IL-10 promotes endothelial progenitor cell infiltration and wound healing via STAT3.

Endothelial progenitor cells (EPCs) contribute to de novo angiogenesis, tissue regeneration, and remodeling. Interleukin 10 (IL-10), an anti-inflammatory cytokine that primarily signals via STAT3, has been shown to drive EPC recruitment to injured tissues. Our previous work demonstrated that overexpression of IL-10 in dermal wounds promotes regenerative tissue repair via STAT3-dependent regulation of fibroblast-specific hyaluronan synthesis. However, IL-10's role and specific mode of action on EPC recruitment, particularly in dermal wound healing and neovascularization in both normal and diabetic wounds, remain to be defined. Therefore, inducible skin-specific STAT3 knockdown mice were studied to determine IL-10's impact on EPCs, dermal wound neovascularization and healing, and whether it is STAT3-dependent. We show that IL-10 overexpression significantly elevated EPC counts in the granulating wound bed, which was associated with robust capillary lumen density and enhanced re-epithelialization of both control and diabetic (db/db) wounds at day 7. We noted increased VEGF and high C-X-C motif chemokine 12 (CXCL12) levels in wounds and a favorable CXCL12 gradient at day 3 that may support EPC mobilization and infiltration from bone marrow to wounds, an effect that was abrogated in STAT3 knockdown wounds. These findings were supported in vitro. IL-10 promoted VEGF and CXCL12 synthesis in primary murine dermal fibroblasts, with blunted VEGF expression upon blocking CXCL12 in the media by antibody binding. IL-10-conditioned fibroblast media also significantly promoted endothelial sprouting and network formation. In conclusion, these studies demonstrate that overexpression of IL-10 in dermal wounds recruits EPCs and leads to increased vascular structures and faster re-epithelialization.

Endogenous Interleukin-10 Contributes to Wound Healing and Regulates Tissue Repair.

INTRODUCTION: Interleukin-10 (IL-10) is essential in fetal regenerative wound healing and likewise promotes a regenerative phenotype in adult dermal wounds. However, the role of endogenous IL-10 in postnatal dermal wound healing is not well-established. We sought to determine the function of endogenous IL-10 in murine full thickness excisional wounds that are splinted to prevent contracture and mimic human patterns of wound closure. METHODS: Full-thickness excisional wounds were made in wildtype (WT) and IL-10-/- mice on a C57BL/6J background (F/M, 8 wk old). In a subset of wounds, contraction was prevented by splinting with silicone stents (stenting) and maintaining a moist wound microenvironment using a semiocclusive dressing. Wounds were examined for re-epithelialization, granulation tissue deposition, and inflammatory cell infiltrate at day 7 and fibrosis and scarring at day 30 postwounding. RESULTS: We observed no difference in wound healing rate between WT and IL-10-/- mice in either the stented or unstented group. At day 7, unstented IL-10-/- wounds had a larger granulation tissue area and more inflammatory infiltrate than their WT counterparts. However, we did observe more F4/80+ cell infiltrate in stented IL-10-/- wounds at day 7. At day 30, stented wounds had increased scar area and epithelial thickness compared to unstented wounds. CONCLUSIONS: These data suggest that endogenous IL-10 expression does not alter closure of full thickness excisional wounds when wound hydration and excessive contraction of murine skin are controlled. However, the loss of IL-10 leads to increased inflammatory cell infiltration and scarring. These new findings suggest that IL-10 contributes to the regulation of inflammation without compromising the healing response. These data combined with previous reports of increased rates of healing in IL-10-/- mice wounds not controlled for hydration and contraction suggest an important role for murine wound healing models used in research studies of molecular mechanisms that regulate healing.

Protecting human amnion and chorion matrices during processing: Performance enhancement in a diabetic mouse model and human co-culture system.

