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Interactions between peripheral blood mononuclear cells and extracellular matrix components in systemic sclerosis
S126
ESDR / JSID I SID Abstracts
0751
0754
INTERACTIONS BETWEEN PERIPHERAL BLOOD MONONUCLEAR CELLS AND EXTRACELLULAR h4ATRlX COMPONENTS IN SYSTEMIC SCLEROSIS. mzvmanska.a Makiela St&ma Elzbl S Jablonska Department of Dermatology, Warsaw School of Medzme, Warsaw, Poland Extracellular matnx (ECM) prorems play an important role in regulanon of cell nugratlon and expressmn of various genes mvolved in actwatmn of mumme reacbons. In our prewous studies we found in paoats with SSc decreased m vzrro adhesmn of penpheral bload mononuclear cells (PBMC) to collagen type IV suggesting zn YZYD acflvauon and extravasatkm of selected PBMC subpopulations. The sun of the present study was to exarmne co-stunulatory effect of ECM CO,,lQO~l~tS (collagentype 1, IV, fibronectin,I&) in 30 patwts with various forms of SSc and in 27 healthy volunteers. PBMC were cultured for 72 hrs m the presence of arm-CD3 manoclonal anobody and ECM components(at the concentratm~ IO-100 nglml) followed by 24 hrs pulse with t&at& thyxudine and counting the radioactivityin the harvested cells. The results showed that fibronectin and ~IlaSen type I had the btgbest co-stun&my actwitm in both SSc patients and the controls (relative increase of cell pmhferation 115% and 58%. p
SERUM LEVEL OF TISSUE INHIBITOR OF METALLOPROTEINASE-2 CORRELATES WITH MELANOMA PROGRESSION, BUT SERUM MATRIX METALLOPROTEINASE-2 LEVEL DOES NOT. p Department of Dermatoloav. Kumemoto Universitv School of Medicine. Kumamoto. Jauan. Since m&x metalloprotelnase-2~ (MMP-2) degrades basement membrane type IV collagen and it is regulated with it’s inhibitors (tissue inhibitor of metalloproteinase-2: TIMP-2). interactions between MMP-2 and TIMP-2 play 817 important roles in the process of tumor invasion and metastasis. The aim of this study are to measure MMP-2, TIMP-1 and -2 in melanoma lesions end these levels in serum of patients with melanoma and to correlate these panmeters with the clinical course of the disease. Frozen 14 primary end 10 metastatic melanoma lesions were stained in immune-peroxidase reactions with anti-MMP-2, TIMP-1 and -2 monoclonal antibody (mAb). Serum levels of 53 melanoma patients and 146 control were measured with Sandwich assay using these mAb. MMP-2 was expressed in melanoma cells as well es stromal fibroblasts. while TIMP-1 and -2 was expressed strongly on blood vessels in melanoma lesions and weakly in melanoma cells. Serum level of MMP-2 was significantly higher than that of control but did not correlate with melanoma progression. On the other hand, serum levels of TIMP-1 and -2 in stage II, Ill and IV was significantly higher than that of stage and control. Moreover, disease-free and over-all su~lval tlme in patients with elevated serum levels of TIMP-1 and -2 were slgnlficantly shorter than those with normal levels. These results suggest that serum level of TIMP-1 and -2 in melanoma patients is new prognostic marker, but that of MMP-2 does not represent a uselul prognostic marker.
0752
0755
ASSESSMENT OF TGFB - MEDIATED STIMULATION OF EXTRACELLULAR MATRIX PROTEIN SYNTHESIS BY AN EASY CELL CULTURE IMMUNOASSAY. C., ~epanmeut of Dermatology, University of Mllnster, MUnster, Germany. TGF-5 is implicated in tissue composition, scar formation and fibrotic processes by upregulation of the synthesis of extmceUuIar ma&. (ECM) and downregulation of mati metallo-proteinases. At present, therapeutic approaches are sought after to modulate these effects of TGF-D. A possible strategy is to use recombinant fragments of small Qroteoglycans to scavenge TGF-!3. Development of this approach requires quantitative wse.wnen~ and we have therefore devised an easy cell culture immunoassay. Cells are grown to early confluency, then incubated with or without TGF-0. After tbe incubation petiod, the medium is harvested and fdtered &rough a nitmcellulose membrane. The cell layer and the membrane are incubated with primary antibodies to an ECM protein. After washing. the binding of the tint antibody is detected by a secondary peroxidme-coupled antibody. A water-soluble chromogen, o-phenylenediamine, is used as peroxidasesubstrate, and the O.D. of the supernarants is measured at 492 “m. The amount of ECM protein in the cell layer or in the medium is expressed in relation to the amount Of total protein. This assay was employed for analysis of the production of laminin 5 and type VII collagen in cultures of human keratinocytes. A 2-3 fold stimulation of the synLhe.w of these ECM-mole.cuIes was observed after TGF-O treatment. This effect could he completely abolished by simultaneous incubation of the cells with neutralizing antibodies against TGF-0 or eliminated in pan by incubation with recombinant decorio f%meulS. The cell culture ELISA is a simple and highly sensitive assay which will aUOw monitoring of the effects of putative TGF-!3 modulators in a controlled biological system.
