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564 Subpopulations of dermal fibroblasts produce distinct extracellular matrices
Wound Healing and Tissue Remodelling | ABSTRACTS 563
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The impact of diabetes and ischemia on Connexin and Pannexin expression in the skin C Faniku1, K Hussey2 and PE Martin1 1 Health and Life Sciences, Glasgow Caledonian University, Glasgow, United Kingdom and 2 Department of Vascular Surgery, Queen Elizabeth Hospital, Glasgow, United Kingdom A major problem of diabetic foot ulcers is ischemia contributing to delayed wound closure. The gap junction protein Connexin43 (Cx43) is differentially remodelled during ‘normal’ and ‘chronic’ wound healing events. The mechanisms by which this protein is involved remains unresolved. This work investigated the expression and post translational modification of Cx43 and Pannexin 1 (Panx1) in skin biopsies from patients undergoing arterial reconstruction or major limb amputation for critical limb ischaemia and in keratinocytes cultured in normoxic (N) and hypoxic (H) (1% oxygen, 5% carbon dioxide and 94 % nitrogen) conditions. Skin tissue biopsies (proximal and distal to the point of venous bypass surgery) from diabetic and non-diabetic patients were processed for immunohistochemistry and stained with antibodies specific to Cx43 and Cx43Ser368. To model the events in vitro HaCaT cells were grown in 12 well plates, scrape wounded and cultured in N or H environments for up to 48 hours (h). Cell movement into the denuded area was recorded. Cells were fixed or protein harvested and expression of hypoxic inducible factor 1-a (HIF-1a), Cx43, Cx43Ser368, Panx1 and Ki67 determined by immunocytochemistry and Western blot analysis. Cx43 and Cx43Ser368 expression was significantly increased in the epidermis of skin biopsies isolated from the distal position (i.e. locations of ischaemia) in both diabetic and non-diabetic tissue. Scratch wound assay determined delayed wound closure in hypoxic conditions, that was confirmed by induction of HIF-1a expression. Cx43 and Cx43Ser368 staining showed translocation of the protein to the nucleus. No changes in Panx1 were observed. In conclusion, post translational phosphorylation of Cx43 occurs in chronically wounded diabetic and ischemic skin. Under H conditions, scrape wound closure is reduced and immunofluorescence suggests that Cx43 and Cx43Ser368 translocate to the nucleus after 24 h hypoxia. Thus, hypoxia impacts on Cx expression but not Panx1 expression in the epidermis.
Subpopulations of dermal fibroblasts produce distinct extracellular matrices M Ghetti1, H Topouzi2, G Theocharidis3, G Cenacchi1, E Bondioli4, P Farrant5, J Connelly3 and C Higgins2 1 Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy, 2 Bioengineering, Imperial College, London, United Kingdom, 3 Cell and Cutaneous Research, Queen Mary University, London, United Kingdom, 4 Burns Centre, Regional Skin Bank, Cesena, Italy and 5 Dermatology, Brighton and Sussex University Hospital, Brighton, United Kingdom Several commercial available dermal scaffolds can promote wound healing, however, achieving complete skin regeneration is a major challenge. Extracellular matrix (ECM), physiologically secreted by cells may prove to be a valuable scaffold. On this basis, this study was focused on characterising ECMs produced by three subpopulations of fibroblasts found in human skin dermis; papillary fibroblasts (Pfi), reticular fibroblasts (Rfi), and dermal papilla fibroblasts (DP). First, scalp skin biopsies from donors were used to isolate epidermal keratinocytes (KC), and dermal fibroblast sub-populations. Fibroblasts were cultured with ascorbic acid to promote matrix synthesis. After 10 days, cells were removed and the remaining ECM was subject to characterisation and analysis by confocal microscopy. In addition, KC were seeded on the top of the ECMs to generate epidermal only skin constructs. When analysing the matrices, we found that Pfi produced ECM with the largest and most randomly oriented fibers. Imaging fluorescence showed that Fibronectin is highly abundant in Pfi, while Thrombospondin is mainly expressed in DP derived ECM. Moreover, epidermal only constructs grown on DP or Pfi matrices exhibited normal markers of differentiation and stratification. Comparatively, we were unable to find expression of ZO-1, a tight junction protein, in constructs supported by Rfi. The differences in matrix and fiber morphology observed in the distinct ECMs reflects physiological differences between the papillary, reticular, and hair follicle dermis. In addition, Rfi were unable to support growth of keratinocytes, reflective of their spatial location, and role in the dermis. We are taking inspiration from these different ECMs, to improve the design of biomimetic materials with therapeutic potential for skin tissue engineering.