Recent evidence suggests that protecting human amnion and chorion matrices (HACM) during processing enhances the performance of HACM for wound repair and tissue regeneration. We utilised a diabetic (db/db) delayed wound healing mouse model. Treatment of db/db full-thickness excisional wounds with HACM, processed with a polyampholyte preservative accentuated the proliferative phase of wound healing that decreased the time necessary to heal wounds. Polyampholyte protection improved the preservation of growth factors and cytokines during room temperature storage following E-beam sterilisation and improved its function in wound healing applications. Our findings indicate protected HACM tissue up-regulated MIP2, NF-kB, TNF-α, KI-67, and Arg1 (0.6-fold to 1.5-fold) but those changes were not statistically significant. Immunofluorescent assessment identifying cell activity illustrated an induction of the proliferative phase of wound healing and a switch from an inflammatory macrophage phenotype (M1) to a pro-regenerative macrophage phenotype (M2a). Genomic profiling of 282 genes was performed using Nanostring from co-cultures of human macrophages and fibroblasts. The polyampholyte + HACM-treated group, compared with the HACM or polyampholyte alone groups, had a statistically significant up-regulation (32-368 fold) of 12 genes primarily involved in macrophage plasticity including CLC7, CD209, CD36, HSD11B1, ICAM1, IL1RN, IL3RA, ITGAX, LSP1, and PLXDC2 (adj. p-value < 0.05). The polyampholyte alone group demonstrated statistically significant down-regulation of four genes ADRA2, COL7A1, CSF3, and PTGS2 (adj. p < 0.05). The HACM alone group up-regulated four genes ATG14, CXCL11, DNMT3A, and THBD, but the results were not statistically significant. Biomechanical measurements indicated that wounds treated with polyampholyte-protected HACM had more tensile integrity compared with wounds treated with HACM alone. These findings indicate that better protection of HACM during processing stabilises the HACM matrix, which may lead to improved wound healing outcomes.

Interleukin-10 Producing T Lymphocytes Attenuate Dermal Scarring.

OBJECTIVE: Demonstrate the impact of IL-10 producing T lymphocytes on mediating dermal scarring. SUMMARY BACKGROUND DATA: We demonstrated that CD4+ cells are essential to improving postinjury wound healing and preventing fibrosis. CD4+ subsets secrete differential cytokine and growth factor profiles, though their role in fibrosis is not known. IL-10, a key anti-inflammatory cytokine shown to promote regenerative wound healing, is secreted by some CD4+ subsets. We, therefore, hypothesize that IL-10 producing CD4+ T lymphocyte subsets selectively attenuate dermal wound fibrosis. METHODS: IL-10-/- and wild-type murine splenocytes were enriched for CD4+ lymphocytes and adoptively transferred into severe combined immunodeficient (SCID) mice that received full-thickness wounds which were analyzed at days 7 and 28 for inflammation and collagen content. We then sorted CD4+CD44int/lowFoxP3-CD62L+ T cells (Tnaive) or CD4+CD44HiFoxP3- type 1 regulatory (Tr1) T cell subsets from 10BiT murine splenocytes, activated them, and transferred them into wounds. In vitro, dermal fibroblasts were cocultured with Tnaive or Tr1 and the effect on extracellular matrix (ECM) regulation was analyzed. RESULTS: The anti-inflammatory and antifibrotic effects of CD4+ cells on SCID wounds were lost with cells from IL-10-/- mice. Adoptive transfer of Tr1 into SCID mice resulted in accelerated wound closure at d7 with reduced fibrosis at d28, with Tr1 favoring hyaluronan production by fibroblasts, an ECM molecule implicated in IL-10-induced regenerative healing. CONCLUSIONS: IL-10 producing T-lymphocytes, specifically Tr1, regulate inflammatory cell cytokine expression to promote HA-rich ECM deposition and attenuate fibrosis. Promoting IL-10 producing lymphocytes in wounds may be a therapeutic target to promote regenerative wound healing.

Novel antimicrobial ointment for infected wound healing in an in vitro and in vivo porcine model.

Microbial contamination of wounds is a significant problem that delays healing, particularly when bacterial biofilms are present. A novel combination of pectinic acid (PG) + caprylic acid (CAP) was previously found in vitro to be highly effective in eradicating various pathogens in biofilms with minimal cytotoxicity. In this study, a novel wound ointment was formulated with PG + CAP and first assessed in vitro using a well-established biofilm eradication model. In vitro, the PG + CAP ointment was shown to be efficacious in reducing the microbial biofilms. This ointment was then tested in vivo in two pilot porcine wound healing models, with and without Staphylococcus aureus microbial challenge. Ointments were applied to each wound daily, and healing by wound closure area measurement was assessed weekly over 4 weeks. After 4 weeks, pigs were sacrificed and wounds were scored for reepithelialization, inflammation, granulation tissue, and collagen deposition. We compared PG + CAP to hydroxyethylcellulose + glycerol ointment base (control) and MediHoney (comparator). In the porcine microbial challenge model, the novel antimicrobial PG + CAP wound ointment rapidly eradicated bacterial organisms embedded in wounds, was safe and well-tolerated, and was associated with enhanced healing compared to ointment base and MediHoney. Specifically, the cumulative histopathology, reepithelialization of epidermis, and mature granulation tissue in the wound bed was significantly better with PG + CAP than with control and MediHoney treatments. This ointment warrants further study as a non-antibiotic ointment for use in treating a wide array of infected wounds.

