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745 Full thickness wound healing in the Lanyu pig
Tissue Regeneration & Wound Healing | ABSTRACTS 740
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Vitamin D protection against acute UV damage e A critical role of autophagy in the skin LM Das, JF Scott and KQ Lu Dermatology, Case Western Reserve University, Cleveland, OH Acute damage of the skin by ultraviolet (UV) exposure initiates an influx of activated immune cells forming a localized TNFa and iNOS-rich inflammatory milieu that further exacerbates skin injury responses and delays tissue repair. Our previous work demonstrates that intervention with vitamin D confers protection from chemical-induced acute tissue injury by attenuating inflammatory factors produced by macrophages in the skin. In this study, we examined the potential role of vitamin D (VD) in suppressing UV-induced skin inflammatory responses in mouse models and through a blinded placebo-controlled trial of 20 human subjects. Vitamin D mediates its health-promoting effects through regulation of autophagy which is in reciprocal balance with TNFa and iNOS. We therefore hypothesize that vitamin D amelioration of UV-induced inflammation is via suppression of macrophage activation through modulation of autophagy. The pivotal findings of the human in vivo study was diminution of UV-induced skin erythema and improved histological features from skin biopsies obtained ex vivo after intervention with a single high dose of oral vitamin D. This correlated with a significant decrease in TNFa (p<0.05) expression, a 3-fold decrease in iNOS, with a concomitant 3-fold increase in LC3+ cells (marker for autophagy). These findings suggest an association between autophagy and vitamin D mediated skin recovery. Parallel studies in UV-exposed mice show an increase in activated inflammatory macrophages (CD11b+F480+Ly-6c hi) with few autophagy positive (LC3+) cells. In contrast vitamin D treatment showed numerous autophagic F480+LC3+ macrophages, confirming an inverse correlation between inflammation and vitamin D-enhanced autophagy. These data introduce a new paradigm in immune response in skin injury. The ability of vitamin D to enhance cell “fitness” by upregulation of autophagy represents potential new avenues that can be translated for clinical use in skin repair.
Identification of a specific subset of monocytes/macrophages that coordinates skin wound healing B Shook1, A Iwasaki2,3 and V Horsley1,3 1 Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, 2 Immunobiology, Yale School of Medicine, New Haven, CT and 3 Dermatology, Yale School of Medicine, New Haven, CT The repair of skin’s barrier function after injury requires temporal coordination of cellular interactions, including immune cells with dermal mesenchymal cells and epidermal epithelial cells. Monocytes/macrophages are abundant throughout wound healing, where they release inflammatory and anti-inflammatory molecules during early and mid-stages, respectively. While a switch in monocyte/macrophage gene expression is critical to initiate repair, little is known about how different subsets of anti-inflammatory wound bed macrophages contribute to healing. Here, we utilized the LysMcre mouse to induce expression of a simian diphtheria toxin receptor in the entire myeloid lineage (LysMcre/iDTR), allowing selective depletion of mid-stage myeloid cells. When mid-stage myeloid cells were depleted, we detected reduced re-epithelialization, wound bed proliferation, fibroblast repopulation and revascularization, similar to wound beds continually depleted of myeloid cells. Further examination of mid-stage macrophages revealed two major populations: CD206+/CD301b+ and CD206+/CD301b-. We observed the CD206+/CD301b+ macrophage pool expands concomitantly with cutaneous repair. Selective depletion of mid-stage CD301b-expressing macrophages, accomplished using the Mgl2DTR/GFP mouse, phenocopied the defects observed in LysMcre/iDTR wound beds. Additionally, when we transplanted multiple FACS isolated subpopulations of 5-day wound bed myeloid cells into 3-day sygeneic wound beds, only CD206+/CD301b+ macrophages were able to significantly increase proliferation and fibroblast repopulation. These data demonstrate that the CD301b-expressing subpopulation of macrophages is critical for activation of reparative processes during the mid-stage of cutaneous repair.