0753 ANT-BI IN’IEGRIN TREA’IMENI SFLECI’IVELY INDUCES APOPTOSIS IN KELOID FIBROBLASTS. M. Choucatr. D La&t. I. Olson. Fivenson. Depts. of Derm and Surgery, Henry Ford Health System. Detroit, MI, USA Cell adhesion to a substrate is mediated by the integrin family of cell surface receptors which are known to elicit intracellular signals upon cell adhesion. A role for the @I integrin family in mediating cell survival has been shown via the effect of Bl blocking antibodies to enhance apoptosis in m-slanocytes attsched to fibronectin (FN) and by the ability of anti- PI anlibadies immobilized on solid supports to suppress apoptosis in melanocytcs. Comparison of iibmnectin levels in tibroblasts derived from keloid vs normal dermis revealed a relative increase in intrac&lular and extracellular FN in k&id derived cells. We have preva~sly couftrmod rhis increased FN production in keloid fibroblasts at both the protein and mRNA levels and furthsr demonstrated fhst f3l integrin is overexpressed in kelold tibroblasfs (KK) compared to fibroblasts from normal skin of kcloid patients (NK). We used tune1 assay and RT/PCR analysis of hc-2 mRNA to study whether kcloid fibroblast apoprosis could be regulated via the @I receptor. KK end NK tibroblasts were plated on FNcoated dishes before and after monoclonal anti- gl inregnn armbody (MoAb) treatment, and analyzed by rune1 assay. Total RNA was also collected for RT/PCR The expression of bc-2 mRNA decrerwd in the MoAb hated FNxoated cuiture dishes only prior to plating for KK>NK. Slide cultures showed slight increase in staining by tune1 assay along with decreased attachment only If Uncoated slides were first exposed to the MoAb. FACS analysis of those attached cells (before or after MoAb) revealed a 2X and IOX increase for KK and NK respectively, if exposed to MoAb Qnor to plating, whereas FNcoaled plate pretreatment with MoAb did not effect apoptosis of those cells which attached (plating effeciency was reduced). Thus NK>KK for the effect of apoptosis induction by sntiBl only if attachment has already occurred to FN (as would be the case in a lesion). Both NK and KK were susceptible to the proapoptotic effects of anti-B1 at the molecular level. Blocking !3l sites could prove to be a methodof preventing k&id formation
1
In Wtro Production of an Endotheliallzed Tissue Engineered Skln Equivalent
Promoting Human Capillary-like
_-
-*
Formation. w
Auoer.. Laboratoire d’Orgaoeg6n8se Experimentate, H6pHal du Saint-Sacrement, QuBbec. Canada GIS 4LB. For patients with extensive burns, the use 01 cultured skin replacements is advantageous and often life saving. Unfortunately, various obstacles have
delayed the widespread use of composite skin equivalents. Insufficient vascularization has been proposed as the most likely reason for their poor survival. Our purpose was to develop a vascular-like network inside tissue engineered skin designed for grafting on deep wounds, to improve graft vascularization. To reach this aim, we fabricated a collaaen biooolvmer in which three human cell types: keratinocytes, dermal fibmbiasts anb umbilical vein endothelial cells were cocultured. We demonstrated that the endothelialized skin equivalent (ESE) promoted spontaneous formation of capillary-like structures inside the biopolymer. lmmunohistochemical analysis and transmission electron microscopy of the ESE showed characteristics associated with the micmvasculature in vita (vanWillebrand factor, Weibel-Palade bodies, basement membrane material and Intercellular junctions). We have developed the first endothelialized human tissue engineered skin, in which a network of capillary-like tubes is formed. The transplantation of this ESE on human should accelerate the graft revascularization by coalescence of its pre-existing vascular-like network with the host’s one. In addition, the ESE turns out to be a promising human in vitro angiogenesis model.
