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920 CCR6-deficient mice have delayed skin wound closure and reduction in peri-wound macrophage infiltration
ABSTRACTS | Tissue Regeneration and Wound Healing 915
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Impaired cutaneous wound healing in tumor necrosis factor stimulated gene-6 deficient mice S Shakya1, J Mack2 and EV Maytin3 1 Cleveland Clinic, Cleveland State University, Cleveland, OH, 2 Cleveland Clinic, Cleveland, OH and 3 Dermatology and Biomedical Engineering, Cleveland Clinic, Cleveland, OH Non-healing, chronic wounds, such as diabetic ulcers, are a health major burden worldwide. Cutaneous wound healing is a complex process that involves a multitude of events occurring concurrently, such as inflammation, epithelial and fibroblast proliferation and migration, angiogenesis, and extracellular matrix (ECM) deposition and remodeling. Hyaluronan (HA) is a key ECM glycosaminoglycan molecule known to regulate wound healing by mediating inflammation, cell proliferation and migration, angiogenesis and fibrosis. Tumor necrosis factor stimulated gene-6 (TSG-6) protein is an enzyme that modifies HA by transferring heavy chains (HC) from Inter-a-Trypsin Inhibitor (IaI) to form complexes of HC and HA (HC.HA), which are considered to stabilize the ECM and are believed to be more adherent to inflammatory cells than HA itself. Our preliminary analysis of wild type (WT) mouse skin showed that TSG-6 and HC.HA is constitutively present in normal skin, and increased in wounds. Little is known about the roles of HC.HA or TSG-6 in cutaneous wound healing. To study this, we made full-thickness excisional wounds in mice lacking TSG-6 (TSG-6 KO mice) and analyzed HC.HA, wound closure and inflammation. We found that compared to WT, HC.HA complexes are absent in TSG-6 KO unwounded and wounded skin indicating there is no redundancy or compensation for lack of TSG-6. Wound closure is significantly delayed in TSG-6 KO mice at days 3, 5 and 7 post-wounding. Finally, neutrophil recruitment is differentially regulated in TSG-6 KO wounds; while neutrophil recruitment was delayed in early wounds, an exacerbated response was seen at later time points. We propose that the delayed neutrophil recruitment in early wounds is due to the absence of HC.HA in the luminal glycocalyx of blood vessels in TSG-6 KO mice. To investigate this further, we will use an in-vitro culture model using freshly isolated neutrophils and an endothelial cell line.
Epidermal regeneration processes are enhanced by carob extract in vitro MJ Flagler, M Hare, J Henry and R Osborne Procter & Gamble, Mason, OH The appearance of aging skin can be improved via cosmetic procedures such as chemical peels and laser treatments which damage the skin surface. Similar to cosmetic procedures, we hypothesize appearance benefits can be obtained from the application of materials which activate elements of the wound healing response (cell migration and proliferation). Two in vitro test methods were leveraged to evaluate a botanical extract (Carob) for the potential to trigger a wound healing response in vitro: (1) Electrical Cell-substrate Impedance Sensing (ECIS; Applied Biophysics, Troy, NY); and (2) Incucyte ZOOM Scratch Wound Assay (Essen Bioscience, Ann Arbor, MI). The ECIS assay utilizes thermal wounding (electrical current) to quantitatively evaluate in vitro cell migration/proliferation enhancement, and cell adhesion (barrier) properties. By contrast, the Incucyte platform uses physical wounding (scratch wound) to quantitatively evaluate in vitro cell migration/proliferation enhancement in response to cellular damage. In the ECIS platform, Carob extract enhanced the rate of in vitro wound healing in hTERT keratinocytes while also increasing cell-to-cell adhesion in a dosedependent manner. Carob extract also significantly increased the rate of in vitro scratch wound closure in the IncuCyte scratch wound assay in hTERT keratinocytes. These results suggest Carob extract is able to significantly improve in vitro skin regeneration via activation of a wound healing response.