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Augmentation of HGF signaling: Potential for enhancing hair follicle neogenesis and development in bioengineered skin R Thangapazham1, O Mungunsukh2, G Sukumar3, C Dalgard3, M Wilkerson3, R Day2 and T Darling1 1 Dermatology, Uniformed Services University of the Health Sciences, Bethesda, MD, 2 Pharmacology and Molecular Therapeutics, Uniformed Services University of the Health Sciences, Bethesda, MD and 3 Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD We previously reported that cultured adult human dermal papilla cells have the ability to induce hair follicle neogenesis when combined with neonatal foreskin keratinocytes in an organotypic skin model. Hair follicle neogenesis was observed in constructs containing dermal papilla cells with alkaline phosphatase (ALP) activity in more than 50% of the cells but not in those with 35% or lower ALP activity positive cells. We hypothesized that trichogenic dermal papilla cells produced soluble factors that regulate epithelial cells and enhance hair follicle neogenesis. RNA-Sequencing analysis of trichogenic (n¼3) and non-trichogenic (n¼3) human dermal papilla cells demonstrated higher ALP expression in trichogenic cells and highlighted signaling pathways relevant for skin and hair follicle regeneration such as WNT and HGF. Quantitative PCR demonstrated 15-fold higher levels of HGF mRNA in trichogenic cells than non-trichogenic cells. The effects of HGF on keratinocytes were analyzed using both recombinant HGF and our recently developed HGF mimetic, a tandem dimer composed of a peptide corresponding to amino acids 36-321 of the Listeria monocytogenes internalin B protein (2InlB). The addition of HGF or 2InlB to monolayer primary cultures of human keratinocytes increased phosphorylation of p42/p44 mitogen-activated protein kinases and stimulated the proliferation of primary and immortalized keratinocytes. Preliminary analysis showed better keratinocyte stratification in bioengineered skin when HGF or 2InlB were incorporated into the dermal equivalent. These studies suggest that HGF contributes to hair follicle neogenesis and provide the groundwork for the use of an HGF mimetic to create superior skin substitutes.
Adipocyte stem cells are maintained by Pdgfa signaling in the skin G Rivera Gonzalez, B Shook, B Holtrup, M Rodeheffer and V Horsley MCDB, Yale University, New Haven, CT During mouse models of skin infection, injury, and hair regrowth, adipogenesis is initiated via both activation of adipocyte precursor cells (APCs) and the growth of pre-existing mature adipocytes. However, the mechanisms that regulate dermal adipose tissue are not well understood. Here, we show that Pdgfa signaling maintains dermal adipose tissue by initiating the proliferation of dermal adipocyte stem cells (dASCs). Prior to the initiation of the hair follicle cycle and generation of new mature adipocytes, Pdgfa is upregulated in the skin and dASCs are activated to proliferate before their preadipocyte progeny. During chronological aging and repeated depilation, when dermal adipose tissue is reduced, dASCs are not maintained, and Pdgfa is downregulated. Conditional deletion of Pdgfa in dermal cells including APCs accelerated these changes, resulting in reduced dermal adipose tissue and dASC cell number. In vitro experiments reveal that Pdgfa signals through PI3K to induce proliferation and elicits a unique transcriptional signature in FACS-purified APCs, regulating genes associated with proliferation and adipocyte differentiation. These studies reveal novel mechanisms by which APCs are maintained in the skin and may be relevant for defects associated with age-related defects in infection, wound healing, and hair growth.
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Role of Sdf1-Cxcr4 signaling in mouse appendage regeneration TH Leung1 and S Kim2 1 Dermatology, University of Pennsylvania, Philadelphia, PA and 2 Developmental Biology, Stanford University, Stanford, CA Regenerative medicine aims to restore normal tissue architecture and function. However, the basis of tissue regeneration in mammalian solid organs remains undefined. Remarkably, mice lacking p21 fully regenerate injured ears without discernable scarring. We show that in wildtype mice following tissue injury, stromal derived factor-1 (Sdf1) is upregulated in the wound epidermis and recruits Cxcr4-expressing leukocytes to the injured area. In p21-deficient mice, this upregulation of Sdf1 and subsequent recruitment of Cxcr4-expressing leukocytes are significantly diminished, thereby permitting appendage regeneration. Lineage tracing demonstrates that this regeneration derives from fate-restricted progenitor cells. Genetic or pharmacologic disruption of Sdf1-Cxcr4 signaling enhances tissue repair in wild-type mice, including full reconstitution of tissue architecture and all cell types. Similarly, we observed ear regeneration following hole punch in leukocyte-deficient nude mice. Our findings provide a cellular and mechanistic basis for tissue regeneration in p21-deficient mice, and identify new therapeutic approaches to induce tissue regeneration in mammals.
