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Potential mechanism of action of insulin as an anti-scarring agent
ECSAPS 2005 Abstracts Objective: This study aimed to investigate if P-cadherin expression in primary and metastatic melanoma related to patient outcome. Methods: Ethical approval was granted for this study. Tissue microarrays of primary (n ¼ 78) and metastatic (n ¼ 96) melanoma specimens were constructed from archived paraffin embedded tissue after histopathological review of the original specimens. Microarrays were sectioned and analysed immunohistochemically for P-cadherin protein expression using a specific monoclonal antibody. Positivity and intensity of staining were assessed by two independent observers without previous knowledge of clinical outcome. Results: Expression of P-cadherin showed a highly statistically significant correlation with outcome (Log rank c2 ¼ 10.1336, p ¼ 0.0015) in primary melanoma following univariate analysis. Patients with tumours that were negative for P-cadherin (n ¼ 24, 31%) correlated with poorer outcomes than those whose tumours expressed P-cadherin (n ¼ 54, 69%) with a 5-year survival of 40% for P-cadherin negative melanoma patients compared with 75% for P-cadherin positive patients. In metastatic melanoma 58% of tumours were positive for P-cadherin (n ¼ 56) and 42% negative (n ¼ 40). P-cadherin expression did not relate to patient outcome in metastatic lesions. Conclusion: Loss P-cadherin expression appears to be associated with poor prognosis in primary melanoma. This is consistent with the view that melanoma progression and metastasis involve down-regulation of receptors such as P-cadherin. Interestingly over half the metastatic lesions analysed were P-cadherin positive. A possible explanation is that this adhesion molecule is re-expressed in metastases, enabling tumour cells to reattach and form new foci of growth in other tissues.
ESR/SPIN-trapping investigation of UV-induced radical production in human skin R. Haywood RAFT, Leopold Muller Building, Mount Vernon Hospital, University College London, UK Clinical problem: People with highly pigmented skin have a lower incidence of skin cancer compared to those with less pigmentation. The damaging effects of the UVA part of sunlight to the skin of Caucasians have been attributed to the production of reactive free radicals, but UVA-induced radicals have only been characterised in pale skin types. Whether UV-irradiation causes radical production in more highly pigmented skin types is unknown. In this study free radical production was investigated in human Caucasian, Asian and Afro-Caribbean ex-vivo skin containing the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) exposed to UVA-irradiation. Materials and methods: Skin samples (n ¼ 8) were obtained from consenting patients undergoing breast reduction surgery. Skin was stored in normal saline soaked gauze at 4 C and used within 24 h. Skin samples (<1 cm2) were pre-incubated with DMPO and irradiated in situ in an electron spin resonance (ESR) spectrometer. Irradiations were undertaken using either a mercury lamp emitting predominantly UVA (UVB wavelengths <320 nm were removed with glass filters) or a solar simulator emitting radiation comparable to sunlight. Results: UVA-irradiation of Caucasian skin with an irradiance equivalent to UK levels of sunlight (from a mercury lamp) resulted in the initial detection of the ascorbate radical (0e15 min). This was followed by the detection of low-molecular-weight spin-adducts of DMPO at 30 min which were initially believed to be trapped lipid radicals consistent with previous reports, but which could also be glutathyil radicals contrary to previous assignments. Similar radicals were detected in
S3 Caucasian skin at higher irradiance, but induction was more rapid. Afro-Caribbean skin was largely protected against the formation of the free radicals detected in Caucasian skin. The intermediate skin types between Caucasian and Afro-Caribbean studied to date have varied in their responses to UVA light; however, the interpretation of these data depends on the assignments of radical species, which is still debatable. Conclusions: Our initial findings from the available data are not inconsistent with a free radical hypothesis of skin ageing and carcinogenesis in Caucasians lacking the protection against radical production provided by melanin pigmentation.