Wound Healing and Wound Care in Neonates: Current Therapies and Novel Options.

GENERAL PURPOSE: To provide wound care information that considers the specific physiology of neonates. TARGET AUDIENCE: This continuing education activity is intended for physicians, physician assistants, nurse practitioners, and nurses with an interest in skin and wound care. LEARNING OBJECTIVES/OUTCOMES: After participating in this educational activity, the participant will:1. Differentiate the use of hydrocolloids, hydrogels, foam dressings, and barrier creams in the neonatal population.2. Identify issues related to the use of solvents, alginates, collagen dressings, and negative-pressure wound therapy in neonates. ABSTRACT: OBJECTIVETo discuss what is known about the wound milieu in premature and full-term neonates, including the unique challenges pediatric clinicians face, the therapies that have proven effective, and the therapies contraindicated for use in neonatal wound healing to guide treatment that accounts for the specific physiological characteristics of this often overlooked population. DATA SOURCES: Data were collected on neonatal wound healing from a wide variety of sources, including PubMed, Google Scholar, journals, and textbooks. STUDY SELECTION: Selection criteria included publications focused on the differences and nuances of wound healing in neonates in comparison with all other age groups. DATA EXTRACTION: Data were extracted based on articles covering wound healing therapies with proven effectiveness in neonates. Terms for neonatal wound care were compiled, and then a comprehensive literature search was performed by the authors. DATA SYNTHESIS: Although many therapies are safe for treatment of older children and adolescents, most have not been explicitly tested for neonatal use. This article reviews therapies with proven effectiveness and/or specific concerns in the neonatal population. CONCLUSION: This review sheds light on the advantages and disadvantages of current standards of care regarding wound healing for neonates to direct researchers and clinicians toward developing treatments specifically for this delicate population.

To discuss what is known about the wound milieu in premature and full-term neonates, including the unique challenges pediatric clinicians face, the therapies that have proven effective, and the therapies contraindicated for use in neonatal wound healing to guide treatment that accounts for the specific physiological characteristics of this often overlooked population. Data were collected on neonatal wound healing from a wide variety of sources, including PubMed, Google Scholar, journals, and textbooks. Selection criteria included publications focused on the differences and nuances of wound healing in neonates in comparison with all other age groups. Data were extracted based on articles covering wound healing therapies with proven effectiveness in neonates. Terms for neonatal wound care were compiled, and then a comprehensive literature search was performed by the authors. Although many therapies are safe for treatment of older children and adolescents, most have not been explicitly tested for neonatal use. This article reviews therapies with proven effectiveness and/or specific concerns in the neonatal population. This review sheds light on the advantages and disadvantages of current standards of care regarding wound healing for neonates to direct researchers and clinicians toward developing treatments specifically for this delicate population.

Sex-specific differences in primary neonatal murine lung fibroblasts exposed to hyperoxia in vitro: implications for bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of the neonate characterized by impaired alveolarization and vascular growth. BPD is more common in premature male infants, but the reasons underlying sexually dimorphic outcomes are not known. It is thought that alterations in fibroblast phenotype in response to environmental stress such as hyperoxia contribute to BPD. Notch signaling creates a profibrotic environment in the lung. However, the role of hyperoxia on differential Notch pathway activation in male vs. female neonatal lung fibroblasts is not known. Primary murine lung fibroblasts from 10-day-old male and female mice were exposed to room air (21% O2, 5% CO2) or hyperoxia (95% O2, 5% CO2), and changes in cell proliferation, viability and expression of fibrosis-related genes and Notch pathway mediators were measured. Upon exposure to hyperoxia, cell proliferation was arrested in male and female fibroblasts, but cell viability was preserved. Increased Notch pathway activation was noted in male fibroblasts along with differential sex-specific modulation of key Notch pathway mediators in response to hyperoxia. α-Smooth muscle actin expression was increased in both male and female fibroblasts upon exposure to hyperoxia. Male and female fibroblasts further demonstrated distinct changes in expression of key fibrosis-related genes upon exposure to hyperoxia. Differential Notch pathway activation and distinct differences in the expression of key fibrosis-related genes might contribute to the sex-specific differences seen in hyperoxia-induced fibrosis and inhibition of lung development in BPD, with more severe implications in male neonates.

Interleukin-10-mediated regenerative postnatal tissue repair is dependent on regulation of hyaluronan metabolism via fibroblast-specific STAT3 signaling.