Full thickness wound healing in the Lanyu pig MW Hughes1,2, E Lin1,3, P Chiu1,2 and C Chuong1,2,4 1 International Laboratory of Wound Repair and Regeneration, National Cheng Kung University, Tainan, Taiwan, 2 Institute of Clinical Medicine, National Cheng Kung University Hospital, Tainan, Taiwan, 3 Basic Medicine, National Cheng Kung University College of Medicine, Tainan, Taiwan and 4 Pathology, University of Southern California School of Medicine, Los Angeles, CA The purpose is to establish an animal model of full thickness wounds to explore skin regeneration. We utilize the Lanyu pig because the architecture and function of the skin is similar to humans, many genetic variants with outcrosses are available, and the genetic structure was studied with 19 microsatellite markers identified. 2x2cm, 2x3cm, 3x4cm, and 4x4cm full thickness wounds were generated on the dorsum of Lanyu pigs. Initial results at post wound day 57 (PWD57) exhibit no wound induced hair follicle neogenesis (WIHN) with no papillary dermis regeneration. Wound contraction is greater in the anterior to posterior axis than medial to lateral. There is a decrease in K10+ suprabasal cells, and an increase in K14+ basal cells in healed wounds. p63, a major regulator of epidermal stratification, exhibits reduced p63 expression at PWD57. Healed wounds exhibit less PCNA than unwounded tissue, and there is no change in epithelial b-catenin expression. The wounds develop more ColI and less ColIII. Elastin protein fails to regenerate after healing at PWD57. Interestingly, an alkaline phosphatase (ALP) + cell population was identified in the basal layer at the base of each rete peg in normal pig and human skin. These K5/K14/ALP+ epithelial cells do not regenerate after full thickness wounding. Our hypothesis is these cells contribute to skin regeneration. In the future, we will isolate the ALP+ cell population to characterize their role. In conclusion, full thickness wounding on the dorsum of Lanyu pigs results in perturbed molecular expression patterns that subsequently leads to abnormal skin structure and function. Our goal is to discover novel molecular mechanisms that can be translated into applications used to help with patients suffering from severe injuries.
www.jidonline.org S131
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Vitamin D protection against acute UV damage e A critical role of autophagy in the skin LM Das, JF Scott and KQ Lu Dermatology, Case Western Reserve University, Cleveland, OH Acute damage of the skin by ultraviolet (UV) exposure initiates an influx of activated immune cells forming a localized TNFa and iNOS-rich inflammatory milieu that further exacerbates skin injury responses and delays tissue repair. Our previous work demonstrates that intervention with vitamin D confers protection from chemical-induced acute tissue injury by attenuating inflammatory factors produced by macrophages in the skin. In this study, we examined the potential role of vitamin D (VD) in suppressing UV-induced skin inflammatory responses in mouse models and through a blinded placebo-controlled trial of 20 human subjects. Vitamin D mediates its health-promoting effects through regulation of autophagy which is in reciprocal balance with TNFa and iNOS. We therefore hypothesize that vitamin D amelioration of UV-induced inflammation is via suppression of macrophage activation through modulation of autophagy. The pivotal findings of the human in vivo study was diminution of UV-induced skin erythema and improved histological features from skin biopsies obtained ex vivo after intervention with a single high dose of oral vitamin D. This correlated with a significant decrease in TNFa (p<0.05) expression, a 3-fold decrease in iNOS, with a concomitant 3-fold increase in LC3+ cells (marker for autophagy). These findings suggest an association between autophagy and vitamin D mediated skin recovery. Parallel studies in UV-exposed mice show an increase in activated inflammatory macrophages (CD11b+F480+Ly-6c hi) with few autophagy positive (LC3+) cells. In contrast vitamin D treatment showed numerous autophagic F480+LC3+ macrophages, confirming an inverse correlation between inflammation and vitamin D-enhanced autophagy. These data introduce a new paradigm in immune response in skin injury. The ability of vitamin D to enhance cell “fitness” by upregulation of autophagy represents potential new avenues that can be translated for clinical use in skin repair.