A novel role for the ETS family transcription factor ERG in hypertrophic scarring B Way, S Brown, N Bulstrode and R O’Shaughnessy Immunobiology, UCL Institute of Child Health, London, United Kingdom A hypertrophic scar is characterized by excessive collagen deposition which gives rise to a raised scar, but not to the degree observed with keloids. Like keloids, they often form at the sites of cuts and burns and as a result of surgery. The investigation of pathological scarring is frequently undermined by a lack of site-matched tissue, as well as the use of different control and test subjects. To eliminate this problem, we examined skin from 30 patients undergoing two-stage surgical ear reconstruction, a procedure that requires sequential incisions in the same body site to harvest cartilage, providing a unique model of site-matched, age-matched, internally controlled unscarred skin and subsequent scar tissue. Crucially this allows us to find predictive markers of hypertrophic scarring in unscarred normal skin Comparison of cultured dermal fibroblasts from the unscarred skin of individuals who go on the scar hypertrophically (n¼7) vs. those who go on to scar normally (n¼23), showed no significant differences in proliferation or motility or expression of key wound healing or TGFb signalling proteins previously implicated in hypertrophic scarring. RNAseq analysis indicated that fibroblasts from skin that hypertrophically scars had a distinct gene expression signature, with expression levels of the pro-angiogenic transcription factor ERG, correlating with scar severity. These initial changes significantly altered the dermal environment in the hypertrophic scar. The resulting hypertrophic and normtrophic scars were analysed after 6 months by label-free mass spectrometry. We identified signficantly decreased levels of the key scar remodelling-associated protein arginase in hypertrophic scars, reflecting loss of arginaseexpressing cells that are required for scar remodelling. We show here that the RNAseq analysis and mass spectometric investigations using this novel surgery model, are complementary approachs to find novel markers of hypertrophic scarring and potentially novel therapies.
The cross-talk between lipid mediators and growth factors in the control of wound healing G Cardinali1, D Kovacs1, V Maresca1, O Sasso2, D Piomelli2 and M Picardo1 1 San Gallicano Dermatologic Institute, Rome, Italy and 2 Drug Discovery and Development, Istituto Italiano di Tecnologia, Genoa, Italy Wound repair is a complex process in which a well-ordered sequence of molecular and cellular events are triggered to regenerate tissue functionality. In the present study, we highlighted the production of amides of fatty acid with taurine, N-acyl-taurines (NATs), whose physiological functions are unknown. These lipids are degradated by the fatty acid amide hydrolase (FAAH) and in a mouse model of wound healing we found that the genetic or pharmacological disruption of FAAH activity accelerates tissue restoration. Since cutaneous healing differs between rodents and humans, we examined the impact of pharmacological FAAH blockade using the inhibitor URB597 and NAT treatment in human primary cultures of keratinocytes and fibroblasts. We found that URB597 and NAT application stimulate keratinocyte migration and promote the differentiation of fibroblast into myofibroblasts, thus facilitating crucial events in tissue repair. To extend the knowledge of the interplay among the pathways triggered by lipid-derived mediators and growth factors, we next investigated the molecular mechanisms underlying the pro-healing effects of the NATs. Treatment with URB597 or NATs produced an increase of EGFR phosphorylation and intracellular calcium levels, two events that are known to be associated with skin repair. Both events were abrogated in the presence of AMG9810, a selective antagonist of the transient receptor potential vanilloid-1 TRPV-1. The results suggest that NATs play a crucial role in the promotion of skin repair acting as mediators in the cross-talk among intracellular pathways triggered by lipids and growth factor receptors, such as EGFR. The knowledge of the generated network might open important therapeutic perspectives to manage conditions of impaired wound healing.