The cytokine IL-10 has potent antifibrotic effects in models of adult fibrosis, but the mechanisms of action are unclear. Here, we report a novel finding that IL-10 triggers a signal transducer and activator of transcription 3 (STAT3)-dependent signaling pathway that regulates hyaluronan (HA) metabolism and drives adult fibroblasts to synthesize an HA-rich pericellular matrix, which mimics the fetal regenerative wound healing phenotype with reduced fibrosis. By using cre-lox-mediated novel, inducible, fibroblast-, keratinocyte-, and wound-specific STAT3-knockdown postnatal mice-plus syngeneic fibroblast cell-transplant models-we demonstrate that the regenerative effects of IL-10 in postnatal wounds are dependent on HA synthesis and fibroblast-specific STAT3-dependent signaling. The importance of IL-10-induced HA synthesis for regenerative wound healing is demonstrated by inhibition of HA synthesis in a murine wound model by administering 4-methylumbelliferone. Although IL-10 and STAT3 signaling were intact, the antifibrotic repair phenotype that is induced by IL-10 overexpression was abrogated in this model. Our data show a novel role for IL-10 beyond its accepted immune-regulatory mechanism. The opportunity for IL-10 to regulate a fibroblast-specific formation of a regenerative, HA-rich wound extracellular matrix may lead to the development of innovative therapies to attenuate postnatal fibrosis in organ systems or diseases in which dysregulated inflammation and HA intersect.-Balaji, S., Wang, X., King, A., Le, L. D., Bhattacharya, S. S., Moles, C. M., Butte, M. J., de Jesus Perez, V. A., Liechty, K. W., Wight, T. N., Crombleholme, T. M., Bollyky, P. L., Keswani, S. G. Interleukin-10-mediated regenerative postnatal tissue repair is dependent on regulation of hyaluronan metabolism via fibroblast-specific STAT3 signaling.

Modified High-Molecular-Weight Hyaluronan Promotes Allergen-Specific Immune Tolerance.

The extracellular matrix in asthmatic lungs contains abundant low-molecular-weight hyaluronan, and this is known to promote antigen presentation and allergic responses. Conversely, high-molecular-weight hyaluronan (HMW-HA), typical of uninflamed tissues, is known to suppress inflammation. We investigated whether HMW-HA can be adapted to promote tolerance to airway allergens. HMW-HA was thiolated to prevent its catabolism and was tethered to allergens via thiol linkages. This platform, which we call "XHA," delivers antigenic payloads in the context of antiinflammatory costimulation. Allergen/XHA was administered intranasally to mice that had been sensitized previously to these allergens. XHA prevents allergic airway inflammation in mice sensitized previously to either ovalbumin or cockroach proteins. Allergen/XHA treatment reduced inflammatory cell counts, airway hyperresponsiveness, allergen-specific IgE, and T helper type 2 cell cytokine production in comparison with allergen alone. These effects were allergen specific and IL-10 dependent. They were durable for weeks after the last challenge, providing a substantial advantage over the current desensitization protocols. Mechanistically, XHA promoted CD44-dependent inhibition of nuclear factor-κB signaling, diminished dendritic cell maturation, and reduced the induction of allergen-specific CD4 T-helper responses. XHA and other potential strategies that target CD44 are promising alternatives for the treatment of asthma and allergic sinusitis.

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.

Comparison of interleukin 10 homologs on dermal wound healing using a novel human skin ex vivo organ culture model.

BACKGROUND: Anti-inflammatory cytokine interleukin (IL)-10 has been shown to induce regenerative healing in postnatal wounds. A viral homolog of IL-10 produced by human cytomegalovirus (CMV IL-10) similarly generates potent immunoregulatory effects, but its effects on wound healing have not been investigated. Currently, there are limited cost-effective methods of screening vulnerary therapeutics. Taken together, we aim to develop and validate a novel human ex vivo dermal wound model and hypothesize that CMV IL-10 will enhance dermal wound healing. METHODS: Full-thickness circular (6-mm) explants were taken from surgical skin samples and 3-mm full-thickness wounds were created. Explants were embedded in collagen I matrix and maintained in specially formulated media with the epidermis at air-liquid interface, and treated with human IL-10 or CMV IL-10 (200 ng/mL). The viability of cultured explants was validated by histology and lactate dehydrogenase (LDH) activity. Epithelial gap, epithelial height, basal keratinocyte migration, vascular endothelial growth factor levels, and neovascularization were measured at days 3 and 7 to determine IL-10 effects on wound healing. RESULTS: Culture explants at day 7 appeared similar to fresh skin in morphology, cell, and vessel density. By day 14, the epidermis separated from the dermis and the cell density diminished. Day 7 wounds appeared viable with advancing epithelial and basal keratinocyte migration with no evidence of necrosis. Cytotoxicity analysis via the quantification of LDH revealed no differences between controls and treated groups. There was a slight increase in the quantity of LDH in media at day 3; however, this decreased at day 5 and continued to decline up to day 21. CMV IL-10 treatment resulted in a significant decrease in the epithelial gap and an increase in epithelial height. There were no differences in the rates of basal keratinocyte migration at day 7 between treated and control groups. Interestingly, human IL-10 increased vascular endothelial growth factor expression and neovascularization compared with controls. CONCLUSIONS: The human ex vivo wound model provides a simple and viable design to study dermal wound healing. Both IL-10 homologs demonstrate vulnerary effects. The viral homolog demonstrates enhanced effects on wound closure compared with human IL-10. These data represent a novel tool that can be used to screen therapeutics, such as CMV IL-10, before preclinical studies.