Identification of a specific subset of monocytes/macrophages that coordinates skin wound healing B Shook1, A Iwasaki2,3 and V Horsley1,3 1 Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, 2 Immunobiology, Yale School of Medicine, New Haven, CT and 3 Dermatology, Yale School of Medicine, New Haven, CT The repair of skin’s barrier function after injury requires temporal coordination of cellular interactions, including immune cells with dermal mesenchymal cells and epidermal epithelial cells. Monocytes/macrophages are abundant throughout wound healing, where they release inflammatory and anti-inflammatory molecules during early and mid-stages, respectively. While a switch in monocyte/macrophage gene expression is critical to initiate repair, little is known about how different subsets of anti-inflammatory wound bed macrophages contribute to healing. Here, we utilized the LysMcre mouse to induce expression of a simian diphtheria toxin receptor in the entire myeloid lineage (LysMcre/iDTR), allowing selective depletion of mid-stage myeloid cells. When mid-stage myeloid cells were depleted, we detected reduced re-epithelialization, wound bed proliferation, fibroblast repopulation and revascularization, similar to wound beds continually depleted of myeloid cells. Further examination of mid-stage macrophages revealed two major populations: CD206+/CD301b+ and CD206+/CD301b-. We observed the CD206+/CD301b+ macrophage pool expands concomitantly with cutaneous repair. Selective depletion of mid-stage CD301b-expressing macrophages, accomplished using the Mgl2DTR/GFP mouse, phenocopied the defects observed in LysMcre/iDTR wound beds. Additionally, when we transplanted multiple FACS isolated subpopulations of 5-day wound bed myeloid cells into 3-day sygeneic wound beds, only CD206+/CD301b+ macrophages were able to significantly increase proliferation and fibroblast repopulation. These data demonstrate that the CD301b-expressing subpopulation of macrophages is critical for activation of reparative processes during the mid-stage of cutaneous repair.
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Augmentation of HGF signaling: Potential for enhancing hair follicle neogenesis and development in bioengineered skin R Thangapazham1, O Mungunsukh2, G Sukumar3, C Dalgard3, M Wilkerson3, R Day2 and T Darling1 1 Dermatology, Uniformed Services University of the Health Sciences, Bethesda, MD, 2 Pharmacology and Molecular Therapeutics, Uniformed Services University of the Health Sciences, Bethesda, MD and 3 Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD We previously reported that cultured adult human dermal papilla cells have the ability to induce hair follicle neogenesis when combined with neonatal foreskin keratinocytes in an organotypic skin model. Hair follicle neogenesis was observed in constructs containing dermal papilla cells with alkaline phosphatase (ALP) activity in more than 50% of the cells but not in those with 35% or lower ALP activity positive cells. We hypothesized that trichogenic dermal papilla cells produced soluble factors that regulate epithelial cells and enhance hair follicle neogenesis. RNA-Sequencing analysis of trichogenic (n¼3) and non-trichogenic (n¼3) human dermal papilla cells demonstrated higher ALP expression in trichogenic cells and highlighted signaling pathways relevant for skin and hair follicle regeneration such as WNT and HGF. Quantitative PCR demonstrated 15-fold higher levels of HGF mRNA in trichogenic cells than non-trichogenic cells. The effects of HGF on keratinocytes were analyzed using both recombinant HGF and our recently developed HGF mimetic, a tandem dimer composed of a peptide corresponding to amino acids 36-321 of the Listeria monocytogenes internalin B protein (2InlB). The addition of HGF or 2InlB to monolayer primary cultures of human keratinocytes increased phosphorylation of p42/p44 mitogen-activated protein kinases and stimulated the proliferation of primary and immortalized keratinocytes. Preliminary analysis showed better keratinocyte stratification in bioengineered skin when HGF or 2InlB were incorporated into the dermal equivalent. These studies suggest that HGF contributes to hair follicle neogenesis and provide the groundwork for the use of an HGF mimetic to create superior skin substitutes.