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CD44 and p38 participate in regulating the pro-fibrotic capability of dermal fibroblasts during skin wound healing Y Wang1, J Mack2 and EV Maytin3 1 Cleveland Clinic Lerner Research Institute, Shaker Heights, OH, 2 Cleveland Clinic, Cleveland, OH and 3 Dermatology and Biomedical Engineering, Cleveland Clinic, Cleveland, OH During wound healing, fibroblasts synthesize extracellular matrix molecules, e.g. collagen and hyaluronan (HA). CD44 is the major cell surface receptor for HA on fibroblasts. Our previous work showed that wound closure is accelerated in double-knockout mice that lack the HA-synthetic enzymes Has1 and Has3, but retain a functional Has2. Morphological analysis (Masson trichrome stains) of post-wounding skin revealed that dermal maturation occurs sooner in Has1/3 null mice than WT mice. To seek mechanistic insights for these observations, skin fibroblasts from Has1/3 null or WT mice were isolated and cultured in vitro. Compared to WT cells, the Has1/3 null fibroblasts had higher Has2 gene expression, larger pericellular HA coat, upregulated TGF-b activity, and more abundant collagen I and asmooth muscle actin (a-SMA). CD44 gene expression was also increased. However, neither the removal of extracellular HA by hyaluronidase, nor knockdown of Has2 gene expression by RNA interference (RNAi) had any impact upon a-SMA gene expression in Has1/3 fibroblasts. On the other hand, CD44 RNAi raised intrinsic TGF-b activity and enhanced TGF-binduced a-SMA gene expression in WT and Has1/3 fibroblasts. The ability of fibroblasts to contract collagen gel was impaired by CD44 RNAi, and cell morphology was altered (changes in focal adhesion size and actin stress fiber arrangement). Chemical inhibition of the activity of p38 MAPK effectively diminished the phenotypic difference in a-SMA protein abundance between WT and Has1/3 null cells, but failed to abrogate induction of a-SMA by CD44 RNAi. Together, these data suggest that the fibrotic capacity of fibroblasts is enhanced in Has1/3 null mice in an HA-independent but p38-dependent manner. CD44 plays a complex role in regulating a-SMA gene expression, actin stress fiber reorganization, and cellular contractility in dermal fibroblasts.
Wnt3a-conditioned media of human bone marrow-derived mesenchymal stem cells induces human dermal fibroblast wnt3a gene expression J McBride, L Rodriguez-Menocal, A Candanedo and E Badiavas University of Miami, Department of Dermatology and Interdisciplinary Stem Cell Institute, Miami, FL Wnts are a family of secreted, lipidated glycoproteins that regulate stem cell self-renewal and differentiation during embryonic development. Wnt ligands are implicated in modulation of skin development and homeostasis during adulthood. Recently, both canonical and “alternative” Wnt pathways have been implicated in processes that both promote and antagonize skin wound repair. Here, we report new evidence that Wnt3a, produced by a donor cell type (bone marrow-derived mesenchymal stem cells), may positively upregulate its own expression in another cell type (dermal fibroblasts). Treatment of primary human dermal fibroblasts with serum-free Wnt3a-conditioned media of primary human bone marrow-derived mesenchymal stem cells (BM-MSC Wnt3a CM) significantly induced human Wnt3a gene expression in the dermal fibroblasts, compared to control BM-MSC CM. This BM-MSC Wnt3a CM stimulates canonical Wnt/T-cell factor/Lymphoid enhancer factor/beta-catenin signaling and stimulates fibroblast proliferation. This model suggests a novel feed-forward mechanism for canonical Wnt signaling between BM-MSCs and dermal fibroblasts. Canonical Wnt signals may promote fibroblast proliferation during the proliferative phase of wound healing, but also promote fibrotic scarring during the remodeling phase of wound healing. This feed-forward amplification signal should be balanced by negative canonical Wnt regulators from numerous autocrine and paracrine sources to reduce wound fibrosis during wound remodeling. This result highlights the complexity of paracrine Wnt signaling regulation between the bone marrow and dermis.
