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721 Evaluation of wound healing in a full-thickness in vitro human skin model
Tissue Regeneration & Wound Healing | ABSTRACTS 716
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The integrin av-TGFb signaling axis is necessary for keratinocyte proliferation during cutaneous wound healing E Duperret, C Natale, A Dahal and TW Ridky Dermatology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA Proliferation and migration of epidermal keratinocytes are essential for proper cutaneous wound closure after injury. av integrins and several of their ligands—vitronectin, TGFb and thrombospondin—are up-regulated in healing wounds. However, the role of av integrins in wound re-epithelialization is unknown. Here we developed a novel 3-dimensional, human organotypic wound re-epithelialization assay, and show that genetic depletion or antibodymediated blockade of pan-integrin av, or the specific heterodimer avb6, in epidermal keratinocytes prevented re-epithelialization of wounded human organotypic skin both in vivo and in vitro, without altering normal skin tissue homeostasis. Further, we demonstrate that integrin av was required for both proliferation and migration of epidermal keratinocytes at the wound edge. Integrin av is required for local activation of latent TGFb, and the wound healing defect in the setting of integrin av loss was rescued by exogenous TGFb, indicating that the av-TGFb signaling axis is a critical component of the normal epidermal wound healing program. As chronic wounds are associated with decreased TGFb signaling, restoration of TGFb activity may have therapeutic utility in some clinical settings.
Therapeutic ablation of pericytes inhibits wound angiogenesis K Strittmatter, H Pomeroy and A Marneros Dermatology, Massachusetts General Hospital/ Harvard Medical School, Boston, MA Novel treatments are needed for conditions with pathologic angiogenesis. Here we tested the consequences of therapeutic ablation of pericytes on induced angiogenesis in a model of laser injury-induced wounding. We have observed in this wound angiogenesis model that a PDGFRb+ scaffold is formed prior to infiltration of neovessels into this scaffold to form neovascular lesions and that this scaffold limits the extent of neovascularization. Based on these observations we hypothesized that ablation of proliferating PDGFRb+ cells to prevent the formation of this scaffold might inhibit neovascular growth and present a novel therapeutic approach for conditions with excessive angiogenesis. To test this hypothesis we targeted proliferating PDGFRb+ cells through (1) utilizing an inducible genetic model that targets specifically proliferating PDGFRb+ cells (GCV-treated PDGFRB-TK mice), (2) administering an anti-PDGFRb aptamer (fovista), (3) and using small chemical inhibitor approaches. We compared inhibition of laser-induced neovascular lesion formation between anti-PDGFR approaches and anti-VEGF-A therapies. We observed that inducible inhibition of proliferation of specifically PDGFRb+ cells after laser injury potently inhibits PDGFRb+ scaffold formation and neovascularization. Similarly, therapeutic inhibition of PDGFRbsignaling through the aptamer fovista significantly inhibited PDGFRb+ scaffold formation and neovascular lesion formation. The small-chemical inhibitor of both PDGFRs and VEGFRs tivozanib also potently inhibited both scaffold formation and neovascularization. Aflibercept anti-VEGF-A therapy inhibited neovascularization, but had no effect on PDGFRb+ scaffold formation. The results demonstrate that therapeutic targeting of proliferating PDGFRb+ cells potently inhibits the formation of the pericyte-like scaffold, with concomitant attenuation of neovascularization to a similar extent as anti-VEGF-A treatments. Thus, targeting of proliferating PDGFRb+ cells may provide a promising therapeutic strategy in the treatment of pathologic neovascularization.