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Adipose derived stem cells act on skin regeneration and ageing via a paracrine effect on fibroblasts E Metral3, C Auxenfans1, W Rachidi2 and O Damour1 1 Banque de Tissus et Cellules, Hospices Civils de Lyon, Lyon, France, 2 INAC/SyMMES/LAN, CEA, Grenoble, France and 3 Recherche et de´veloppement, Gattefosse´, Saint Priest, France Following the benefic action of Adipose derived stem cells (ASC) on skin quality observed in vivo in esthetic surgery, their influence was investigated on an in vitro skin equivalent (SE) model, confirming their positive effect on skin. Indeed, a thicker epidermis correlated to a higher keratinocyte proliferation and differentiation, as well as a matrix-richer dermis were obtained in SE containing ASC mixed with fibroblasts in their dermis. In addition, ASC also had an influence on skin ageing by slowing the senescence of the SE when culture-time was extended, and by regenerating a model of aged skin cultured with cells from aged donors. In order to explain the mechanisms of action of ASC on skin, their influence on cutaneous cell proliferations (fibroblasts and keratinocytes) was investigated in a 2D co-culture system. A higher growth rate was obtained for fibroblast from young donors co-cultured with ASC, explaining the richer dermis, but no action on keratinocytes was observed. On another hand, GFP-transduced-ASC showed that ASC were not able to trans-differentiate into epithelial cells. All these results suggest a paracrine effect of ASC on fibroblasts that increase their proliferation leading to a richer dermis in a 3D SE model, and indirectly acting on keratinocytes to result in a thicker epidermis. The importance of ASC and fibroblast on the epidermis was then confirmed in 3D models with several combination of ASC/fibroblast from different donors. Finally, by studying their secretome, it will may be possible to determine the factors or pathways responsible for their action in cosmetic and pharmacologic application.
p63 regulation of the iRHOM2/ADAM17 pathway in keratinocytes P Arcidiacono, A Chikh and DP Kelsell Centre for Cell Biology & Cutaneous Research, Blizard nstitute Queen Mary University, London, United Kingdom We have recently associated dominant RHBDF2 mutations, the gene encoding inactive rhomboid protein 2 (iRHOM2), with the inherited syndrome tylosis (Palmoplantar keratoderma) with oesophageal cancer (TOC). iRHOM2, is a key regulator of ADAM17 (TNFa converting enzyme; TACE), with TOC-associated iRHOM2 mutations leading to increased ADAM17 activity and associated substrate “shedding” including growth factors and proinflammatory cytokines. Towards identifying the molecular mechanisms controlling the iRHOM2-ADAM17 pathway, we investigated putative regulators using bioinformatic software (UCSC Genome browser). We found that both iRHOM2 and ADAM17 contain putative p63like responsive consensus binding sites in their respective promoters, which were confirmed by chromatin immunoprecipitations (ChIP) experiments. Furthermore, overexpression of p63 revealed transactivation of ADAM17 and iRHOM2 at the protein level. Immunohistochemical analysis showed an increased expression of p63 in the nuclei of the basal and suprabasal layers of TOC epidermis compared to control. Similarly, in the immortalized TOC keratinocytes p63 expression was increased compared to normal control keratinocytes, in addition to its known target genes, Caspase 1 and ZNF750, as shown by immunoblot analysis. Silencing of p63 by small interfering RNA (siRNA) significantly reduced endogenous iRHOM2 and ADAM17 proteins in TOC keratinocytes and also the shedding of ADAM17 substrates evaluated by ELISA. Moreover at a cell biology level, siRNA p63 led to a decrease in cell proliferation and migration, which were correlated with the induction of apoptosis in TOC keratinocytes. In addition, shRNA of iRHOM2 or small molecule inhibition of ADAM17 also affected p63 protein and gene expression in TOC keratinocytes suggesting a reciprocal regulation between iRHOM2/ADAM17 and p63. These data identify p63 as a regulator of iRHOM2 and its key role in epidermal differentiation and barrier function. Our work helps to identify future therapeutic approaches in inflammatory skin disease and in oesophageal carcinogenesis.