S158 Journal of Investigative Dermatology (2017), Volume 137
Tissue engineered human hair follicles from genetically, environmentally and extrinsically reprogrammed dermal papilla cells A Coffman1, H Abaci1, JC Chen2, E Wang1, Y Doucet2, Z Guo2 and AM Christiano2 1 Columbia University Medical Center, New York, NY and 2 Columbia University, New York, NY Human skin equivalents (HSEs) have provided an effective therapy for patients with significant skin loss, however they still have limitations including poor viability and lack of skin appendages. We recently improved the viability of skin grafts by establishing a method to micropattern vasculature in HSEs. However, it still remains a prevailing challenge to engineer functional HSEs with hair follicles; since cultured human dermal papilla cells (DPs) in vitro lose their hair-inducing capacity. We have previously shown that the molecular memory of human DPs can be partially restored when DPs are grown as 3D spheroids. Here, we aimed to expand our previous work and achieve complete restoration of DP phenotype and functionality by leveraging microenvironmental, genetic, and extrinsic approaches. We developed an innovative tissue engineering strategy and used 3D-printing technology to create hair follicle-like microchannels on HSEs. These microchannels allowed DPs to spontaneously form 3D spheroids and generate an optimal conformation of cells to initiate epidermalmesenchymal interactions. To promote the hair inductivity of the DPs, we overexpressed Lef1, which we previously identified as a master regulator of the inductive DP gene signature. Incorporating Lef-1 transfected DPs in HSEs significantly promoted hair follicle differentiation. Prolonged culture of these constructs yielded well-defined hair follicle layers, subsequently resulting in the growth of hair fibers. We further enhanced hair follicle induction in HSEs testing various exogenous small molecules targeting Wnt - and Jak-STAT signaling. This ability to regenerate an entire hair follicle from cultured human cells will have an overwhelming impact on the medical management of patients with significant skin loss.
CCR6-deficient mice have delayed skin wound closure and reduction in peri-wound macrophage infiltration L Anderson1, X Wu2, S Simon3 and S Hwang4 1 UC Davis, Sacramento, CA, 2 UCDavis, Sacramento, CA, 3 UC Davis, Davis, CA and 4 UC Davis School of Medicine, Sacramento, CA Psoriasis is a chronic inflammatory disease afflicting up to 3% of Caucasians worldwide. A murine model of psoriasis shows that CCR6+ gd T cells are recruited to psoriatic skin in part by CCL20 and that these cells produce proinflammatory cytokines, IL-17 and IL-20, which promotes disease pathogenesis. Notably, CCR6-/- mice fail to develop psoriasis in response to induction by exogenous IL-23, raising interest in CCR6 as a relevant biological target for this disease. Because the CCR6/CCL20 pathway has been shown to regulate corneal wound repair, we sought to determine if CCR6-deficient mice had defects in epithelial repair in the skin. Utilizing a 6mm full thickness skin wound on the dorsum of CCR6-/- or congenic WT mice, we investigated the importance of CCR6/CCL20 signaling in wound healing. Specifically, we examined changes in wound size over time and determined changes in the recruitment of CCR6+gd T cells and macrophages as wounds healed. A reduction in rate of wound closure was observed in CCR6-/- compared to WT mice with the greatest difference observed at day 6, where CCR6-/- wounds were 80% of initial wound size whereas WT wounds were 20% of initial wound size (P-/- mice compared to WT at days 1, 3 and 5 postwounding. In addition, a 2-fold increase in the number of circulating gd T cells was detected in CCR6-/- mice compared to WT mice at baseline (p-/- wounds correlates with less macrophage invasion. While all wounds in CCR6-/- mice did eventually heal, these preliminary studies reveal a potential side effect of CCL20/CCR6 inhibition as a therapy for psoriasis and thus may be important in the design of future clinical trials. Additional studies are underway to elucidate the signaling mechanisms that link gd T cells to recruitment of macrophages to wounds.