Clinical parameters, assessment and treatment of burn scarring ´ E. Van den Kerckhove, R. Hierner, W. Boeckx, P. Massage Department of Plastic, Reconstructive and Aesthetic Surgery, K.U. Leuven, University Hospitals, UZ Gasthuisberg, Leuven, Belgium In this presentation some of the basic principles in hypertrophic scar healing will be briefly summarised. More specifically, the clinical parameters (as thickness, erythema and pigmentation, pliability) that specify this type of aberrant wound healing are discussed. Furthermore, subjective (questionnaires) and objective tools (colourimeters, elastometers, ultrasound, etc.) to assess these parameters will be critically analysed with regard to their scientifical or added value in a possible test battery for scar assessment. Finally, based on evidence based articles a short overview will be given of all different conservative therapeutical strategies. Also own research in the field of the use of pressure on burn related is shown. Although the working mechanism is still questionable both pressure therapy and silicone contact media seem to have the best scientifical evidence for efficacy in the conservative treatment on prevention of hypertrophic (burn) scars.
Potential mechanism of action of insulin as an anti-scarring agent R. Baker, C. Vigor, J. Richardson, K. Rolfe, A. Grobbelaar, C. Linge RAFT, Leopold Muller Building, Mount Vernon Hospital, University College London, UK Introduction: Previously we have identified insulin but not the closely related insulin-growth factor 1 (IGF-I) as an inhibitor of the fibrosis-associated fibroblast phenotype, the myofibroblast. Insulin’s efficacy was demonstrated in vitro on normal tissuederived (dermis and normal scar), and pathological (keloid and hypertrophic scar) fibroblasts. In a murine model a single subcutaneous infiltration of low dose insulin significantly reduced the presence of myofibroblasts in the healing wound without reducing its ultimate breaking strength. We have attempted to further dissect insulin’s mechanism of action in this respect, an understanding of which will allow us to design more specific pharmaceutical agents to use in anti-scarring therapeutics. Methods: Human dermal fibroblast strains (n ¼ 6) were used up to passage 8. Cells were grown on coverslips under myofibroblast-differentiation-inducing conditions (growth factor deactivated medium) either with or without mannose-6-phosphate (a known inhibitor of endogenous transforming growth factor beta, TGFb, activation). Cells were also treated with exogenous TGFb, with or without insulin. Coverslips were harvested over a two week time course and immunohistochemically stained
S4 for b-smooth muscle actin (a myofibroblast marker) to determine myofibroblast/fibroblast ratios. Total TGFb protein was measured in cell-conditioned media and to explore TGFb isoform expression, RNA was harvested from the cells and quantitative RTPCR was performed for profibrotic TGFb1 and 2 and antifibrotic TGFb3. Results: Addition of mannose-6-phosphate and/or insulin to cultured human fibroblasts significantly inhibited myofibroblast differentiation. Optimal levels of insulin did not reduce the induction of myofibroblasts by added exogenous TGFb, nor did it significantly reduce the secretion (protein) or expression (RNA) levels of autocrine total TGFb. Furthermore, initial results suggest that the ratio of TGFb isoforms remains unchanged after insulin treatment. Conclusion: These results suggest that insulin inhibits myofibroblast differentiation by affecting autocrine TGFb activation and not active TGFb’s action nor the myofibroblast differentiation process directly. Insulin’s action is also not via alteration of the total expression nor the ratio of profibrotic to anti-scarring isoforms of TGFb.