0756 N-METHYlJ.-SBRINlI STIMULATES WALURONAN PRODUCTION IN HUMAN SKIN FIBROBLASTS. Sbingg S&j% TV suvs a0 Kan-wara, Japan Hyaluronan has been reported to play an important role in inflammation, cell locomotion and wound healing. Its half-life time is estimated to be 2-4.5 days in mammalian ski, suggesting that hyaluronan synthesis and degradation are controlled strictly and dynamically in the skin. Decrease of hyaluronan content with aging is considered to be one of the factors which decrease the elasticity of the skin. Hyaluronan synthesis has been reported to be stimulated by growth factors, phorbol ester and retinoic acid. In this study, we found that N-methyl-L-serine (NMS), an amino acid derivative, stimulated hyahuonan production by human skin tibroblasts. The addition of NMS, but not N-methyl-D-wine. stimulated the incorporation of l?Ilelucosamine into hvaluronan dose-decendentlv un to i.S-fold at IOmM compared with control. This eft&t was sQ&ic for ‘hya~uronan synthesis because synthesis of other glycosaminoglycans remained un&nged. Gel filtration showed that NMS stimulated the production of high-molecular-mass hyaluronan (> IO” Da) without changing fhe size distribution of low-molecular-mass hvaluronsn. NMS required 24h to stimulate membrane-associated hysluronan synthase followed by increased hyahuonan production, suggesting that a second messenger is involved, in the NMS-induced stimulation of hyaluronun production. These results showed that NMS stereochemically stimulates the production of high-molecular-mass hyaluronsn in human skin fibroblssts. NMS is expected to be an anti-aging reagent capable of increasing the elasticity of the skin by stimulation of hyaluronan synthesis.
s&&intaro Inow. Basic Research Laboratory,
. .-
ESDR / JSID I SID Abstracts
0751
0754
INTERACTIONS BETWEEN PERIPHERAL BLOOD MONONUCLEAR CELLS AND EXTRACELLULAR h4ATRlX COMPONENTS IN SYSTEMIC SCLEROSIS. mzvmanska.a Makiela St&ma Elzbl S Jablonska Department of Dermatology, Warsaw School of Medzme, Warsaw, Poland Extracellular matnx (ECM) prorems play an important role in regulanon of cell nugratlon and expressmn of various genes mvolved in actwatmn of mumme reacbons. In our prewous studies we found in paoats with SSc decreased m vzrro adhesmn of penpheral bload mononuclear cells (PBMC) to collagen type IV suggesting zn YZYD acflvauon and extravasatkm of selected PBMC subpopulations. The sun of the present study was to exarmne co-stunulatory effect of ECM CO,,lQO~l~tS (collagentype 1, IV, fibronectin,I&) in 30 patwts with various forms of SSc and in 27 healthy volunteers. PBMC were cultured for 72 hrs m the presence of arm-CD3 manoclonal anobody and ECM components(at the concentratm~ IO-100 nglml) followed by 24 hrs pulse with t&at& thyxudine and counting the radioactivityin the harvested cells. The results showed that fibronectin and ~IlaSen type I had the btgbest co-stun&my actwitm in both SSc patients and the controls (relative increase of cell pmhferation 115% and 58%. p
SERUM LEVEL OF TISSUE INHIBITOR OF METALLOPROTEINASE-2 CORRELATES WITH MELANOMA PROGRESSION, BUT SERUM MATRIX METALLOPROTEINASE-2 LEVEL DOES NOT. p Department of Dermatoloav. Kumemoto Universitv School of Medicine. Kumamoto. Jauan. Since m&x metalloprotelnase-2~ (MMP-2) degrades basement membrane type IV collagen and it is regulated with it’s inhibitors (tissue inhibitor of metalloproteinase-2: TIMP-2). interactions between MMP-2 and TIMP-2 play 817 important roles in the process of tumor invasion and metastasis. The aim of this study are to measure MMP-2, TIMP-1 and -2 in melanoma lesions end these levels in serum of patients with melanoma and to correlate these panmeters with the clinical course of the disease. Frozen 14 primary end 10 metastatic melanoma lesions were stained in immune-peroxidase reactions with anti-MMP-2, TIMP-1 and -2 monoclonal antibody (mAb). Serum levels of 53 melanoma patients and 146 control were measured with Sandwich assay using these mAb. MMP-2 was expressed in melanoma cells as well es stromal fibroblasts. while TIMP-1 and -2 was expressed strongly on blood vessels in melanoma lesions and weakly in melanoma cells. Serum level of MMP-2 was significantly higher than that of control but did not correlate with melanoma progression. On the other hand, serum levels of TIMP-1 and -2 in stage II, Ill and IV was significantly higher than that of stage and control. Moreover, disease-free and over-all su~lval tlme in patients with elevated serum levels of TIMP-1 and -2 were slgnlficantly shorter than those with normal levels. These results suggest that serum level of TIMP-1 and -2 in melanoma patients is new prognostic marker, but that of MMP-2 does not represent a uselul prognostic marker.