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Impaired cutaneous wound healing in tumor necrosis factor stimulated gene-6 deficient mice S Shakya1, J Mack2 and EV Maytin3 1 Cleveland Clinic, Cleveland State University, Cleveland, OH, 2 Cleveland Clinic, Cleveland, OH and 3 Dermatology and Biomedical Engineering, Cleveland Clinic, Cleveland, OH Non-healing, chronic wounds, such as diabetic ulcers, are a health major burden worldwide. Cutaneous wound healing is a complex process that involves a multitude of events occurring concurrently, such as inflammation, epithelial and fibroblast proliferation and migration, angiogenesis, and extracellular matrix (ECM) deposition and remodeling. Hyaluronan (HA) is a key ECM glycosaminoglycan molecule known to regulate wound healing by mediating inflammation, cell proliferation and migration, angiogenesis and fibrosis. Tumor necrosis factor stimulated gene-6 (TSG-6) protein is an enzyme that modifies HA by transferring heavy chains (HC) from Inter-a-Trypsin Inhibitor (IaI) to form complexes of HC and HA (HC.HA), which are considered to stabilize the ECM and are believed to be more adherent to inflammatory cells than HA itself. Our preliminary analysis of wild type (WT) mouse skin showed that TSG-6 and HC.HA is constitutively present in normal skin, and increased in wounds. Little is known about the roles of HC.HA or TSG-6 in cutaneous wound healing. To study this, we made full-thickness excisional wounds in mice lacking TSG-6 (TSG-6 KO mice) and analyzed HC.HA, wound closure and inflammation. We found that compared to WT, HC.HA complexes are absent in TSG-6 KO unwounded and wounded skin indicating there is no redundancy or compensation for lack of TSG-6. Wound closure is significantly delayed in TSG-6 KO mice at days 3, 5 and 7 post-wounding. Finally, neutrophil recruitment is differentially regulated in TSG-6 KO wounds; while neutrophil recruitment was delayed in early wounds, an exacerbated response was seen at later time points. We propose that the delayed neutrophil recruitment in early wounds is due to the absence of HC.HA in the luminal glycocalyx of blood vessels in TSG-6 KO mice. To investigate this further, we will use an in-vitro culture model using freshly isolated neutrophils and an endothelial cell line.
Epidermal regeneration processes are enhanced by carob extract in vitro MJ Flagler, M Hare, J Henry and R Osborne Procter & Gamble, Mason, OH The appearance of aging skin can be improved via cosmetic procedures such as chemical peels and laser treatments which damage the skin surface. Similar to cosmetic procedures, we hypothesize appearance benefits can be obtained from the application of materials which activate elements of the wound healing response (cell migration and proliferation). Two in vitro test methods were leveraged to evaluate a botanical extract (Carob) for the potential to trigger a wound healing response in vitro: (1) Electrical Cell-substrate Impedance Sensing (ECIS; Applied Biophysics, Troy, NY); and (2) Incucyte ZOOM Scratch Wound Assay (Essen Bioscience, Ann Arbor, MI). The ECIS assay utilizes thermal wounding (electrical current) to quantitatively evaluate in vitro cell migration/proliferation enhancement, and cell adhesion (barrier) properties. By contrast, the Incucyte platform uses physical wounding (scratch wound) to quantitatively evaluate in vitro cell migration/proliferation enhancement in response to cellular damage. In the ECIS platform, Carob extract enhanced the rate of in vitro wound healing in hTERT keratinocytes while also increasing cell-to-cell adhesion in a dosedependent manner. Carob extract also significantly increased the rate of in vitro scratch wound closure in the IncuCyte scratch wound assay in hTERT keratinocytes. These results suggest Carob extract is able to significantly improve in vitro skin regeneration via activation of a wound healing response.