TGF-b1 signalling in scars K. Rolfe, J. Richardson, C. Vigor, A.O. Grobbelaar, C. Linge RAFT, Leopold Muller Building, Mount Vernon Hospital, University College London, UK Introduction: Hypertrophic scars (HTSs) are severe excessive scars that are characterised by an abundance of collagenous scar tissue and hypercellularity (mainly activated myofibroblasts), although their exact aetiology remains unknown. TGF-b1 is known to play a role in orchestrating a number of cellular processes e.g. cell proliferation, differentiation, migration and apoptosis and is known to influence the deposition and remodelling of the extracellular matrix. Deregulation of this signalling pathway has been shown to play a role in fibrosis or scarring. TGF-b1 is known to signal through Smads but has also been shown to signal through certain MAP kinases (e.g. JNK and ERK). Clinical aim: That with a greater understanding of how scar cells respond to TGF-b1 we may be able to determine a pharmaceutical agent which reduces fibrosis. Materials and methods: Three normal scars (NS) and three HTS cell lines were established. Intracellular signalling pathways: Cells were seeded at 2 105 and placed in serum free media for 72 h. Cells were treated with 2 ng/ml TGF-b1 over a time course of hours. Protein lysates were made and run on sodium dodecyl sulphate (SDS)-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and these were probed with commercially available antibodies. All westerns were repeated three times. Bands were quantified with UVP densitometry. Immunofluorescence: Cells were seeded onto sterile coverslips and placed in serum free media for 72 h; they were then treated with TGFb1. Nuclear translocation was assessed by using commercial available antibodies and immunofluorescence. Results: NS showed a statistically significant increase in the phosphorylation of Smad2/3 compared to HTS over a long time course. Though NS showed increase in Smad4 this was not statistically significant from HTS. There was no statistical significance demonstrated in Smad7. MAPK pathways will also be compared. Both HTS and NS showed nuclear translocation of p-Smad2/3 by 20 min and this was also seen in Smad4. Conclusion: TGF-b1 appears to signal through the phosphorylation of Smads, in scar cells. NS showed an increase in the phosphorylation of Smad2/3 and 4. Further work is underway to assess other pathways.
ECSAPS 2005 Abstracts The involvement of the ECM and RGD peptides in apoptosis induction during wound healing C. Vigor, K.J. Rolfe, J. Richardson, R. Baker, A. Grobbelaar, C. Linge RAFT, Leopold Muller Building, Mount Vernon Hospital, University College London, UK At the start of wound healing a chemotactic fibrin clot forms, which is ultimately superseded by a collagen matrix scaffold. As the wound matrix environment matures towards a mainly type I collagen composition, fibroblasts differentiate into their active phenotype (myofibroblast) and the wound closes. The redundant myofibroblasts are then signalled to undergo apoptosis by as yet unknown mechanisms. Nevertheless, Fluck et al. (1998) and Grinnell et al. (1999) have provided evidence for a potential role of fibrillar collagen in the regulation of apoptosis. It was found that when normal dermal fibroblasts were embedded into a collagen type I matrix and allowed to remodel it they underwent apoptosis. We hypothesise that during the proteolytic remodelling of the extra cellular matrix (ECM), small soluble RGD-containing peptides are released into the matrix milieu. Soluble RGD-peptides, have previously been found to be internalised into a cell in an integrin-independent manner and are able to directly bind to and activate caspase-3, thus inducing apoptosis (Buckley et al., 1999). With an understanding of the cellular mechanisms involved in granulation tissue remodelling and apoptosis during wound repair, this may allow us to manipulate signalling pathways that are potentially the cause of aberrant wound healing. In order to investigate this hypothesis we embedded dermal fibroblasts (n > 6) into collagen type I or fibrin matrices. We assessed cell viability in both contractile (relaxed) and anchored (stressed) matrices by in situ haematoxylin staining and trypan blue viable staining. Apoptotic events were detected using DNA nick-end labelling (ApoBrdU). The mechanisms of ‘collagen remodelling induced apoptosis’ were examined by the use of exogenous RGD-containing peptides and through attempts to induce cell death with breakdown products of collagen matrices. Alongside this we studied the activation and transcription levels of matrix metalloproteinases by semi-quantitative RT-PCR and zymography. Results show that apoptosis is induced specifically in response to that of collagen matrix remodelling, correlating with an increase in matrix degrading enzymes namely ‘matrix metalloproteinases’. Further experiments have alluded to the involvement of small (<10 kDa) soluble RGD-containing peptides produced by enzymatic cleavage of collagen, which induces apoptosis of dermal fibroblasts through specific caspase-3 cleavage. This suggests that collagen type I could play an important part in the specific mechanism of apoptosis during wound healing. The quantitative assessment of flexor tendon scarring e Novel models to evaluate new therapies O. Branford, D. Lee, D. Bader, A.O. Grobbelaar University College London, Department of Medical Engineering, Queen Mary University of London, UK Introduction: Twenty percent of digital flexor tendon repairs results in poor outcome, often due to adhesions. Adhesion assessment has frequently been secondary to that of tendon healing. The methods used tell us little about how the scars form, how they may be characterised or prevented. Evaluation has involved basic mechanical or histological assessment. Our aim was to develop a quantitative assessment of adhesions to provide a rigorous basis for the comparison of anti-adhesive therapies.
