Transcriptional changes in human palate and skin healing.

Most human tissue injuries lead to the formation of a fibrous scar and result in the loss of functional tissue. One adult tissue that exhibits a more regenerative response to injury with minimal scarring is the oral mucosa. We generated a microarray gene expression dataset to examine the response to injury in human palate and skin excisional biopsies spanning the first 7 days after wounding. Differential expression analyses were performed in each tissue to identify genes overexpressed or underexpressed over time when compared to baseline unwounded tissue gene expression levels. To attribute biological processes of interest to these gene expression changes, gene set enrichment analysis was used to identify core gene sets that are enriched over the time-course of the wound healing process with respect to unwounded tissue. This analysis identified gene sets uniquely enriched in either palate or skin wounds and gene sets that are enriched in both tissues in at least one time point after injury. Finally, a cell type enrichment analysis was performed to better understand the cell type distribution in these tissues and how it changes over the time course of wound healing. This work provides a source of human wound gene expression data that includes two tissue types with distinct regenerative and scarring phenotypes.

Dermal fibroblast phagocytosis of apoptotic cells: A novel pathway for wound resolution.

The effective clearance of apoptotic cells is an essential step in the resolution of healing wounds. In particular, blood vessel regression during wound resolution produces a significant number of apoptotic endothelial cells (ApoEC) that must be cleared. In considering the fate of ApoEC and the presence of fibroblasts during wound resolution, we hypothesized that fibroblasts might serve as phagocytes involved in endothelial cell removal. The current study investigated whether dermal fibroblasts engulf ApoEC, whether this uptake alters the phenotype of dermal fibroblasts, and the biological molecules involved. In both in vitro and in vivo studies, following ApoEC engulfment, fibroblasts acquired a pro-healing phenotype (increased cell migration, contractility, α-smooth muscle actin expression, and collagen deposition). In addition, fibroblast uptake of ApoEC was shown to be mediated in part by the milk fat globule-EGF factor 8 protein/integrin αv ß5 pathway. Our study demonstrates a novel function of fibroblasts in the clearance of ApoEC and suggests that this capability has significant implications for tissue repair and fibrosis.

Macrophages in Healing Wounds: Paradoxes and Paradigms.

Macrophages are prominent cells in normally healing adult skin wounds, yet their exact functions and functional significance to healing outcomes remain enigmatic. Many functional attributes are ascribed to wound macrophages, including host defense and support of the proliferation of new tissue to replace that lost by injury. Indeed, the depletion of macrophages is unmistakably detrimental to normal skin healing in adult mammals. Yet in certain systems, dermal wounds seem to heal well with limited or even no functional macrophages, creating an apparent paradox regarding the function of this cell in wounds. Recent advances in our understanding of wound macrophage phenotypes, along with new information about cellular plasticity in wounds, may provide some explanation for the apparently contradictory findings and suggest new paradigms regarding macrophage function in wounds. Continued study of this remarkable cell is needed to develop effective therapeutic options to improve healing outcomes.

LDL induces cholesterol loading and inhibits endothelial proliferation and angiogenesis in Matrigels: correlation with impaired angiogenesis during wound healing.

Hypercholesterolemia is a major risk factor for adverse cardiovascular outcomes, but its effect on angiogenesis and wound healing is not well understood. In this study, using a combination of mass spectrometry and laurdan two-photon imaging, we show that elevated levels of low-density lipoprotein (LDL), like those seen in hypercholesterolemic patients, lead to an increase in both free cholesterol and cholesterol esters, as well as increase in lipid order of endothelial cell membranes. Notably, these effects are distinct and opposite to the lack of cholesterol loading and the disruption of lipid order observed in our earlier studies in response to oxidized LDL (oxLDL). The same pathological level of LDL leads to a significant inhibition of endothelial proliferation and cell cycle arrest in G2/M phase, whereas oxLDL enhances endothelial proliferation in S phase of the cycle. LDL but not oxLDL suppresses the expression of vascular endothelial growth factor receptor-2 while enhancing the expression of vascular endothelial growth factor (VEGF). Furthermore, we show that aged (8-10 mo) hypercholesterolemic apolipoprotein E-deficient (ApoE-/-) mice display delayed wound closure compared with age-matched C57/BL6 wild-type controls following a skin punch biopsy. The delay in wound healing is associated with a decreased expression of cluster of differentiation 31 platelet endothelial cell adhesion molecule endothelial marker and decreased angiogenesis within the wound bed. Furthermore, decreased endothelial responsiveness to the growth factors VEGF and basic fibroblast growth factor is observed in ApoE-/- mice in Matrigel plugs and in Matrigels with high levels of LDL in wild-type mice. We propose that plasma hypercholesterolemia is antiangiogenic due to elevated levels of LDL.