Adenoviral-mediated gene transfer of insulin-like growth factor 1 enhances wound healing and induces angiogenesis.

BACKGROUND: Chronic wounds are characterized by a wound healing and neovascularization deficit. Strategies to increase neovascularization can significantly improve chronic wound healing. Insulin-like growth factor (IGF)-1 is reported to be a keratinocyte mitogen and is believed to induce angiogenesis via a vascular endothelial growth factor (VEGF)-dependent pathway. Using a novel ex vivo human dermal wound model and a diabetic-impaired wound healing murine model, we hypothesized that adenoviral overexpression of IGF-1 (Ad-IGF-1) will enhance wound healing and induce angiogenesis through a VEGF-dependent pathway. METHODS: Ex vivo: 6-mm full-thickness punch biopsies were obtained from normal human skin, and 3-mm full-thickness wounds were created at the center. Skin explants were maintained at air liquid interface. Db/db murine model: 8-mm full-thickness dorsal wounds in diabetic (db/db) mice were created. Treatment groups in both human ex vivo and in vivo db/db wound models include 1×10(8) particle forming units of Ad-IGF-1 or Ad-LacZ, and phosphate buffered saline (n=4-5/group). Cytotoxicity (lactate dehydrogenase) was quantified at days 3, 5, and 7 for the human ex vivo wound model. Epithelial gap closure (hematoxylin and eosin; Trichrome), VEGF expression (enzyme-linked immunosorbent assay), and capillary density (CD 31+CAPS/HPF) were analyzed at day 7. RESULTS: In the human ex vivo organ culture, the adenoviral vectors did not demonstrate any significant difference in cytotoxicity compared with phosphate buffered saline. Ad-IGF-1 overexpression significantly increases basal keratinocyte migration, with no significant effect on epithelial gap closure. There was a significant increase in capillary density in the Ad-IGF-1 wounds. However, there was no effect on VEGF levels in Ad-IGF-1 samples compared with controls. In db/db wounds, Ad-IGF-1 overexpression significantly improves epithelial gap closure and granulation tissue with a dense cellular infiltrate compared with controls. Ad-IGF-1 also increases capillary density, again with no significant difference in VEGF levels in the wounds compared with control treatments. CONCLUSIONS: In two different models, our data demonstrate that adenoviral-mediated gene transfer of IGF-1 results in enhanced wound healing and induces angiogenesis via a VEGF-independent pathway. Understanding the underlying mechanisms of IGF-1 effects on angiogenesis may help produce novel therapeutics for chronic wounds or diseases characterized by a deficit in neovascularization.

Pseudotyped adeno-associated viral vectors for gene transfer in dermal fibroblasts: implications for wound-healing applications.