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CD44 and p38 participate in regulating the pro-fibrotic capability of dermal fibroblasts during skin wound healing Y Wang1, J Mack2 and EV Maytin3 1 Cleveland Clinic Lerner Research Institute, Shaker Heights, OH, 2 Cleveland Clinic, Cleveland, OH and 3 Dermatology and Biomedical Engineering, Cleveland Clinic, Cleveland, OH During wound healing, fibroblasts synthesize extracellular matrix molecules, e.g. collagen and hyaluronan (HA). CD44 is the major cell surface receptor for HA on fibroblasts. Our previous work showed that wound closure is accelerated in double-knockout mice that lack the HA-synthetic enzymes Has1 and Has3, but retain a functional Has2. Morphological analysis (Masson trichrome stains) of post-wounding skin revealed that dermal maturation occurs sooner in Has1/3 null mice than WT mice. To seek mechanistic insights for these observations, skin fibroblasts from Has1/3 null or WT mice were isolated and cultured in vitro. Compared to WT cells, the Has1/3 null fibroblasts had higher Has2 gene expression, larger pericellular HA coat, upregulated TGF-b activity, and more abundant collagen I and asmooth muscle actin (a-SMA). CD44 gene expression was also increased. However, neither the removal of extracellular HA by hyaluronidase, nor knockdown of Has2 gene expression by RNA interference (RNAi) had any impact upon a-SMA gene expression in Has1/3 fibroblasts. On the other hand, CD44 RNAi raised intrinsic TGF-b activity and enhanced TGF-binduced a-SMA gene expression in WT and Has1/3 fibroblasts. The ability of fibroblasts to contract collagen gel was impaired by CD44 RNAi, and cell morphology was altered (changes in focal adhesion size and actin stress fiber arrangement). Chemical inhibition of the activity of p38 MAPK effectively diminished the phenotypic difference in a-SMA protein abundance between WT and Has1/3 null cells, but failed to abrogate induction of a-SMA by CD44 RNAi. Together, these data suggest that the fibrotic capacity of fibroblasts is enhanced in Has1/3 null mice in an HA-independent but p38-dependent manner. CD44 plays a complex role in regulating a-SMA gene expression, actin stress fiber reorganization, and cellular contractility in dermal fibroblasts.
Wnt3a-conditioned media of human bone marrow-derived mesenchymal stem cells induces human dermal fibroblast wnt3a gene expression J McBride, L Rodriguez-Menocal, A Candanedo and E Badiavas University of Miami, Department of Dermatology and Interdisciplinary Stem Cell Institute, Miami, FL Wnts are a family of secreted, lipidated glycoproteins that regulate stem cell self-renewal and differentiation during embryonic development. Wnt ligands are implicated in modulation of skin development and homeostasis during adulthood. Recently, both canonical and “alternative” Wnt pathways have been implicated in processes that both promote and antagonize skin wound repair. Here, we report new evidence that Wnt3a, produced by a donor cell type (bone marrow-derived mesenchymal stem cells), may positively upregulate its own expression in another cell type (dermal fibroblasts). Treatment of primary human dermal fibroblasts with serum-free Wnt3a-conditioned media of primary human bone marrow-derived mesenchymal stem cells (BM-MSC Wnt3a CM) significantly induced human Wnt3a gene expression in the dermal fibroblasts, compared to control BM-MSC CM. This BM-MSC Wnt3a CM stimulates canonical Wnt/T-cell factor/Lymphoid enhancer factor/beta-catenin signaling and stimulates fibroblast proliferation. This model suggests a novel feed-forward mechanism for canonical Wnt signaling between BM-MSCs and dermal fibroblasts. Canonical Wnt signals may promote fibroblast proliferation during the proliferative phase of wound healing, but also promote fibrotic scarring during the remodeling phase of wound healing. This feed-forward amplification signal should be balanced by negative canonical Wnt regulators from numerous autocrine and paracrine sources to reduce wound fibrosis during wound remodeling. This result highlights the complexity of paracrine Wnt signaling regulation between the bone marrow and dermis.