0752
0755
ASSESSMENT OF TGFB - MEDIATED STIMULATION OF EXTRACELLULAR MATRIX PROTEIN SYNTHESIS BY AN EASY CELL CULTURE IMMUNOASSAY. C., ~epanmeut of Dermatology, University of Mllnster, MUnster, Germany. TGF-5 is implicated in tissue composition, scar formation and fibrotic processes by upregulation of the synthesis of extmceUuIar ma&. (ECM) and downregulation of mati metallo-proteinases. At present, therapeutic approaches are sought after to modulate these effects of TGF-D. A possible strategy is to use recombinant fragments of small Qroteoglycans to scavenge TGF-!3. Development of this approach requires quantitative wse.wnen~ and we have therefore devised an easy cell culture immunoassay. Cells are grown to early confluency, then incubated with or without TGF-0. After tbe incubation petiod, the medium is harvested and fdtered &rough a nitmcellulose membrane. The cell layer and the membrane are incubated with primary antibodies to an ECM protein. After washing. the binding of the tint antibody is detected by a secondary peroxidme-coupled antibody. A water-soluble chromogen, o-phenylenediamine, is used as peroxidasesubstrate, and the O.D. of the supernarants is measured at 492 “m. The amount of ECM protein in the cell layer or in the medium is expressed in relation to the amount Of total protein. This assay was employed for analysis of the production of laminin 5 and type VII collagen in cultures of human keratinocytes. A 2-3 fold stimulation of the synLhe.w of these ECM-mole.cuIes was observed after TGF-O treatment. This effect could he completely abolished by simultaneous incubation of the cells with neutralizing antibodies against TGF-0 or eliminated in pan by incubation with recombinant decorio f%meulS. The cell culture ELISA is a simple and highly sensitive assay which will aUOw monitoring of the effects of putative TGF-!3 modulators in a controlled biological system.