ESR/SPIN-trapping investigation of UV-induced radical production in human skin R. Haywood RAFT, Leopold Muller Building, Mount Vernon Hospital, University College London, UK Clinical problem: People with highly pigmented skin have a lower incidence of skin cancer compared to those with less pigmentation. The damaging effects of the UVA part of sunlight to the skin of Caucasians have been attributed to the production of reactive free radicals, but UVA-induced radicals have only been characterised in pale skin types. Whether UV-irradiation causes radical production in more highly pigmented skin types is unknown. In this study free radical production was investigated in human Caucasian, Asian and Afro-Caribbean ex-vivo skin containing the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) exposed to UVA-irradiation. Materials and methods: Skin samples (n ¼ 8) were obtained from consenting patients undergoing breast reduction surgery. Skin was stored in normal saline soaked gauze at 4 C and used within 24 h. Skin samples (<1 cm2) were pre-incubated with DMPO and irradiated in situ in an electron spin resonance (ESR) spectrometer. Irradiations were undertaken using either a mercury lamp emitting predominantly UVA (UVB wavelengths <320 nm were removed with glass filters) or a solar simulator emitting radiation comparable to sunlight. Results: UVA-irradiation of Caucasian skin with an irradiance equivalent to UK levels of sunlight (from a mercury lamp) resulted in the initial detection of the ascorbate radical (0e15 min). This was followed by the detection of low-molecular-weight spin-adducts of DMPO at 30 min which were initially believed to be trapped lipid radicals consistent with previous reports, but which could also be glutathyil radicals contrary to previous assignments. Similar radicals were detected in
S3 Caucasian skin at higher irradiance, but induction was more rapid. Afro-Caribbean skin was largely protected against the formation of the free radicals detected in Caucasian skin. The intermediate skin types between Caucasian and Afro-Caribbean studied to date have varied in their responses to UVA light; however, the interpretation of these data depends on the assignments of radical species, which is still debatable. Conclusions: Our initial findings from the available data are not inconsistent with a free radical hypothesis of skin ageing and carcinogenesis in Caucasians lacking the protection against radical production provided by melanin pigmentation.
Clinical parameters, assessment and treatment of burn scarring ´ E. Van den Kerckhove, R. Hierner, W. Boeckx, P. Massage Department of Plastic, Reconstructive and Aesthetic Surgery, K.U. Leuven, University Hospitals, UZ Gasthuisberg, Leuven, Belgium In this presentation some of the basic principles in hypertrophic scar healing will be briefly summarised. More specifically, the clinical parameters (as thickness, erythema and pigmentation, pliability) that specify this type of aberrant wound healing are discussed. Furthermore, subjective (questionnaires) and objective tools (colourimeters, elastometers, ultrasound, etc.) to assess these parameters will be critically analysed with regard to their scientifical or added value in a possible test battery for scar assessment. Finally, based on evidence based articles a short overview will be given of all different conservative therapeutical strategies. Also own research in the field of the use of pressure on burn related is shown. Although the working mechanism is still questionable both pressure therapy and silicone contact media seem to have the best scientifical evidence for efficacy in the conservative treatment on prevention of hypertrophic (burn) scars.