Adipocyte stem cells are maintained by Pdgfa signaling in the skin G Rivera Gonzalez, B Shook, B Holtrup, M Rodeheffer and V Horsley MCDB, Yale University, New Haven, CT During mouse models of skin infection, injury, and hair regrowth, adipogenesis is initiated via both activation of adipocyte precursor cells (APCs) and the growth of pre-existing mature adipocytes. However, the mechanisms that regulate dermal adipose tissue are not well understood. Here, we show that Pdgfa signaling maintains dermal adipose tissue by initiating the proliferation of dermal adipocyte stem cells (dASCs). Prior to the initiation of the hair follicle cycle and generation of new mature adipocytes, Pdgfa is upregulated in the skin and dASCs are activated to proliferate before their preadipocyte progeny. During chronological aging and repeated depilation, when dermal adipose tissue is reduced, dASCs are not maintained, and Pdgfa is downregulated. Conditional deletion of Pdgfa in dermal cells including APCs accelerated these changes, resulting in reduced dermal adipose tissue and dASC cell number. In vitro experiments reveal that Pdgfa signals through PI3K to induce proliferation and elicits a unique transcriptional signature in FACS-purified APCs, regulating genes associated with proliferation and adipocyte differentiation. These studies reveal novel mechanisms by which APCs are maintained in the skin and may be relevant for defects associated with age-related defects in infection, wound healing, and hair growth.
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Role of Sdf1-Cxcr4 signaling in mouse appendage regeneration TH Leung1 and S Kim2 1 Dermatology, University of Pennsylvania, Philadelphia, PA and 2 Developmental Biology, Stanford University, Stanford, CA Regenerative medicine aims to restore normal tissue architecture and function. However, the basis of tissue regeneration in mammalian solid organs remains undefined. Remarkably, mice lacking p21 fully regenerate injured ears without discernable scarring. We show that in wildtype mice following tissue injury, stromal derived factor-1 (Sdf1) is upregulated in the wound epidermis and recruits Cxcr4-expressing leukocytes to the injured area. In p21-deficient mice, this upregulation of Sdf1 and subsequent recruitment of Cxcr4-expressing leukocytes are significantly diminished, thereby permitting appendage regeneration. Lineage tracing demonstrates that this regeneration derives from fate-restricted progenitor cells. Genetic or pharmacologic disruption of Sdf1-Cxcr4 signaling enhances tissue repair in wild-type mice, including full reconstitution of tissue architecture and all cell types. Similarly, we observed ear regeneration following hole punch in leukocyte-deficient nude mice. Our findings provide a cellular and mechanistic basis for tissue regeneration in p21-deficient mice, and identify new therapeutic approaches to induce tissue regeneration in mammals.
Full thickness wound healing in the Lanyu pig MW Hughes1,2, E Lin1,3, P Chiu1,2 and C Chuong1,2,4 1 International Laboratory of Wound Repair and Regeneration, National Cheng Kung University, Tainan, Taiwan, 2 Institute of Clinical Medicine, National Cheng Kung University Hospital, Tainan, Taiwan, 3 Basic Medicine, National Cheng Kung University College of Medicine, Tainan, Taiwan and 4 Pathology, University of Southern California School of Medicine, Los Angeles, CA The purpose is to establish an animal model of full thickness wounds to explore skin regeneration. We utilize the Lanyu pig because the architecture and function of the skin is similar to humans, many genetic variants with outcrosses are available, and the genetic structure was studied with 19 microsatellite markers identified. 2x2cm, 2x3cm, 3x4cm, and 4x4cm full thickness wounds were generated on the dorsum of Lanyu pigs. Initial results at post wound day 57 (PWD57) exhibit no wound induced hair follicle neogenesis (WIHN) with no papillary dermis regeneration. Wound contraction is greater in the anterior to posterior axis than medial to lateral. There is a decrease in K10+ suprabasal cells, and an increase in K14+ basal cells in healed wounds. p63, a major regulator of epidermal stratification, exhibits reduced p63 expression at PWD57. Healed wounds exhibit less PCNA than unwounded tissue, and there is no change in epithelial b-catenin expression. The wounds develop more ColI and less ColIII. Elastin protein fails to regenerate after healing at PWD57. Interestingly, an alkaline phosphatase (ALP) + cell population was identified in the basal layer at the base of each rete peg in normal pig and human skin. These K5/K14/ALP+ epithelial cells do not regenerate after full thickness wounding. Our hypothesis is these cells contribute to skin regeneration. In the future, we will isolate the ALP+ cell population to characterize their role. In conclusion, full thickness wounding on the dorsum of Lanyu pigs results in perturbed molecular expression patterns that subsequently leads to abnormal skin structure and function. Our goal is to discover novel molecular mechanisms that can be translated into applications used to help with patients suffering from severe injuries.
www.jidonline.org S131