulates epithelial wound healing, inflammatory response, and nerve regeneration in the corneas in an Shh-dependent manner, suggesting a therapeutic potential for these molecules in treating diabetic keratopathy.

Potential Effect of Plasma Rich in Growth Factors-Endoret in Stromal Wound Healing in Additive Surgery

Purpose: To compare the clinical and histological outcomes after intrastromal corneal ring segment (ICRS) implantation with and without plasma rich in growth factors (PRGF) in an experimental animal model. Materials and Methods: First, the toxicity of PRGF was tested in hen’s keratocyte cultures. Then, an animal model with 18 hens was randomly divided into 2 groups. In the first group, one ICRS was implanted in each eye (ICRS group). In the second group, the ICRS was firstly immersed 30 min in PRGF-Endoret solution, then implanted and, finally, PRGF-Endoret was inoculated into the channel (PRGF-ICRS group). Animals of each group were also separated into 3 groups regarding the time they were sacrificed, and corneal tissue was fixed for histological analysis at 2, 7 and 30 days. Cell death was detected by terminal uridine nick end labelling (TUNEL) assay. Proliferation was labelled by 5-bromo-2-deoxyuridine (BrdU) incorporation and myofibroblast differentiation by alpha-smooth muscle actin (αSMA) immunodetection. Clinical examination, analyzing epithelial wound closure, deposits and stromal haze, was carried out at the different study times. Results: No toxic effect was observed by PRGF in hen stromal cell cultures. Clinically, in PRGF-ICRS corneas at 7 days, there were more deposits with higher intensity than in ICRS group. Histologically, at day 2 there was less epithelial damage over the segment in the PRGF-ICRS group, corneal oedema around the segment disappeared earlier and, at day 7, there was also double the number of cells around the segment than in the ICRS group displaying different morphologies. The number of TUNEL-positive cells was statistically higher in the PRGF-ICRS group at 7 and 30 days, and the number of BrdU-positive cells was statistically higher at all analyzed times. However, there were no differences in the number of αSMA-positive cells at 30 days between both groups. Conclusions: The ICRS immersion in PRGF-Endoret prior and after to its corneal implantation, in an experimental animal model, enhances clinical deposits and histological cell turnover without increasing myofibroblast differentiation reducing stromal wound-healing time after surgery.

Neurokinin-1 Receptor Antagonism Ameliorates Dry Eye Disease by Inhibiting Antigen-Presenting Cell Maturation and T Helper 17 Cell Activation

Neuroinflammation plays an important role in the pathogenesis of ocular surface disease, including dry eye disease (DED), but little is known about the contribution of substance P (SP) to DED. In this study, we investigated the expression of SP at the ocular surface and evaluated its effect on maturation of antigen-presenting cells (APCs), the key cell component involved in the induction of type 17 helper T-cell (Th17) response in DED. The effect of topical blockade of SP signaling was further investigated using neurokinin-1 receptor (NK1R) inhibitors on APC maturation, Th17 cell activation, and disease severity in a mouse model of DED. The results demonstrate that SP is constitutively expressed at the ocular surface, and trigeminal ganglion neurons are the major source of SP in DED. SP derived from trigeminal ganglion enhanced the expression of major histocompatibility complex class II maturation marker by bone marrow-derived dendritic cells, an effect that is abrogated by blockade of SP signaling using NK1R antagonist spantide. Finally, using a well-established murine model of DED, topical treatment of DED mice with NK1R antagonists CP-99,994 and L-733,060 suppressed APC acquisition of major histocompatibility complex class II, reduced Th17 cell activity, and ameliorated DED severity. These findings are of translational value, as they suggest that antagonizing NK1R-mediated SP signaling may be an effective strategy in suppressing Th17-mediated ocular surface disease.