Pigment epithelium-derived factor attenuates angiogenesis and collagen deposition in hypertrophic scars.

Scar forming wounds are often characterized by higher levels of vascularity than non-scarring wounds and normal skin, and inhibition of angiogenesis has been shown to inhibit scar formation in some model systems. The rabbit ear hypertrophic scar (HS) model has been widely used to study the mechanisms that underlie the development of HS as well as the effectiveness of various treatments. Although the rabbit ear HS model is well characterized in terms of scar formation, the rate and level of angiogenesis has not been investigated in this model, and the cause-effect relationship between angiogenesis and rabbit HSs has not been examined. In the current study, full-thickness excisional wounds were created on the ventral side of New Zealand White rabbit ears to induce HS formation, and the dynamic pattern of angiogenesis and the expression of angiogenic regulatory factors were examined over time. Blood vessel density was found to peak at 2.7% on day 14 post-wounding, decreasing to 1.7% by day 28. mRNA levels of the proangiogenic factor VEGF-A peaked at day 14, while the expression of the antiangiogenic factors pigment epithelium-derived factor (PEDF) and thrombospondin 1 (TSP1) peaked at day 28 post-wounding. To examine whether inhibition of angiogenesis influences HS formation in this model, wounds were treated with exogenous soluble antiangiogenic agents including recombinant PEDF (rPEDF) and a functional PEDF peptide (PEDF-335). rPEDF and PEDF-335 were administered intradermally from day 4 post-wounding every 3 days until day 19. Intradermal injection of rPEDF or PEDF-335 both led to decreased angiogenesis and decreased collagen deposition at the wound site. The results support the utility of antiangiogenic therapies, including rPEDF/PEDF-335, as a potential new strategy for the prevention or treatment of HSs.

Site-Specific Expression Pattern of PIWI-Interacting RNA in Skin and Oral Mucosal Wound Healing.

The oral mucosa exhibits exceptional healing capability when compared to skin. Recent studies suggest that intrinsic differences in coding genes and regulatory small non-coding RNA (sncRNA) genes (e.g., microRNAs) may underlie the exceptional healing that occurs in the oral mucosa. Here, we investigate the role of a novel class of sncRNA-Piwi-interacting RNA (piRNA)-in the tissue-specific differential response to injury. An abundance of piRNAs was detected in both skin and oral mucosal epithelium during wound healing. The expression of PIWI genes (the obligate binding partners of piRNAs) was also detected in skin and oral wound healing. This data suggested that PIWI-piRNA machinery may serve an unknown function in the highly orchestrated wound healing process. Furthermore, unique tissue-specific piRNA profiles were obtained in the skin and oral mucosal epithelium, and substantially more changes in piRNA expression were observed during skin wound healing than oral mucosal wound healing. Thus, we present the first clue suggesting a role of piRNA in wound healing, and provide the first site-specific piRNA profile of skin and oral mucosal wound healing. These results serve as a foundation for the future investigation of the functional contribution(s) of piRNA in wound repair and tissue regeneration.

Predictive Analysis of Mechanistic Triggers and Mitigation Strategies for Pathological Scarring in Skin Wounds.

Wound fibrosis (i.e., excessive scar formation) is a medical problem of increasing prevalence, with poorly understood mechanistic triggers and limited therapeutic options. In this study, we employed an integrated approach that combines computational predictions with new experimental studies in mice to identify plausible mechanistic triggers of pathological scarring in skin wounds. We developed a computational model that predicts the time courses for six essential cell types, 18 essential molecular mediators, and collagen, which are involved in inflammation and proliferation during wound healing. By performing global sensitivity analyses using thousands of model-simulated wound-healing scenarios, we identified five key processes (among the 90 modeled processes) whose dysregulation may lead to pathological scarring in wounds. By modulating a subset of these key processes, we simulated fibrosis in wounds. Moreover, among the 18 modeled molecular mediators, we identified TGF-ß and the matrix metalloproteinases as therapeutic targets whose modulation may reduce fibrosis. The model predicted that simultaneous modulation of TGF-ß and matrix metalloproteinases would be more effective in treating excessive scarring than modulation of either therapeutic target alone. Our model was validated with previously published and newly generated experimental data, and suggested new in vivo experiments.