0753 ANT-BI IN’IEGRIN TREA’IMENI SFLECI’IVELY INDUCES APOPTOSIS IN KELOID FIBROBLASTS. M. Choucatr. D La&t. I. Olson. Fivenson. Depts. of Derm and Surgery, Henry Ford Health System. Detroit, MI, USA Cell adhesion to a substrate is mediated by the integrin family of cell surface receptors which are known to elicit intracellular signals upon cell adhesion. A role for the @I integrin family in mediating cell survival has been shown via the effect of Bl blocking antibodies to enhance apoptosis in m-slanocytes attsched to fibronectin (FN) and by the ability of anti- PI anlibadies immobilized on solid supports to suppress apoptosis in melanocytcs. Comparison of iibmnectin levels in tibroblasts derived from keloid vs normal dermis revealed a relative increase in intrac&lular and extracellular FN in k&id derived cells. We have preva~sly couftrmod rhis increased FN production in keloid fibroblasts at both the protein and mRNA levels and furthsr demonstrated fhst f3l integrin is overexpressed in kelold tibroblasfs (KK) compared to fibroblasts from normal skin of kcloid patients (NK). We used tune1 assay and RT/PCR analysis of hc-2 mRNA to study whether kcloid fibroblast apoprosis could be regulated via the @I receptor. KK end NK tibroblasts were plated on FNcoated dishes before and after monoclonal anti- gl inregnn armbody (MoAb) treatment, and analyzed by rune1 assay. Total RNA was also collected for RT/PCR The expression of bc-2 mRNA decrerwd in the MoAb hated FNxoated cuiture dishes only prior to plating for KK>NK. Slide cultures showed slight increase in staining by tune1 assay along with decreased attachment only If Uncoated slides were first exposed to the MoAb. FACS analysis of those attached cells (before or after MoAb) revealed a 2X and IOX increase for KK and NK respectively, if exposed to MoAb Qnor to plating, whereas FNcoaled plate pretreatment with MoAb did not effect apoptosis of those cells which attached (plating effeciency was reduced). Thus NK>KK for the effect of apoptosis induction by sntiBl only if attachment has already occurred to FN (as would be the case in a lesion). Both NK and KK were susceptible to the proapoptotic effects of anti-B1 at the molecular level. Blocking !3l sites could prove to be a methodof preventing k&id formation
1
In Wtro Production of an Endotheliallzed Tissue Engineered Skln Equivalent
Promoting Human Capillary-like
_-
-*
Formation. w
Auoer.. Laboratoire d’Orgaoeg6n8se Experimentate, H6pHal du Saint-Sacrement, QuBbec. Canada GIS 4LB. For patients with extensive burns, the use 01 cultured skin replacements is advantageous and often life saving. Unfortunately, various obstacles have
delayed the widespread use of composite skin equivalents. Insufficient vascularization has been proposed as the most likely reason for their poor survival. Our purpose was to develop a vascular-like network inside tissue engineered skin designed for grafting on deep wounds, to improve graft vascularization. To reach this aim, we fabricated a collaaen biooolvmer in which three human cell types: keratinocytes, dermal fibmbiasts anb umbilical vein endothelial cells were cocultured. We demonstrated that the endothelialized skin equivalent (ESE) promoted spontaneous formation of capillary-like structures inside the biopolymer. lmmunohistochemical analysis and transmission electron microscopy of the ESE showed characteristics associated with the micmvasculature in vita (vanWillebrand factor, Weibel-Palade bodies, basement membrane material and Intercellular junctions). We have developed the first endothelialized human tissue engineered skin, in which a network of capillary-like tubes is formed. The transplantation of this ESE on human should accelerate the graft revascularization by coalescence of its pre-existing vascular-like network with the host’s one. In addition, the ESE turns out to be a promising human in vitro angiogenesis model.
0756 N-METHYlJ.-SBRINlI STIMULATES WALURONAN PRODUCTION IN HUMAN SKIN FIBROBLASTS. Sbingg S&j% TV suvs a0 Kan-wara, Japan Hyaluronan has been reported to play an important role in inflammation, cell locomotion and wound healing. Its half-life time is estimated to be 2-4.5 days in mammalian ski, suggesting that hyaluronan synthesis and degradation are controlled strictly and dynamically in the skin. Decrease of hyaluronan content with aging is considered to be one of the factors which decrease the elasticity of the skin. Hyaluronan synthesis has been reported to be stimulated by growth factors, phorbol ester and retinoic acid. In this study, we found that N-methyl-L-serine (NMS), an amino acid derivative, stimulated hyahuonan production by human skin tibroblasts. The addition of NMS, but not N-methyl-D-wine. stimulated the incorporation of l?Ilelucosamine into hvaluronan dose-decendentlv un to i.S-fold at IOmM compared with control. This eft&t was sQ&ic for ‘hya~uronan synthesis because synthesis of other glycosaminoglycans remained un&nged. Gel filtration showed that NMS stimulated the production of high-molecular-mass hyaluronan (> IO” Da) without changing fhe size distribution of low-molecular-mass hvaluronsn. NMS required 24h to stimulate membrane-associated hysluronan synthase followed by increased hyahuonan production, suggesting that a second messenger is involved, in the NMS-induced stimulation of hyaluronun production. These results showed that NMS stereochemically stimulates the production of high-molecular-mass hyaluronsn in human skin fibroblssts. NMS is expected to be an anti-aging reagent capable of increasing the elasticity of the skin by stimulation of hyaluronan synthesis.
s&&intaro Inow. Basic Research Laboratory,
. .-