Potential mechanism of action of insulin as an anti-scarring agent R. Baker, C. Vigor, J. Richardson, K. Rolfe, A. Grobbelaar, C. Linge RAFT, Leopold Muller Building, Mount Vernon Hospital, University College London, UK Introduction: Previously we have identified insulin but not the closely related insulin-growth factor 1 (IGF-I) as an inhibitor of the fibrosis-associated fibroblast phenotype, the myofibroblast. Insulin’s efficacy was demonstrated in vitro on normal tissuederived (dermis and normal scar), and pathological (keloid and hypertrophic scar) fibroblasts. In a murine model a single subcutaneous infiltration of low dose insulin significantly reduced the presence of myofibroblasts in the healing wound without reducing its ultimate breaking strength. We have attempted to further dissect insulin’s mechanism of action in this respect, an understanding of which will allow us to design more specific pharmaceutical agents to use in anti-scarring therapeutics. Methods: Human dermal fibroblast strains (n ¼ 6) were used up to passage 8. Cells were grown on coverslips under myofibroblast-differentiation-inducing conditions (growth factor deactivated medium) either with or without mannose-6-phosphate (a known inhibitor of endogenous transforming growth factor beta, TGFb, activation). Cells were also treated with exogenous TGFb, with or without insulin. Coverslips were harvested over a two week time course and immunohistochemically stained
S4 for b-smooth muscle actin (a myofibroblast marker) to determine myofibroblast/fibroblast ratios. Total TGFb protein was measured in cell-conditioned media and to explore TGFb isoform expression, RNA was harvested from the cells and quantitative RTPCR was performed for profibrotic TGFb1 and 2 and antifibrotic TGFb3. Results: Addition of mannose-6-phosphate and/or insulin to cultured human fibroblasts significantly inhibited myofibroblast differentiation. Optimal levels of insulin did not reduce the induction of myofibroblasts by added exogenous TGFb, nor did it significantly reduce the secretion (protein) or expression (RNA) levels of autocrine total TGFb. Furthermore, initial results suggest that the ratio of TGFb isoforms remains unchanged after insulin treatment. Conclusion: These results suggest that insulin inhibits myofibroblast differentiation by affecting autocrine TGFb activation and not active TGFb’s action nor the myofibroblast differentiation process directly. Insulin’s action is also not via alteration of the total expression nor the ratio of profibrotic to anti-scarring isoforms of TGFb.
TGF-b1 signalling in scars K. Rolfe, J. Richardson, C. Vigor, A.O. Grobbelaar, C. Linge RAFT, Leopold Muller Building, Mount Vernon Hospital, University College London, UK Introduction: Hypertrophic scars (HTSs) are severe excessive scars that are characterised by an abundance of collagenous scar tissue and hypercellularity (mainly activated myofibroblasts), although their exact aetiology remains unknown. TGF-b1 is known to play a role in orchestrating a number of cellular processes e.g. cell proliferation, differentiation, migration and apoptosis and is known to influence the deposition and remodelling of the extracellular matrix. Deregulation of this signalling pathway has been shown to play a role in fibrosis or scarring. TGF-b1 is known to signal through Smads but has also been shown to signal through certain MAP kinases (e.g. JNK and ERK). Clinical aim: That with a greater understanding of how scar cells respond to TGF-b1 we may be able to determine a pharmaceutical agent which reduces fibrosis. Materials and methods: Three normal scars (NS) and three HTS cell lines were established. Intracellular signalling pathways: Cells were seeded at 2 105 and placed in serum free media for 72 h. Cells were treated with 2 ng/ml TGF-b1 over a time course of hours. Protein lysates were made and run on sodium dodecyl sulphate (SDS)-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and these were probed with commercially available antibodies. All westerns were repeated three times. Bands were quantified with UVP densitometry. Immunofluorescence: Cells were seeded onto sterile coverslips and placed in serum free media for 72 h; they were then treated with TGFb1. Nuclear translocation was assessed by using commercial available antibodies and immunofluorescence. Results: NS showed a statistically significant increase in the phosphorylation of Smad2/3 compared to HTS over a long time course. Though NS showed increase in Smad4 this was not statistically significant from HTS. There was no statistical significance demonstrated in Smad7. MAPK pathways will also be compared. Both HTS and NS showed nuclear translocation of p-Smad2/3 by 20 min and this was also seen in Smad4. Conclusion: TGF-b1 appears to signal through the phosphorylation of Smads, in scar cells. NS showed an increase in the phosphorylation of Smad2/3 and 4. Further work is underway to assess other pathways.