Small molecule FAK activator promotes human intestinal epithelial monolayer wound closure and mouse ulcer healing

GI mucosal healing requires epithelial sheet migration. The non-receptor tyrosine kinase focal adhesion kinase (FAK) stimulates epithelial motility. A virtual screen identified the small drug-like FAK mimic ZINC40099027, which activates FAK. We assessed whether ZINC40099027 promotes FAK-Tyr-397 phosphorylation and wound healing in Caco-2 monolayers and two mouse intestinal injury models. Murine small bowel ulcers were generated by topical serosal acetic acid or subcutaneous indomethacin in C57BL/6J mice. One day later, we began treatment with ZINC40099027 or DMSO, staining the mucosa for phosphorylated FAK and Ki-67 and measuring mucosal ulcer area, serum creatinine, ALT, and body weight at day 4. ZINC40099027 (10–1000 nM) dose-dependently activated FAK phosphorylation, without activating Pyk2-Tyr-402 or Src-Tyr-419. ZINC40099027 did not stimulate proliferation, and stimulated wound closure independently of proliferation. The FAK inhibitor PF-573228 prevented ZINC40099027-stimulated wound closure. In both mouse ulcer models, ZINC40099027accelerated mucosal wound healing. FAK phosphorylation was increased in jejunal epithelium at the ulcer edge, and Ki-67 staining was unchanged in jejunal mucosa. ZINC40099027 serum concentration at sacrifice resembled the effective concentration in vitro. Weight, creatinine and ALT did not differ between groups. Small molecule FAK activators can specifically promote epithelial restitution and mucosal healing and may be useful to treat gut mucosal injury.

Bioactive peptide amphiphile nanofiber gels enhance burn wound healing

BackgroundBurns are physically debilitating and potentially fatal injuries. The standard-of-care for burn wounds is the coverage with gauze dressings designed to minimize trauma to the regenerating epidermis and dermis during dressing changes. However, deep partial- and full-thickness burns always heal slowly when standard wound care alone is performed. We have previously reported that peptide amphiphile (PA) gels, pH-induced self-assembling nanostructured fibrous scaffolds, promote cell proliferation and have great potential in regenerative medicine for rapid repair of tissues. In this study, we hypothesized that the PA gels are capable of accelerating wound healing in burn injury. MethodsArtificially generated thermally damaged fibroblasts and human umbilical vein endothelial cells were seeded onto the various PA nanofiber gels including bioactive and nonbioactive peptide sequences. Cell proliferation was assessed at different time points, and thermally damaged fibroblasts and HUVECs manifested increased proliferation with time when cultured with various PA gels. To determine in vivo effects, burn wounds of rats were treated with the bioactive Arg-Gly-Asp-Ser (RGDS)-modified gel that showed greater cell proliferation in vitro. The wound closure was observed, and skin samples were harvested for histologic evaluation. ResultsCell proliferation using the RGDS-PA gel was significantly higher than that observed in other gels. The RGDS-PA gel significantly enhanced re-epithelialization during the burn wound healing process between days 7 and 28. Application of PA gels accelerates the recovery of deep partial-thickness burn wounds by stimulation of fibroblasts and the creation of an environment conducive to epithelial cell proliferation and wound closure. ConclusionsThis biomaterial represents a new therapeutic strategy to overcome current clinical challenges in the treatment of injuries resulting from burns.

Functional Role of Microbiota‐derived Metabolites in the GPCR‐mediated Regulation of Intestinal Wound Healing and Barrier Function