BACKGROUND: Cell-specific gene transfer and sustained transgene expression are goals of cutaneous gene therapy. Pseudotyping strategy with adeno-associated viral (AAV) vectors has the potential to confer unique cellular tropism and transduction efficiency. We hypothesize that pseudotyped AAV vectors have differential tropism and transduction efficiency under normal and wound conditions in dermal fibroblasts. MATERIALS AND METHODS: We packaged AAV2 genome with green fluorescent protein reporter in capsids of other serotypes, AAV5, AAV7, and AAV8, producing pseudotyped vectors AAV2/5, AAV2/7, and AAV2/8, respectively. Murine and human dermal fibroblasts were transduced by the different pseudotypes for 24 h at multiplicities of infection 10(2), 10(3), 10(4), and 10(5). We assessed transduction efficiency at days 3 and 7. Experiments were repeated in a simulated wound environment by adding 10 ng/mL platelet-derived growth factor-B to culture media. RESULTS: Transduction efficiency of the pseudotyped AAV vectors was dose dependent. Multiplicity of infection 10(5) resulted in significantly higher gene transfer. Under normal culture conditions, the pseudotyping strategy conferred differential transduction of dermal fibroblasts, with significantly enhanced transduction of murine cells by AAV2/5 and AAV2/8 compared with AAV2/2. Adeno-associated virus 2/8 was more efficacious in transducing human cells. Under wound conditions, transduction efficiency of AAV2/2, 2/5, and 2/8 was significantly lower in murine fibroblasts. At day 3 under wound conditions, all vectors demonstrated similar transduction efficiency, but by day 7, the three pseudotyped vectors transduced significantly more murine cells compared with AAV2/2. However, in human cells, there was no significant difference in the transduction efficiency of each pseudotype between normal and wound conditions at both 3 and 7 d. CONCLUSIONS: The AAV pseudotyping strategy represents a gene transfer technology that can result in differential transduction of dermal fibroblasts. The differences in transduction efficiency in murine and human dermal fibroblasts in both the normal and wound environment highlight issues with translatability of gene transfer techniques. These data provide a template for using pseudotyped AAV vectors in cutaneous applications.

Interleukin-10 regulates the fetal hyaluronan-rich extracellular matrix via a STAT3-dependent mechanism.

BACKGROUND: The midgestational fetus is capable of regenerative healing. We have recently demonstrated a novel role for the anti-inflammatory cytokine interleukin 10 (IL-10) as a regulator of hyaluronan (HA) in the extracellular matrix. The signaling pathway of IL-10 has been studied in monocytes but is unknown in dermal fibroblasts. We hypothesized IL-10 signals through its primary receptor, IL-10R1, to activate STAT3, resulting in HA synthesis. METHODS: Murine midgestational (E14.5) fetal fibroblasts were evaluated in vitro. Pericellular matrix was quantified using a particle exclusion assay. STAT3 levels and cellular localization were evaluated by Western blot/band densitometry and immunocytochemistry/confocal microscopy. HA levels were quantified by enzyme-linked immunosorbent assay. The effects of IL-10R1 signal blockade by a neutralizing antibody and STAT3 inhibition were evaluated. An ex vivo midgestation fetal forearm culture incisional wound model in control and transgenic IL-10-/- mice was used to evaluate the role of STAT3 on the extracellular matrix. RESULTS: Fetal fibroblasts produce a robust hyaluronan-rich pericellular matrix that is IL-10R1 and STAT3 dependent. Inhibition of IL-10R1 signaling results in decreased phosphorylated STAT3 levels and inhibition of nuclear localization. Inhibition of STAT3 results in decreased HA production. At day 3, midgestation fetal wounds have efficient re-epithelialization, which is significantly slowed in IL-10-/- wounds at the same gestation and with inhibition of STAT3. CONCLUSIONS: Our data demonstrate that IL-10 regulates HA synthesis through its primary receptor IL-10R1 and STAT3 activation. This supports a novel nonimmunoregulatory mechanism of IL-10 in its role in fetal regenerative wound healing.

Role of salivary vascular endothelial growth factor (VEGF) in palatal mucosal wound healing.

The mucosa of alimentary tract heals more rapidly than cutaneous wounds. The underlying mechanisms of this enhanced healing have not been completely elucidated. Constant exposure to salivary growth factors has been shown to play a critical role in mucosal homeostasis and tissue repair. Angiogenesis also has an essential role in successful wound repair. One of the main angiogenic growth factors, vascular endothelial growth factor (VEGF), has a pleiotropic role in tissue repair via neovascularization, reepithelialization, and regulation of extracellular matrix. We have previously reported a critical role for salivary VEGF in bowel adaptation after small bowel resection. We hypothesize that salivary VEGF is an essential stimulus for oral mucosal tissue repair, and use the murine palatal wound model to test our hypothesis. In a loss-of-function experiment, we removed the primary source of VEGF production through selective submandibular gland (SMG) sialoadenectomy in a murine model and observed the effects on wound closure and neovascularization. We then performed a selective loss-of-function experiment using the protein VEGF-Trap to inhibit salivary VEGF. In a gain-of-function experiment, we supplemented oral VEGF following SMG sialoadenectomy. After SMG sialoadenectomy, there was significant reduction in salivary VEGF level, wound closure, and vessel density. Lower levels of salivary VEGF were correlated with impaired neovascularization and reepithelialization. The selective blockade of VEGF using VEGF-Trap resulted in a similar impairment in wound healing and neovascularization. The sole supplementation of oral VEGF after SMG sialoadenectomy rescued the impaired wound healing phenotype and restored neovascularization to normal levels. These data show a novel role for salivary-VEGF in mucosal wound healing, and provide a basis for the development of novel therapeutics aimed at augmenting wound repair of the oral mucosa, as well as wounds at other sites in the alimentary tract.