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Angiogenic sprout formation is promoted by a fibronectin (FN) peptide (P-1) from the first type III repeat that binds hepatocyte growth factor (HGF) M McTigue1, MG Tonnesen1,2 and RA Clark1 1 Dermatology/BME, Stony Brook Univ, Stony Brook, NY and 2 Dermatology, VAMC, Northport, NY Chronic wounds and burns contain a paucity of growth factors and no fibronectin (FN) secondary to degradation by inflammatory endopeptidases. This leads to slow or non healing wounds. Angiogenesis is one of the key elements of tissue repair and regeneration. FN and angiogenic growth factors, including HGF, play critical roles in neovascularization. In fact, knock-out of either molecule is embryonic lethal. The interaction of these pleiotropic molecules was not appreciated until recently when Rahman et al.(2005) found that FN- HGF complex formation facilitated endothelial cell migration. Here we demonstrate for the first time that a 25 amino acid peptide (P-1) from the FN first type III repeat binds HGF with high affinity using surface plasmon resonance spectroscopy (SPR, BIAcore T 200). As judged by SPR spectroscopy, P-1 binds additional (PDGF-BB, FGF-2) but not all (VEGFA, angiopoietin-1, angiopoietin-2) angiogenic growth factors. Using a 3-dimensional fibrin gel, endothelial cell (EC)-bead assay previously described by our laboratory, we found that as little as 1uM P-1 stimulated more EC-coated beads to generate angiogenic sprouts in the presence of suboptimal HGF concentrations (10ng/ml) than those stimulated with optimal HGF concentrations (30ng/ml). Application of such FN-derived peptides to wounds would potentiate the activity of low concentrations of growth factors in chronic wounds or burns and thus would speed healing. Three-dimensional molecular models of FN peptide interactions with growth factor docking sites have been created. These models provide a basis for screening small molecules for binding site fit and subsequent in vitro screening for biological activity. Such strategy should ultimately provide enzyme-resistant small molecules that bind and enhance growth factors required for healing wounds and burns.
Pleiotrophin is downregulated in human keloids D Lee1,2,3, C Jin2,3,4, M Kim1,2,3, M Lee1,2,3, Y Kim1,2,3, J Chung1,2,3 and S Cho1,2,5 1 Dermatology, Seoul National University College of Medicine, Seoul, Korea (the Republic of), 2 Laboratory of Cutaneous Aging and Hair Research, Biomedical Research Institute, SNU Hospital, Seoul, Korea (the Republic of), 3 Institute of Human-Environmental Interface Biology, Medical Research Center, SNU, Seoul, Korea (the Republic of), 4 Dermatology, Yanbian University Hospital, Yanji, China and 5 Dermatology, SNU Boramae Medical Center, Seoul, Korea (the Republic of) Keloid is an abnormal hyperproliferative scarring process with involvement of complex genetic and triggering environmental factors. Previously published dysregulated gene expression profile of keloids includes genes involved in tumor formation. Pleiotrophin (PTN) is a secreted, heparin-binding growth factor which is involved in various biological functions such as cell growth, differentiation, and tumor progression. Although PTN expression was reported to be increased in hypertrophic scars, there is no study on PTN expression in keloids, and previous microarray results are controversial. To clarify differential expression of PTN in keloids, we investigated the expression of PTN and its interacting molecules in keloid and control fibroblasts, and performed immunohistochemical staining of PTN using tissue arrays. The expressions of PTN, its upstream regulator platelet-derived growth factor subunit B (PDGF-B) and corresponding PDGF receptors were significantly downregulated in keloid fibroblasts compared to normal human fibroblasts, and the decreased PTN protein expression was confirmed by immunohistochemistry. Moreover, expression of functional downstream receptor protein tyrosine phosphatase b/z was significantly increased in keloid fibroblasts, supporting overall down-regulation of PTN signaling pathway. The lowered PTN expression in keloids suggests a different pathomechanism from that of hypertrophic scars.
720
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Loss of b-PIX in keratinocytes activates Rac1 and promotes collective cell migration S Hiroyasu, SB Hopkinson, G Stimac and JC Jones School of Molecular Biosciences, Washington State University, Pullman, WA During the repair of the epidermal layers of wounded skin, the cytoskeleton of a moving keratinocyte and its attachments to connective tissue elements, including focal adhesions (FAs), undergo reorganization. These changes are coordinated by molecules such as the guanine nucleotide exchange factor b-PIX, a protein involved in activation of small GTPases. In fibroblasts, b-PIX activates Rac1, thereby increasing migration speed, and also negatively regulates FA maturation. However, the function of b-PIX in keratinocytes is unknown. In HaCaT cells, b-PIX localizes to the distal side of actin-associated nascent FAs along free cell edges, but is absent from mature FAs located closer to the cell center. To study its functions, we generated b-PIX knockdown (b-PIX KD) HaCaT cells using shRNA techniques. FA size is increased in b-PIX KD cells compared to their control counterparts, consistent with the known functions of b-PIX in regulating FA maturation. Surprisingly, the frequency of lamellipodial protrusions in b-PIX KD cells is significantly greater than that in controls. Moreover, the magnitude of traction forces generated by b-PIX KD cells is increased relative to control cells. Consistent with these data, both the collective cell migration and the speed of individual of b-PIX KD cells are enhanced compared to control cells. Since it has been established that activity of small GTPases regulates motility and lamellipodial protrusion, these data suggest that a deficiency of b-PIX changes the activation states of small GTPases. Indeed, surprisingly, Rac1 activity is enhanced in b-PIX KD cells undergoing collective cell migration compared to controls. In summary, we have uncovered an unexpected role for b-PIX in keratinocytes. In such cells, b-PIX prevents FA maturation, and negatively regulates the activity of Rac1. Therefore, b-PIX inhibits, rather than promotes, lamellipodial protrusion, traction force generation, and cell migration. Hence, targeting b-PIX may be a novel means to promote epithelialization of wounds in vivo.