Overexpression of the Oral Mucosa-Specific microRNA-31 Promotes Skin Wound Closure.

Wounds within the oral mucosa are known to heal more rapidly than skin wounds. Recent studies suggest that differences in the microRNAome profiles may underlie the exceptional healing that occurs in oral mucosa. Here, we test whether skin wound-healing can be accelerating by increasing the levels of oral mucosa-specific microRNAs. A panel of 57 differentially expressed high expresser microRNAs were identified based on our previously published miR-seq dataset of paired skin and oral mucosal wound-healing [Sci. Rep. (2019) 9:7160]. These microRNAs were further grouped into 5 clusters based on their expression patterns, and their differential expression was confirmed by TaqMan-based quantification of LCM-captured epithelial cells from the wound edges. Of these 5 clusters, Cluster IV (consisting of 8 microRNAs, including miR-31) is most intriguing due to its tissue-specific expression pattern and temporal changes during wound-healing. The in vitro functional assays show that ectopic transfection of miR-31 consistently enhanced keratinocyte proliferation and migration. In vivo, miR-31 mimic treatment led to a statistically significant acceleration of wound closure. Our results demonstrate that wound-healing can be enhanced in skin through the overexpression of microRNAs that are highly expressed in the privileged healing response of the oral mucosa.

Computational analysis identifies putative prognostic biomarkers of pathological scarring in skin wounds.

BACKGROUND: Pathological scarring in wounds is a prevalent clinical outcome with limited prognostic options. The objective of this study was to investigate whether cellular signaling proteins could be used as prognostic biomarkers of pathological scarring in traumatic skin wounds. METHODS: We used our previously developed and validated computational model of injury-initiated wound healing to simulate the time courses for platelets, 6 cell types, and 21 proteins involved in the inflammatory and proliferative phases of wound healing. Next, we analysed thousands of simulated wound-healing scenarios to identify those that resulted in pathological (i.e., excessive) scarring. Then, we identified candidate proteins that were elevated (or decreased) at the early stages of wound healing in those simulations and could therefore serve as predictive biomarkers of pathological scarring outcomes. Finally, we performed logistic regression analysis and calculated the area under the receiver operating characteristic curve to quantitatively assess the predictive accuracy of the model-identified putative biomarkers. RESULTS: We identified three proteins (interleukin-10, tissue inhibitor of matrix metalloproteinase-1, and fibronectin) whose levels were elevated in pathological scars as early as 2 weeks post-wounding and could predict a pathological scarring outcome occurring 40 days after wounding with 80% accuracy. CONCLUSION: Our method for predicting putative prognostic wound-outcome biomarkers may serve as an effective means to guide the identification of proteins predictive of pathological scarring.

Pore Diameter of Mesoporous Silica Modulates Oxidation of H2O2-Sensing Chromophore in a Porous Matrix.

Hydrogen peroxide (H2O2) is an attractive chemical because of its bleaching properties in paper and pulp industry and as a disinfectant in the food, water, and medical industries. However, it is important to monitor the residual H2O2 level after its usage and prevent any unintended health problems or chemical reactions. Most H2O2 sensors often utilize fluorophores or electrical circuitry that requires an additional irradiation or a digital display. To this end, this study presents a 3,3',5,5'-tetramethylbenzidine (TMB)/horseradish peroxidase (HRP)-loaded patch that alerts the presence of high H2O2 levels by generating a visible blue color. We hypothesized that water-insoluble TMB immobilized within mesoporous silica particles of proper pore diameter and structure would act as a colorimetric indicator through the H2O2-mediated oxidation within a cross-linked patch. We examined this hypothesis by immobilizing TMB molecules in mesoporous silica particles with 2 and 7 nm diameter cylindrical pores as well as on nonporous silica particles. Then, we loaded these TMB-silica particles and HRP in a porous alginate patch via sequential in situ cross-linking reaction and lyophilization. In the presence of 25-5000 µM H2O2, which simulate H2O2 concentrations found in residual disinfecting fluids, the patch loaded with TMB-mesoporous silica particles with 7 nm diameter pores generated a distinct blue color with varying intensities depending on the H2O2 concentration. The design principles demonstrated in this study should be applicable to a broad array of sensors to be integrated into a moldable, three-dimensional matrix.