Microfibril-associated protein 5 and the regulation of skin scar formation.

Many factors regulate scar formation, which yields a modified extracellular matrix (ECM). Among ECM components, microfibril-associated proteins have been minimally explored in the context of skin wound repair. Microfibril-associated protein 5 (MFAP5), a small 25 kD serine and threonine rich microfibril-associated protein, influences microfibril function and modulates major extracellular signaling pathways. Though known to be associated with fibrosis and angiogenesis in certain pathologies, MFAP5's role in wound healing is unknown. Using a murine model of skin wound repair, we found that MFAP5 is significantly expressed during the proliferative and remodeling phases of healing. Analysis of existing single-cell RNA-sequencing data from mouse skin wounds identified two fibroblast subpopulations as the main expressors of MFAP5 during wound healing. Furthermore, neutralization of MFAP5 in healing mouse wounds decreased collagen deposition and refined angiogenesis without altering wound closure. In vitro, recombinant MFAP5 significantly enhanced dermal fibroblast migration, collagen contractility, and expression of pro-fibrotic genes. Additionally, TGF-ß1 increased MFAP5 expression and production in dermal fibroblasts. Our findings suggest that MFAP5 regulates fibroblast function and influences scar formation in healing wounds. Our work demonstrates a previously undescribed role for MFAP5 and suggests that microfibril-associated proteins may be significant modulators of wound healing outcomes and scarring.

Impact of Chronic Steroid Use on Plastic Surgery Outcomes: Analysis of 94,140 Cases.

BACKGROUND: Chronic steroid use suppresses inflammation, edema, and autoimmunity, and delays wound healing. Using data from the American College of Surgeons National Surgical Quality Improvement Program, this study characterizes the risk of perioperative chronic steroid use for complications in plastic surgery cases. METHODS: A retrospective study was performed on 94,140 plastic surgery cases from the American College of Surgeons National Surgical Quality Improvement Program database for the years 2006 to 2015. CPT codes were used to categorize operations. Patients were separated into two cohorts based on chronic steroid use status. Univariate analysis was performed using chi-square, Fisher's exact, or Wilcoxon rank sum test. Logistic regression models were fitted to evaluate the association between chronic steroid use and postoperative complications. Total hospital length of stay was compared. Odds ratios were computed at the 95 percent confidence interval. RESULTS: Chronic steroid users were more likely to develop surgical complications (OR, 1.3; p = 0.0452) and medical complications (OR, 1.8; p = 0.0002) compared with nonusers. Among the 10 most frequent procedures performed on chronic steroid users, steroid use was a significant risk factor for postoperative complications after reduction mammaplasty (OR, 2.2; p = 0.001); delayed insertion of breast prosthesis following mastopexy or mastectomy or during reconstruction (OR, 2.2; p = 0.049); and in trunk muscle, myocutaneous, or fasciocutaneous flap surgery (OR, 7.2; p = 0.0029). CONCLUSION: With this information in hand, plastic surgeons will be better equipped to counsel patients and adequately design perioperative protocols for chronic steroid users. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Blockade of mast cell activation reduces cutaneous scar formation.

Damage to the skin initiates a cascade of well-orchestrated events that ultimately leads to repair of the wound. The inflammatory response is key to wound healing both through preventing infection and stimulating proliferation and remodeling of the skin. Mast cells within the tissue are one of the first immune cells to respond to trauma, and upon activation they release pro-inflammatory molecules to initiate recruitment of leukocytes and promote a vascular response in the tissue. Additionally, mast cells stimulate collagen synthesis by dermal fibroblasts, suggesting they may also influence scar formation. To examine the contribution of mast cells in tissue repair, we determined the effects the mast cell inhibitor, disodium cromoglycate (DSCG), on several parameters of dermal repair including, inflammation, re-epithelialization, collagen fiber organization, collagen ultrastructure, scar width and wound breaking strength. Mice treated with DSCG had significantly reduced levels of the inflammatory cytokines IL-1α, IL-1ß, and CXCL1. Although DSCG treatment reduced the production of inflammatory mediators, the rate of re-epithelialization was not affected. Compared to control, inhibition of mast cell activity caused a significant decrease in scar width along with accelerated collagen re-organization. Despite the reduced scar width, DSCG treatment did not affect the breaking strength of the healed tissue. Tryptase ß1 exclusively produced by mast cells was found to increase significantly in the course of wound healing. However, DSCG treatment did not change its level in the wounds. These results indicate that blockade of mast cell activation reduces scar formation and inflammation without further weakening the healed wound.

Toll-like receptor 4 has an essential role in early skin wound healing.