Evaluation of wound healing in a full-thickness in vitro human skin model MA Bachelor, J Oldach, G Stolper, A Armento and P Hayden MatTek Corporation, Ashland, MA Wound healing is a fundamental process to re-establish tissue integrity and skin barrier function. A model of wound healing was created by introducing epidermal wounds in a full thickness in vitro human skin model (EpiDermFT) using a 3-mm punch biopsy and subsequently evaluated at multiple recovery time points. EpiDermFT exhibits stratified epidermal components and a fully developed basement membrane resembling in vivo skin in regard to both morphology and barrier function. Historically, EpiDermFT has been used to evaluate re-epithelization of the wound by: a) manually bisecting the tissues through the center of the wound, b) staining with hematoxylin and eosin, and c) quantifying migration from the wound origin. Accurate bisection of the wound is difficult and often leads to variability in assay results. Here we describe a novel method of visualizing wound re-epithelization in situ simplifying analysis and reducing introduction of variables inherent in tissue processing that could potentially confound data. Following wounding, tissues were fixed and immunostained with markers of epidermal differentiation as well as a marker of fibroblasts allowing simultaneous visualization of migrating keratinocytes (keratin 14), differentiated suprabasal cells (involucrin), and dermal fibroblasts (vimentin) within the wound. Histological and immunohistochemical analysis showed keratinocyte migration at 2 days following wounding. In both methods, wounded tissues cultured without growth factors (2% human serum) had a reduced healing rate in which keratinocytes did not cover the entire wound within a 6 day timeframe. In contrast, wounded tissues cultured with growth factors demonstrated a dramatic increase in healing rate as keratinocyte migration completely covered the wounded area by day 6. In conclusion, this novel method of evaluating re-epithelization by utilizing immunohistochemical markers of differentiation is a quicker and more reproducible method of analyzing wound healing.
www.jidonline.org S127
717
The integrin av-TGFb signaling axis is necessary for keratinocyte proliferation during cutaneous wound healing E Duperret, C Natale, A Dahal and TW Ridky Dermatology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA Proliferation and migration of epidermal keratinocytes are essential for proper cutaneous wound closure after injury. av integrins and several of their ligands—vitronectin, TGFb and thrombospondin—are up-regulated in healing wounds. However, the role of av integrins in wound re-epithelialization is unknown. Here we developed a novel 3-dimensional, human organotypic wound re-epithelialization assay, and show that genetic depletion or antibodymediated blockade of pan-integrin av, or the specific heterodimer avb6, in epidermal keratinocytes prevented re-epithelialization of wounded human organotypic skin both in vivo and in vitro, without altering normal skin tissue homeostasis. Further, we demonstrate that integrin av was required for both proliferation and migration of epidermal keratinocytes at the wound edge. Integrin av is required for local activation of latent TGFb, and the wound healing defect in the setting of integrin av loss was rescued by exogenous TGFb, indicating that the av-TGFb signaling axis is a critical component of the normal epidermal wound healing program. As chronic wounds are associated with decreased TGFb signaling, restoration of TGFb activity may have therapeutic utility in some clinical settings.