ECSAPS 2005 Abstracts The involvement of the ECM and RGD peptides in apoptosis induction during wound healing C. Vigor, K.J. Rolfe, J. Richardson, R. Baker, A. Grobbelaar, C. Linge RAFT, Leopold Muller Building, Mount Vernon Hospital, University College London, UK At the start of wound healing a chemotactic fibrin clot forms, which is ultimately superseded by a collagen matrix scaffold. As the wound matrix environment matures towards a mainly type I collagen composition, fibroblasts differentiate into their active phenotype (myofibroblast) and the wound closes. The redundant myofibroblasts are then signalled to undergo apoptosis by as yet unknown mechanisms. Nevertheless, Fluck et al. (1998) and Grinnell et al. (1999) have provided evidence for a potential role of fibrillar collagen in the regulation of apoptosis. It was found that when normal dermal fibroblasts were embedded into a collagen type I matrix and allowed to remodel it they underwent apoptosis. We hypothesise that during the proteolytic remodelling of the extra cellular matrix (ECM), small soluble RGD-containing peptides are released into the matrix milieu. Soluble RGD-peptides, have previously been found to be internalised into a cell in an integrin-independent manner and are able to directly bind to and activate caspase-3, thus inducing apoptosis (Buckley et al., 1999). With an understanding of the cellular mechanisms involved in granulation tissue remodelling and apoptosis during wound repair, this may allow us to manipulate signalling pathways that are potentially the cause of aberrant wound healing. In order to investigate this hypothesis we embedded dermal fibroblasts (n > 6) into collagen type I or fibrin matrices. We assessed cell viability in both contractile (relaxed) and anchored (stressed) matrices by in situ haematoxylin staining and trypan blue viable staining. Apoptotic events were detected using DNA nick-end labelling (ApoBrdU). The mechanisms of ‘collagen remodelling induced apoptosis’ were examined by the use of exogenous RGD-containing peptides and through attempts to induce cell death with breakdown products of collagen matrices. Alongside this we studied the activation and transcription levels of matrix metalloproteinases by semi-quantitative RT-PCR and zymography. Results show that apoptosis is induced specifically in response to that of collagen matrix remodelling, correlating with an increase in matrix degrading enzymes namely ‘matrix metalloproteinases’. Further experiments have alluded to the involvement of small (<10 kDa) soluble RGD-containing peptides produced by enzymatic cleavage of collagen, which induces apoptosis of dermal fibroblasts through specific caspase-3 cleavage. This suggests that collagen type I could play an important part in the specific mechanism of apoptosis during wound healing. The quantitative assessment of flexor tendon scarring e Novel models to evaluate new therapies O. Branford, D. Lee, D. Bader, A.O. Grobbelaar University College London, Department of Medical Engineering, Queen Mary University of London, UK Introduction: Twenty percent of digital flexor tendon repairs results in poor outcome, often due to adhesions. Adhesion assessment has frequently been secondary to that of tendon healing. The methods used tell us little about how the scars form, how they may be characterised or prevented. Evaluation has involved basic mechanical or histological assessment. Our aim was to develop a quantitative assessment of adhesions to provide a rigorous basis for the comparison of anti-adhesive therapies.