Toll-like receptor 4 (TLR4) has a key role in the initiation of innate immunity and in the regulation of adaptive immune responses. Using microarray analysis and PCR, TLR4 expression was observed to increase in murine skin wounds at the early stages. The cellular location of TLR4 was primarily in keratinocytes at the wound edges. The closure of excisional wounds was significantly delayed in TLR4-deficient (C3H/HeJ) as compared with wild-type mice, and both IL-1ß and IL-6 production were significantly lower in the wounds of TLR4-deficient mice. EGF also markedly decreased in the wound edge of epidermis in TLR4-deficient mice. In vitro studies confirmed that a wound stimulus induces TLR4 mRNA expression in primary normal human epidermal keratinocytes (NHEK). In vitro injury also induced the phosphorylation of p38 and JNK MAPK (Jun N-terminal kinase mitogen-activated protein kinase) and the expression of IL-1ß and tumor necrosis factor-α by NHEK. Blockade of TLR4 delayed NHEK migration and abolished the phosphorylation of p38 and JNK MAPK, and blockade of TLR4 and/or p38/JNK abolished IL-1ß production. The results suggest that inflammatory cytokine production by injured NHEK is stimulated via the TLR4-p38 and JNK MAPK signaling pathway. Together, the results provide evidence for a role of TLR4 at sites of injury, and suggest that TLR4 is an important regulator of wound inflammation.

Fibroblast function and wound breaking strength is impaired by acute ethanol intoxication.

BACKGROUND: Alcohol intoxication occurs in nearly half of all trauma patients and increases the morbidity, mortality, and healing complications of these patients. Prior studies in our laboratory and elsewhere have demonstrated impairments in re-epithelialization, angiogenesis, and inflammation in wounds following acute ethanol exposure. Clinically, acute ethanol exposure has been shown to cause an increased breakdown of wounds. To date, the mechanisms by which acute ethanol exposure modifies wound strength have received little experimental attention. METHODS: To examine how ethanol influences functions critical to the development of wound strength, the effect of ethanol exposure on fibroblast proliferation and extracellular matrix production was examined. Normal human dermal fibroblasts (NHDF) were exposed to ethanol (100 mg/dl) and then examined for proliferative capacity and mRNA production of collagen I, collagen III, and lysyl oxidase (LOX). In in vivo studies, the wound breaking strength, LOX activity, collagen, and hyaluronic acid (HA) contents of wounds of ethanol-exposed (100 mg/dl) mice were examined. RESULTS: At 24, 48, and 72 hours after acute ethanol exposure (8 hours duration), NHDF displayed a significant impairment in proliferative capacity (up to 50% at 24 hours p < 0.001). After ethanol exposure, NHDF produced less collagen I and LOX mRNA, but more collagen III mRNA than control fibroblasts (p < 0.05). Ethanol exposure in vivo caused a reduction in wound breaking strength of up to 40% when compared to control mice (p < 0.01). LOX activity, collagen, and HA contents in the wounds of ethanol-exposed mice were significantly reduced (p < 0.01). CONCLUSIONS: These studies reveal that a single exposure to ethanol prior to injury can cause a significant decrease in wound breaking strength. Our studies suggest that ethanol directly impairs fibroblast function, leading to decreased collagen production. The results provide a possible explanation for how acute ethanol exposure might increase in wound complications and wound failure.

Sprouty2 downregulates angiogenesis during mouse skin wound healing.

Angiogenesis is regulated by signals received by receptor tyrosine kinases such as vascular endothelial growth factor receptors. Mammalian Sprouty (Spry) proteins are known to function by specifically antagonizing the activation of the mitogen-activated protein kinase signaling pathway by receptor tyrosine kinases, a pathway known to promote angiogenesis. To examine the role of Spry2 in the regulation of angiogenesis during wound repair, we used a model of murine dermal wound healing. Full-thickness excisional wounds (3 mm) were made on the dorsum of anesthetized adult female FVB mice. Samples were harvested at multiple time points postwounding and analyzed using real-time RT-PCR, Western blot analysis, and immunofluorescent histochemistry. Spry2 mRNA and protein levels in the wound bed increased significantly during the resolving phases of healing, coincident with the onset of vascular regression in this wound model. In another experiment, intracellular levels of Spry2 or its dominant-negative mutant (Y55F) were elevated by a topical application to the wounds of controlled-release gel containing cell permeable, transactivator of transcription-tagged Spry2, Spry2Y55F, or green fluorescent protein (as control). Wound samples were analyzed for vascularity using CD31 immunofluorescent histochemistry as well as for total and phospho-Erk1/2 protein content. The treatment of wounds with Spry2 resulted in a significant decrease in vascularity and a reduced abundance of phospho-Erk1/2 compared with wounds treated with the green fluorescent protein control. In contrast, the wounds treated with the dominant-negative Spry2Y55F exhibited a moderate increase in vascularity and elevated phospho-Erk1/2 content. These results indicate that endogenous Spry2 functions to downregulate angiogenesis in the healing murine skin wound, potentially by inhibiting the mitogen-activated protein kinase signaling pathway.

Brewing complications: the effect of acute ethanol exposure on wound healing.

Ethanol consumption is linked to a higher incidence of traumatic wounds and increases the risk for morbidity and mortality following surgical or traumatic injury. One of the most profound effects of acute ethanol exposure on wound healing occurs during the inflammatory response, and altered cytokine production is a primary component. Acute ethanol exposure also impairs the proliferative response during healing, causing delays in epithelial coverage, collagen synthesis, and blood vessel regrowth. The accumulated data support the paradigm that acute ethanol intoxication prior to injury significantly diminishes a patient's ability to heal efficiently.