Therapeutic ablation of pericytes inhibits wound angiogenesis K Strittmatter, H Pomeroy and A Marneros Dermatology, Massachusetts General Hospital/ Harvard Medical School, Boston, MA Novel treatments are needed for conditions with pathologic angiogenesis. Here we tested the consequences of therapeutic ablation of pericytes on induced angiogenesis in a model of laser injury-induced wounding. We have observed in this wound angiogenesis model that a PDGFRb+ scaffold is formed prior to infiltration of neovessels into this scaffold to form neovascular lesions and that this scaffold limits the extent of neovascularization. Based on these observations we hypothesized that ablation of proliferating PDGFRb+ cells to prevent the formation of this scaffold might inhibit neovascular growth and present a novel therapeutic approach for conditions with excessive angiogenesis. To test this hypothesis we targeted proliferating PDGFRb+ cells through (1) utilizing an inducible genetic model that targets specifically proliferating PDGFRb+ cells (GCV-treated PDGFRB-TK mice), (2) administering an anti-PDGFRb aptamer (fovista), (3) and using small chemical inhibitor approaches. We compared inhibition of laser-induced neovascular lesion formation between anti-PDGFR approaches and anti-VEGF-A therapies. We observed that inducible inhibition of proliferation of specifically PDGFRb+ cells after laser injury potently inhibits PDGFRb+ scaffold formation and neovascularization. Similarly, therapeutic inhibition of PDGFRbsignaling through the aptamer fovista significantly inhibited PDGFRb+ scaffold formation and neovascular lesion formation. The small-chemical inhibitor of both PDGFRs and VEGFRs tivozanib also potently inhibited both scaffold formation and neovascularization. Aflibercept anti-VEGF-A therapy inhibited neovascularization, but had no effect on PDGFRb+ scaffold formation. The results demonstrate that therapeutic targeting of proliferating PDGFRb+ cells potently inhibits the formation of the pericyte-like scaffold, with concomitant attenuation of neovascularization to a similar extent as anti-VEGF-A treatments. Thus, targeting of proliferating PDGFRb+ cells may provide a promising therapeutic strategy in the treatment of pathologic neovascularization.
718
719
Angiogenic sprout formation is promoted by a fibronectin (FN) peptide (P-1) from the first type III repeat that binds hepatocyte growth factor (HGF) M McTigue1, MG Tonnesen1,2 and RA Clark1 1 Dermatology/BME, Stony Brook Univ, Stony Brook, NY and 2 Dermatology, VAMC, Northport, NY Chronic wounds and burns contain a paucity of growth factors and no fibronectin (FN) secondary to degradation by inflammatory endopeptidases. This leads to slow or non healing wounds. Angiogenesis is one of the key elements of tissue repair and regeneration. FN and angiogenic growth factors, including HGF, play critical roles in neovascularization. In fact, knock-out of either molecule is embryonic lethal. The interaction of these pleiotropic molecules was not appreciated until recently when Rahman et al.(2005) found that FN- HGF complex formation facilitated endothelial cell migration. Here we demonstrate for the first time that a 25 amino acid peptide (P-1) from the FN first type III repeat binds HGF with high affinity using surface plasmon resonance spectroscopy (SPR, BIAcore T 200). As judged by SPR spectroscopy, P-1 binds additional (PDGF-BB, FGF-2) but not all (VEGFA, angiopoietin-1, angiopoietin-2) angiogenic growth factors. Using a 3-dimensional fibrin gel, endothelial cell (EC)-bead assay previously described by our laboratory, we found that as little as 1uM P-1 stimulated more EC-coated beads to generate angiogenic sprouts in the presence of suboptimal HGF concentrations (10ng/ml) than those stimulated with optimal HGF concentrations (30ng/ml). Application of such FN-derived peptides to wounds would potentiate the activity of low concentrations of growth factors in chronic wounds or burns and thus would speed healing. Three-dimensional molecular models of FN peptide interactions with growth factor docking sites have been created. These models provide a basis for screening small molecules for binding site fit and subsequent in vitro screening for biological activity. Such strategy should ultimately provide enzyme-resistant small molecules that bind and enhance growth factors required for healing wounds and burns.
Pleiotrophin is downregulated in human keloids D Lee1,2,3, C Jin2,3,4, M Kim1,2,3, M Lee1,2,3, Y Kim1,2,3, J Chung1,2,3 and S Cho1,2,5 1 Dermatology, Seoul National University College of Medicine, Seoul, Korea (the Republic of), 2 Laboratory of Cutaneous Aging and Hair Research, Biomedical Research Institute, SNU Hospital, Seoul, Korea (the Republic of), 3 Institute of Human-Environmental Interface Biology, Medical Research Center, SNU, Seoul, Korea (the Republic of), 4 Dermatology, Yanbian University Hospital, Yanji, China and 5 Dermatology, SNU Boramae Medical Center, Seoul, Korea (the Republic of) Keloid is an abnormal hyperproliferative scarring process with involvement of complex genetic and triggering environmental factors. Previously published dysregulated gene expression profile of keloids includes genes involved in tumor formation. Pleiotrophin (PTN) is a secreted, heparin-binding growth factor which is involved in various biological functions such as cell growth, differentiation, and tumor progression. Although PTN expression was reported to be increased in hypertrophic scars, there is no study on PTN expression in keloids, and previous microarray results are controversial. To clarify differential expression of PTN in keloids, we investigated the expression of PTN and its interacting molecules in keloid and control fibroblasts, and performed immunohistochemical staining of PTN using tissue arrays. The expressions of PTN, its upstream regulator platelet-derived growth factor subunit B (PDGF-B) and corresponding PDGF receptors were significantly downregulated in keloid fibroblasts compared to normal human fibroblasts, and the decreased PTN protein expression was confirmed by immunohistochemistry. Moreover, expression of functional downstream receptor protein tyrosine phosphatase b/z was significantly increased in keloid fibroblasts, supporting overall down-regulation of PTN signaling pathway. The lowered PTN expression in keloids suggests a different pathomechanism from that of hypertrophic scars.