www.jidonline.org S257
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The impact of diabetes and ischemia on Connexin and Pannexin expression in the skin C Faniku1, K Hussey2 and PE Martin1 1 Health and Life Sciences, Glasgow Caledonian University, Glasgow, United Kingdom and 2 Department of Vascular Surgery, Queen Elizabeth Hospital, Glasgow, United Kingdom A major problem of diabetic foot ulcers is ischemia contributing to delayed wound closure. The gap junction protein Connexin43 (Cx43) is differentially remodelled during ‘normal’ and ‘chronic’ wound healing events. The mechanisms by which this protein is involved remains unresolved. This work investigated the expression and post translational modification of Cx43 and Pannexin 1 (Panx1) in skin biopsies from patients undergoing arterial reconstruction or major limb amputation for critical limb ischaemia and in keratinocytes cultured in normoxic (N) and hypoxic (H) (1% oxygen, 5% carbon dioxide and 94 % nitrogen) conditions. Skin tissue biopsies (proximal and distal to the point of venous bypass surgery) from diabetic and non-diabetic patients were processed for immunohistochemistry and stained with antibodies specific to Cx43 and Cx43Ser368. To model the events in vitro HaCaT cells were grown in 12 well plates, scrape wounded and cultured in N or H environments for up to 48 hours (h). Cell movement into the denuded area was recorded. Cells were fixed or protein harvested and expression of hypoxic inducible factor 1-a (HIF-1a), Cx43, Cx43Ser368, Panx1 and Ki67 determined by immunocytochemistry and Western blot analysis. Cx43 and Cx43Ser368 expression was significantly increased in the epidermis of skin biopsies isolated from the distal position (i.e. locations of ischaemia) in both diabetic and non-diabetic tissue. Scratch wound assay determined delayed wound closure in hypoxic conditions, that was confirmed by induction of HIF-1a expression. Cx43 and Cx43Ser368 staining showed translocation of the protein to the nucleus. No changes in Panx1 were observed. In conclusion, post translational phosphorylation of Cx43 occurs in chronically wounded diabetic and ischemic skin. Under H conditions, scrape wound closure is reduced and immunofluorescence suggests that Cx43 and Cx43Ser368 translocate to the nucleus after 24 h hypoxia. Thus, hypoxia impacts on Cx expression but not Panx1 expression in the epidermis.
Subpopulations of dermal fibroblasts produce distinct extracellular matrices M Ghetti1, H Topouzi2, G Theocharidis3, G Cenacchi1, E Bondioli4, P Farrant5, J Connelly3 and C Higgins2 1 Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy, 2 Bioengineering, Imperial College, London, United Kingdom, 3 Cell and Cutaneous Research, Queen Mary University, London, United Kingdom, 4 Burns Centre, Regional Skin Bank, Cesena, Italy and 5 Dermatology, Brighton and Sussex University Hospital, Brighton, United Kingdom Several commercial available dermal scaffolds can promote wound healing, however, achieving complete skin regeneration is a major challenge. Extracellular matrix (ECM), physiologically secreted by cells may prove to be a valuable scaffold. On this basis, this study was focused on characterising ECMs produced by three subpopulations of fibroblasts found in human skin dermis; papillary fibroblasts (Pfi), reticular fibroblasts (Rfi), and dermal papilla fibroblasts (DP). First, scalp skin biopsies from donors were used to isolate epidermal keratinocytes (KC), and dermal fibroblast sub-populations. Fibroblasts were cultured with ascorbic acid to promote matrix synthesis. After 10 days, cells were removed and the remaining ECM was subject to characterisation and analysis by confocal microscopy. In addition, KC were seeded on the top of the ECMs to generate epidermal only skin constructs. When analysing the matrices, we found that Pfi produced ECM with the largest and most randomly oriented fibers. Imaging fluorescence showed that Fibronectin is highly abundant in Pfi, while Thrombospondin is mainly expressed in DP derived ECM. Moreover, epidermal only constructs grown on DP or Pfi matrices exhibited normal markers of differentiation and stratification. Comparatively, we were unable to find expression of ZO-1, a tight junction protein, in constructs supported by Rfi. The differences in matrix and fiber morphology observed in the distinct ECMs reflects physiological differences between the papillary, reticular, and hair follicle dermis. In addition, Rfi were unable to support growth of keratinocytes, reflective of their spatial location, and role in the dermis. We are taking inspiration from these different ECMs, to improve the design of biomimetic materials with therapeutic potential for skin tissue engineering.