S158 Journal of Investigative Dermatology (2017), Volume 137
Tissue engineered human hair follicles from genetically, environmentally and extrinsically reprogrammed dermal papilla cells A Coffman1, H Abaci1, JC Chen2, E Wang1, Y Doucet2, Z Guo2 and AM Christiano2 1 Columbia University Medical Center, New York, NY and 2 Columbia University, New York, NY Human skin equivalents (HSEs) have provided an effective therapy for patients with significant skin loss, however they still have limitations including poor viability and lack of skin appendages. We recently improved the viability of skin grafts by establishing a method to micropattern vasculature in HSEs. However, it still remains a prevailing challenge to engineer functional HSEs with hair follicles; since cultured human dermal papilla cells (DPs) in vitro lose their hair-inducing capacity. We have previously shown that the molecular memory of human DPs can be partially restored when DPs are grown as 3D spheroids. Here, we aimed to expand our previous work and achieve complete restoration of DP phenotype and functionality by leveraging microenvironmental, genetic, and extrinsic approaches. We developed an innovative tissue engineering strategy and used 3D-printing technology to create hair follicle-like microchannels on HSEs. These microchannels allowed DPs to spontaneously form 3D spheroids and generate an optimal conformation of cells to initiate epidermalmesenchymal interactions. To promote the hair inductivity of the DPs, we overexpressed Lef1, which we previously identified as a master regulator of the inductive DP gene signature. Incorporating Lef-1 transfected DPs in HSEs significantly promoted hair follicle differentiation. Prolonged culture of these constructs yielded well-defined hair follicle layers, subsequently resulting in the growth of hair fibers. We further enhanced hair follicle induction in HSEs testing various exogenous small molecules targeting Wnt - and Jak-STAT signaling. This ability to regenerate an entire hair follicle from cultured human cells will have an overwhelming impact on the medical management of patients with significant skin loss.
CCR6-deficient mice have delayed skin wound closure and reduction in peri-wound macrophage infiltration L Anderson1, X Wu2, S Simon3 and S Hwang4 1 UC Davis, Sacramento, CA, 2 UCDavis, Sacramento, CA, 3 UC Davis, Davis, CA and 4 UC Davis School of Medicine, Sacramento, CA Psoriasis is a chronic inflammatory disease afflicting up to 3% of Caucasians worldwide. A murine model of psoriasis shows that CCR6+ gd T cells are recruited to psoriatic skin in part by CCL20 and that these cells produce proinflammatory cytokines, IL-17 and IL-20, which promotes disease pathogenesis. Notably, CCR6-/- mice fail to develop psoriasis in response to induction by exogenous IL-23, raising interest in CCR6 as a relevant biological target for this disease. Because the CCR6/CCL20 pathway has been shown to regulate corneal wound repair, we sought to determine if CCR6-deficient mice had defects in epithelial repair in the skin. Utilizing a 6mm full thickness skin wound on the dorsum of CCR6-/- or congenic WT mice, we investigated the importance of CCR6/CCL20 signaling in wound healing. Specifically, we examined changes in wound size over time and determined changes in the recruitment of CCR6+gd T cells and macrophages as wounds healed. A reduction in rate of wound closure was observed in CCR6-/- compared to WT mice with the greatest difference observed at day 6, where CCR6-/- wounds were 80% of initial wound size whereas WT wounds were 20% of initial wound size (P-/- mice compared to WT at days 1, 3 and 5 postwounding. In addition, a 2-fold increase in the number of circulating gd T cells was detected in CCR6-/- mice compared to WT mice at baseline (p-/- wounds correlates with less macrophage invasion. While all wounds in CCR6-/- mice did eventually heal, these preliminary studies reveal a potential side effect of CCL20/CCR6 inhibition as a therapy for psoriasis and thus may be important in the design of future clinical trials. Additional studies are underway to elucidate the signaling mechanisms that link gd T cells to recruitment of macrophages to wounds.