720
721
Loss of b-PIX in keratinocytes activates Rac1 and promotes collective cell migration S Hiroyasu, SB Hopkinson, G Stimac and JC Jones School of Molecular Biosciences, Washington State University, Pullman, WA During the repair of the epidermal layers of wounded skin, the cytoskeleton of a moving keratinocyte and its attachments to connective tissue elements, including focal adhesions (FAs), undergo reorganization. These changes are coordinated by molecules such as the guanine nucleotide exchange factor b-PIX, a protein involved in activation of small GTPases. In fibroblasts, b-PIX activates Rac1, thereby increasing migration speed, and also negatively regulates FA maturation. However, the function of b-PIX in keratinocytes is unknown. In HaCaT cells, b-PIX localizes to the distal side of actin-associated nascent FAs along free cell edges, but is absent from mature FAs located closer to the cell center. To study its functions, we generated b-PIX knockdown (b-PIX KD) HaCaT cells using shRNA techniques. FA size is increased in b-PIX KD cells compared to their control counterparts, consistent with the known functions of b-PIX in regulating FA maturation. Surprisingly, the frequency of lamellipodial protrusions in b-PIX KD cells is significantly greater than that in controls. Moreover, the magnitude of traction forces generated by b-PIX KD cells is increased relative to control cells. Consistent with these data, both the collective cell migration and the speed of individual of b-PIX KD cells are enhanced compared to control cells. Since it has been established that activity of small GTPases regulates motility and lamellipodial protrusion, these data suggest that a deficiency of b-PIX changes the activation states of small GTPases. Indeed, surprisingly, Rac1 activity is enhanced in b-PIX KD cells undergoing collective cell migration compared to controls. In summary, we have uncovered an unexpected role for b-PIX in keratinocytes. In such cells, b-PIX prevents FA maturation, and negatively regulates the activity of Rac1. Therefore, b-PIX inhibits, rather than promotes, lamellipodial protrusion, traction force generation, and cell migration. Hence, targeting b-PIX may be a novel means to promote epithelialization of wounds in vivo.
Evaluation of wound healing in a full-thickness in vitro human skin model MA Bachelor, J Oldach, G Stolper, A Armento and P Hayden MatTek Corporation, Ashland, MA Wound healing is a fundamental process to re-establish tissue integrity and skin barrier function. A model of wound healing was created by introducing epidermal wounds in a full thickness in vitro human skin model (EpiDermFT) using a 3-mm punch biopsy and subsequently evaluated at multiple recovery time points. EpiDermFT exhibits stratified epidermal components and a fully developed basement membrane resembling in vivo skin in regard to both morphology and barrier function. Historically, EpiDermFT has been used to evaluate re-epithelization of the wound by: a) manually bisecting the tissues through the center of the wound, b) staining with hematoxylin and eosin, and c) quantifying migration from the wound origin. Accurate bisection of the wound is difficult and often leads to variability in assay results. Here we describe a novel method of visualizing wound re-epithelization in situ simplifying analysis and reducing introduction of variables inherent in tissue processing that could potentially confound data. Following wounding, tissues were fixed and immunostained with markers of epidermal differentiation as well as a marker of fibroblasts allowing simultaneous visualization of migrating keratinocytes (keratin 14), differentiated suprabasal cells (involucrin), and dermal fibroblasts (vimentin) within the wound. Histological and immunohistochemical analysis showed keratinocyte migration at 2 days following wounding. In both methods, wounded tissues cultured without growth factors (2% human serum) had a reduced healing rate in which keratinocytes did not cover the entire wound within a 6 day timeframe. In contrast, wounded tissues cultured with growth factors demonstrated a dramatic increase in healing rate as keratinocyte migration completely covered the wounded area by day 6. In conclusion, this novel method of evaluating re-epithelization by utilizing immunohistochemical markers of differentiation is a quicker and more reproducible method of analyzing wound healing.
www.jidonline.org S127