A novel role for the ETS family transcription factor ERG in hypertrophic scarring B Way, S Brown, N Bulstrode and R O’Shaughnessy Immunobiology, UCL Institute of Child Health, London, United Kingdom A hypertrophic scar is characterized by excessive collagen deposition which gives rise to a raised scar, but not to the degree observed with keloids. Like keloids, they often form at the sites of cuts and burns and as a result of surgery. The investigation of pathological scarring is frequently undermined by a lack of site-matched tissue, as well as the use of different control and test subjects. To eliminate this problem, we examined skin from 30 patients undergoing two-stage surgical ear reconstruction, a procedure that requires sequential incisions in the same body site to harvest cartilage, providing a unique model of site-matched, age-matched, internally controlled unscarred skin and subsequent scar tissue. Crucially this allows us to find predictive markers of hypertrophic scarring in unscarred normal skin Comparison of cultured dermal fibroblasts from the unscarred skin of individuals who go on the scar hypertrophically (n¼7) vs. those who go on to scar normally (n¼23), showed no significant differences in proliferation or motility or expression of key wound healing or TGFb signalling proteins previously implicated in hypertrophic scarring. RNAseq analysis indicated that fibroblasts from skin that hypertrophically scars had a distinct gene expression signature, with expression levels of the pro-angiogenic transcription factor ERG, correlating with scar severity. These initial changes significantly altered the dermal environment in the hypertrophic scar. The resulting hypertrophic and normtrophic scars were analysed after 6 months by label-free mass spectrometry. We identified signficantly decreased levels of the key scar remodelling-associated protein arginase in hypertrophic scars, reflecting loss of arginaseexpressing cells that are required for scar remodelling. We show here that the RNAseq analysis and mass spectometric investigations using this novel surgery model, are complementary approachs to find novel markers of hypertrophic scarring and potentially novel therapies.
The cross-talk between lipid mediators and growth factors in the control of wound healing G Cardinali1, D Kovacs1, V Maresca1, O Sasso2, D Piomelli2 and M Picardo1 1 San Gallicano Dermatologic Institute, Rome, Italy and 2 Drug Discovery and Development, Istituto Italiano di Tecnologia, Genoa, Italy Wound repair is a complex process in which a well-ordered sequence of molecular and cellular events are triggered to regenerate tissue functionality. In the present study, we highlighted the production of amides of fatty acid with taurine, N-acyl-taurines (NATs), whose physiological functions are unknown. These lipids are degradated by the fatty acid amide hydrolase (FAAH) and in a mouse model of wound healing we found that the genetic or pharmacological disruption of FAAH activity accelerates tissue restoration. Since cutaneous healing differs between rodents and humans, we examined the impact of pharmacological FAAH blockade using the inhibitor URB597 and NAT treatment in human primary cultures of keratinocytes and fibroblasts. We found that URB597 and NAT application stimulate keratinocyte migration and promote the differentiation of fibroblast into myofibroblasts, thus facilitating crucial events in tissue repair. To extend the knowledge of the interplay among the pathways triggered by lipid-derived mediators and growth factors, we next investigated the molecular mechanisms underlying the pro-healing effects of the NATs. Treatment with URB597 or NATs produced an increase of EGFR phosphorylation and intracellular calcium levels, two events that are known to be associated with skin repair. Both events were abrogated in the presence of AMG9810, a selective antagonist of the transient receptor potential vanilloid-1 TRPV-1. The results suggest that NATs play a crucial role in the promotion of skin repair acting as mediators in the cross-talk among intracellular pathways triggered by lipids and growth factor receptors, such as EGFR. The knowledge of the generated network might open important therapeutic perspectives to manage conditions of impaired wound healing.