Cellular and extracellular vaginal changes following murine ovarian removal.

Loss of estrogen as a result of aging, pelvic cancer therapy, genetics, or eating disorders affects numerous body systems including the reproductive tract. Specifically, a chronic hypoestrogenic state fosters debilitating vaginal symptoms like atrophy, dryness, and dyspareunia. Current treatment options, including vaginal estrogen and hyaluronan (HA), anecdotally improve symptoms, but rectifying mechanisms are largely understudied. In order to study the hypoestrogenic vaginal environment, in particular the extracellular matrix (ECM), as well as understand the mechanisms behind current treatments and develop new therapies, we characterized a reliable and reproducible animal model. Bilateral ovariectomies (OVX) were performed on 9-week-old CD1 mice. After 1 month of estrogen loss due to ovarian removal, a phenotype that is similar to human vaginal tissue in an estrogen reduced state was noted in mice compared to sham-operated controls. The uterine to body weight ratio decreased by 80% and vaginal epithelium was significantly thinner in OVX compared to sham mice. Estrogen signaling was altered in OVX, but submucosal ERα localization did not reach statistical differences. HA localization in the submucosal area was altered and CD44 expression decreased in OVX mice. Collagen turn-over was altered following OVX. The inflammation profile was also disrupted, and submucosal vaginal CD45+ and F4/80+ cell populations were significantly reduced in the OVX mice. These results show altered cellular and molecular changes due to reduced estrogen levels. Developing new treatments for hypoestrogenic vaginal symptoms rely on better understanding of not only the cellular changes, but also the altered vaginal ECM environment. Further studies using this mouse model has the potential to advance women's vaginal health treatments and aid in understanding the interplay between organ systems in both healthy, aged, and diseased states.

Unveiling Vaginal Fibrosis: A Novel Murine Model Using Bleomycin and Epithelial Disruption.

Objective: Develop, validate, and characterize a fibrotic murine vaginal wound healing model using bleomycin instillations and epithelial disruption. Approach: We tested the effect of repeated bleomycin instillations with mucosal layer disruption on induction of vaginal fibrosis. Tissue samples collected at various time points were analyzed for fibrosis-related gene expression changes and collagen content. Results: Low (1.5U/kg) and high-dose (2.5U/kg) bleomycin instillations alone did not induce fibrosis, but when high-dose bleomycin was combined with epithelial disruption, increased pro-fibrotic gene expression and trichrome staining were observed. To evaluate spatial and temporal changes in the ECM structure and gene expression, tissue samples were collected at 1 day, 3 weeks, and 6 weeks after bleomycin and epithelial disruption. Data analyses revealed a significant decrease in matrix metabolizing genes and an increase in pro-fibrotic genes and inhibitors of matrix metabolizing genes in the bleomycin plus epithelial disruption group at 3 weeks. Elevated levels of the profibrotic genes Acta2 , Col1a1 , and Col3a were exclusively detected in this group at 3 weeks, and trichrome staining confirmed increased collagen content after 3 weeks. Hydroxyproline levels showed a tendency towards elevation at 3 weeks (p=0.12) and 6 weeks (p=0.14), indicating fibrosis manifestation at 3 weeks and resolution by 6 weeks post-instillation and epithelial disruption. Innovation: We combined bleomycin instillations with epithelial disruption to induce fibrosis and understand the mechanisms of the vaginal repair process. Conclusions: Epithelial disruption combined with bleomycin induces murine vaginal fibrosis within three weeks, characterized by increased collagen synthesis. Remarkably, the vaginal tissue fully recovers within six weeks, elucidating the regenerative capacity of the vagina.

Injectable Antioxidant and Oxygen-Releasing Lignin Composites to Promote Wound Healing.