The mammalian intestine houses a complex and abundant ensemble of commensal bacteria, which are in constant cross‐talk with the intestinal epithelial cells, and thereby, ensure gut homeostasis, development, permeability, and repair. Wound regeneration is a complex but coordinated cellular events to repair injured gut mucosa, which involves both cellular proliferation and migration. Gut barrier function is central to the health and is critical in a wide variety of diseases including inflammatory bowel diseases (IBD). Our recent metagenomics study discovered a consortium of novel gut mucosa‐associated proregenerative symbiotic bacteria, which preferentially enrich in and colonize the injured mucosa to augment epithelial repair. The resident microbiota of the gut is endowed with over a hundred times more genes than those in our genome. However, the microbiota's more than nine million genes, and their metabolic functions remained mostly unrecognized. We performed an unbiased screening analysis by mass spectrometry‐based metabolomic profiling of intact and wounded mucosa in germ‐free and conventional mice. Unlike the germ‐free animals, the colon of the conventional mice demonstrated increased amounts of acidic microbial metabolites, which established an extracellular pH gradient with more acidic pH at the apex of the colonic crypt. We found that the murine and human intestinal epithelial cells express the proton‐sensing G‐protein coupled‐receptor GPCR68. Intriguingly, the murine colonic contents of the conventional mouse or a specific metabolite, 2‐hydroxy‐4‐methylpentanoic acid (HMP) decreases the extracellular pH to modulate claudin‐4 dependent paracellular permeability and to regulate the proliferation of intestinal epithelial cells in a GPCR68‐dependent manner. Importantly, our findings highlighted that the HMP‐stimulated intestinal epithelial wound closure requires GPR68. Collectively, our data underscore the significance of intestinal metabolites in modulating the extracellular proton concentration in the gut mucosal microenvironment. Furthermore, our findings demonstrate that the GPCR68 functions as the sensor of microbially modulated proton gradient that is potentially involved in the maintenance of gut mucosal homeostasis, wound healing, and restoration of the intestinal barrier function.Support or Funding InformationK01DK114391 (National Institute of Diabetes and Digestive and Kidney Diseases), R01DK089763 (National Institute of Diabetes and Digestive and Kidney Diseases), R01AI064462 (National Institute of Allergy and Infectious Diseases)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Staphylococcus aureus alpha-hemolysin impairs corneal epithelial wound healing and promotes intracellular bacterial invasion

Colonization by Staphylococcus aureus (S. aureus) has been implicated in many infectious and wound healing disorders. This study was performed to characterize the pathogenic role of S. aureus alpha-hemolysin (alpha-toxin) in corneal epithelial wound healing and infectious keratitis in the setting of a corneal wound. The effect of wild-type and isogenic Hla mutant (α-hemolysin gene deleted) S. aureus bacteria and conditioned media on corneal epithelial wound healing was tested in vitro using a scratch assay and in vivo using a murine epithelial debridement model. The invasiveness of wild-type and Hla mutant S. aureus was evaluated in vitro in human corneal epithelial cells and in vivo in a murine model of infectious keratitis following total epithelial debridement. S. aureus and its conditioned media significantly delayed epithelial wound closure both in vitro (P < 0.05) and in vivo (P < 0.05). The effect of S. aureus on wound healing was significantly diminished with the Hla mutant strain (P < 0.05). Likewise, compared to the wild-type strain, the Hla mutant strain demonstrated significantly reduced ability to invade corneal epithelial cells in vitro (P < 0.05) and infect murine corneas following total epithelial debridement in vivo (P < 0.05). In conclusion, S. aureus alpha-hemolysin plays a major role in the pathologic modulation of corneal epithelial wound healing and the intracellular invasion of the bacteria. Limiting colonization by S. aureus and/or blocking alpha-hemolysin may provide a therapeutic approach for corneal wound healing and infectious disorders.

Opposing Effects of Neuropilin-1 and -2 on Sensory Nerve Regeneration in Wounded Corneas: Role of Sema3C in Ameliorating Diabetic Neurotrophic Keratopathy.

The diabetic cornea exhibits pathological alterations, such as delayed epithelial wound healing and nerve regeneration. We investigated the role of semaphorin (SEMA) 3C in corneal wound healing and reinnervation in normal and diabetic B6 mice. Wounding induced the expression of SEMA3A, SEMA3C, and their receptor neuropilin-2 (NRP2), but not NRP1, in normal corneal epithelial cells; this upregulation was suppressed for SEMA3C and NRP2 in diabetic corneas. Injections of Sema3C-specific small interfering RNA and NRP2-neutralizing antibodies in wounded mice resulted in a decrease in the rate of wound healing and regenerating nerve fibers, whereas exogenous SEMA3C had opposing effects in diabetic corneas. NRP1 neutralization, on the other hand, decreased epithelial wound closure but increased sensory nerve regeneration in diabetic corneas, suggesting a detrimental role in nerve regeneration. Taken together, epithelium-expressed SEMA3C plays a role in corneal epithelial wound closure and sensory nerve regeneration. The hyperglycemia-suppressed SEMA3C/NRP2 signaling may contribute to the pathogenesis of diabetic neurotrophic keratopathy, and SEMA3C might be used as an adjunctive therapeutic for treating the disease.