567
568
Adipose derived stem cells act on skin regeneration and ageing via a paracrine effect on fibroblasts E Metral3, C Auxenfans1, W Rachidi2 and O Damour1 1 Banque de Tissus et Cellules, Hospices Civils de Lyon, Lyon, France, 2 INAC/SyMMES/LAN, CEA, Grenoble, France and 3 Recherche et de´veloppement, Gattefosse´, Saint Priest, France Following the benefic action of Adipose derived stem cells (ASC) on skin quality observed in vivo in esthetic surgery, their influence was investigated on an in vitro skin equivalent (SE) model, confirming their positive effect on skin. Indeed, a thicker epidermis correlated to a higher keratinocyte proliferation and differentiation, as well as a matrix-richer dermis were obtained in SE containing ASC mixed with fibroblasts in their dermis. In addition, ASC also had an influence on skin ageing by slowing the senescence of the SE when culture-time was extended, and by regenerating a model of aged skin cultured with cells from aged donors. In order to explain the mechanisms of action of ASC on skin, their influence on cutaneous cell proliferations (fibroblasts and keratinocytes) was investigated in a 2D co-culture system. A higher growth rate was obtained for fibroblast from young donors co-cultured with ASC, explaining the richer dermis, but no action on keratinocytes was observed. On another hand, GFP-transduced-ASC showed that ASC were not able to trans-differentiate into epithelial cells. All these results suggest a paracrine effect of ASC on fibroblasts that increase their proliferation leading to a richer dermis in a 3D SE model, and indirectly acting on keratinocytes to result in a thicker epidermis. The importance of ASC and fibroblast on the epidermis was then confirmed in 3D models with several combination of ASC/fibroblast from different donors. Finally, by studying their secretome, it will may be possible to determine the factors or pathways responsible for their action in cosmetic and pharmacologic application.
p63 regulation of the iRHOM2/ADAM17 pathway in keratinocytes P Arcidiacono, A Chikh and DP Kelsell Centre for Cell Biology & Cutaneous Research, Blizard nstitute Queen Mary University, London, United Kingdom We have recently associated dominant RHBDF2 mutations, the gene encoding inactive rhomboid protein 2 (iRHOM2), with the inherited syndrome tylosis (Palmoplantar keratoderma) with oesophageal cancer (TOC). iRHOM2, is a key regulator of ADAM17 (TNFa converting enzyme; TACE), with TOC-associated iRHOM2 mutations leading to increased ADAM17 activity and associated substrate “shedding” including growth factors and proinflammatory cytokines. Towards identifying the molecular mechanisms controlling the iRHOM2-ADAM17 pathway, we investigated putative regulators using bioinformatic software (UCSC Genome browser). We found that both iRHOM2 and ADAM17 contain putative p63like responsive consensus binding sites in their respective promoters, which were confirmed by chromatin immunoprecipitations (ChIP) experiments. Furthermore, overexpression of p63 revealed transactivation of ADAM17 and iRHOM2 at the protein level. Immunohistochemical analysis showed an increased expression of p63 in the nuclei of the basal and suprabasal layers of TOC epidermis compared to control. Similarly, in the immortalized TOC keratinocytes p63 expression was increased compared to normal control keratinocytes, in addition to its known target genes, Caspase 1 and ZNF750, as shown by immunoblot analysis. Silencing of p63 by small interfering RNA (siRNA) significantly reduced endogenous iRHOM2 and ADAM17 proteins in TOC keratinocytes and also the shedding of ADAM17 substrates evaluated by ELISA. Moreover at a cell biology level, siRNA p63 led to a decrease in cell proliferation and migration, which were correlated with the induction of apoptosis in TOC keratinocytes. In addition, shRNA of iRHOM2 or small molecule inhibition of ADAM17 also affected p63 protein and gene expression in TOC keratinocytes suggesting a reciprocal regulation between iRHOM2/ADAM17 and p63. These data identify p63 as a regulator of iRHOM2 and its key role in epidermal differentiation and barrier function. Our work helps to identify future therapeutic approaches in inflammatory skin disease and in oesophageal carcinogenesis.
www.jidonline.org S257