The application of engineered biomaterials for wound healing has been pursued since the beginning of tissue engineering. Here, we attempt to apply functionalized lignin to confer antioxidation to the extracellular microenvironments of wounds and to deliver oxygen from the dissociation of calcium peroxide for enhanced vascularization and healing responses without eliciting inflammatory responses. Elemental analysis showed 17 times higher quantity of calcium in the oxygen-releasing nanoparticles. Lignin composites including the oxygen-generating nanoparticles released around 700 ppm oxygen per day at least for 7 days. By modulating the concentration of the methacrylated gelatin, we were able to maintain the injectability of lignin composite precursors and the stiffness of lignin composites suitable for wound healing after photo-cross-linking. In situ formation of lignin composites with the oxygen-releasing nanoparticles enhanced the rate of tissue granulation, the formation of blood vessels, and the infiltration of α-smooth muscle actin+ fibroblasts into the wounds over 7 days. At 28 days after surgery, the lignin composite with oxygen-generating nanoparticles remodeled the collagen architecture, resembling the basket-weave pattern of unwounded collagen with minimal scar formation. Thus, our study shows the potential of functionalized lignin for wound-healing applications requiring balanced antioxidation and controlled release of oxygen for enhanced tissue granulation, vascularization, and maturation of collagen.

Tissue-engineered provisional matrix as a novel approach to enhance diabetic wound healing.

Inherent pathologies associated with diabetic wound microenvironment including increased proteolysis, inflammatory dysregulation, and impaired neovascularization prevent timely resolution of chronic diabetic ulcers. It is hypothesized that augmentation of local wound microenvironment with a stable provisional matrix formed by proteolysis-resistant angiogenic peptide nanofibers (NFs) will create permissive environment for attenuated inflammation, enhanced neovascularization, and improved diabetic wound healing. Using murine excisional wound healing models, full-thickness dorsal skin wounds were treated with either NFs or control solutions (phosphate buffered saline; hyaluronic acid) and analyzed for morphology, inflammatory response, neovascularization, and biomechanical properties. NF treatment of diabetic wounds stimulated formation of a robust pro-angiogenic in situ tissue-engineered provisional matrix leading to a significant decrease in wound inflammatory cell infiltration and proinflammatory interleukin-6 levels, a significant increase in endothelial and endothelial progenitor cell infiltration, vascular endothelial growth factor levels, and neovascularization (day 7), as well as improved wound morphology, accelerated wound closure, and significantly stronger repair tissue (day 28). These results suggest that appropriate design of provisional matrix may compensate for some of the complex disruptions in diabetic wound microenvironment and provide missing cues to cells and direct in situ responses toward improved healing, which is promising for future development of new therapies for diabetic ulcers.

Pseudotyped adeno-associated viral vector tropism and transduction efficiencies in murine wound healing.

Cell specific gene transfer and sustained transgene expression are goals of cutaneous gene therapy for tissue repair and regeneration. Adeno-associated virus serotype 2 (AAV2/2) mediated gene transfer to the skin results in stable transgene expression in the muscle fascicles of the panniculus carnosus in mice, with minimal gene transfer to the dermal or epidermal elements. We hypothesized that pseudotyped AAV vectors may have a unique and characteristic tropism and transduction efficiency profile for specific cells in the cutaneous wounds. We compared transduction efficiencies of cells in the epidermis, cells in the dermis, and the fascicles of the panniculus carnosus by AAV2/2 and three pseudotyped AAV vectors, AAV2/5, AAV2/7, and AAV2/8 in a murine excisional wound model. AAV2/5 and AAV2/8 result in significantly enhanced transduction of cells both in the epidermis and the dermis compared to AAV2/2. AAV2/5 transduces both the basilar and supra-basilar keratinocytes. In contrast, AAV2/8 transduces mainly supra-basilar keratinocytes. Both AAV2/7 and AAV2/8 result in more efficient gene transfer to the muscular panniculus carnosus compared to AAV2/2. The capsid of the different pseudotyped AAV vectors produces distinct tropism and efficiency profiles in the murine wound healing model. Both AAV2/5 and AAV2/8 administration result in significantly enhanced gene transfer. To further characterize cell specific transduction and tropism profiles of the AAV pseudotyped vectors, we performed in vitro experiments using human and mouse primary dermal fibroblasts. Our data demonstrate that pseudotyping strategy confers a differential transduction of dermal fibroblasts, with higher transduction of both human and murine cells by AAV2/5 and AAV2/8 at early and later time points. At later time points, AAV2/2 demonstrates increased transduction. Interestingly, AAV2/8 appears to be more efficacious in transducing human cells as compared to AAV2/5. The pseudotype-specific pattern of transduction and tropism observed both in vivo and in vitro suggests that choice of AAV vectors should be based on the desired target cell and the timing of transgene expression in wound healing for gene transfer therapy in dermal wounds.