RESUMO
Islet transplantation represents a viable treatment for type 1 diabetes. However, due to loss of substantial mass of islets early after transplantation, islets from two or more donors are required to achieve insulin independence. Islet-extracellular matrix disengagement, which occurs during islet isolation process, leads to subsequent islet cell apoptosis and is an important contributing factor to early islet loss. In this study, we developed a fibroblast populated collagen matrix (FPCM) as a novel scaffold to improve islet cell viability and function post-transplantation. FPCM was developed by embedding fibroblasts within type-I collagen and used as scaffold for islet grafts. Viability and insulin secretory function of islets embedded within FPCM was evaluated in vitro and in a syngeneic murine islet transplantation model. Islets embedded within acellular matrix or naked islets were used as control. Islet cell survival and function was markedly improved particularly after embedding within FPCM. The composite scaffold significantly promoted islet isograft survival and reduced the critical islet mass required for diabetes reversal by half (from 200 to 100 islets per recipient). Fibroblast embedded within FPCM produced fibronectin and growth factors and induced islet cell proliferation. No evidence of fibroblast over-growth within composite grafts was noticed. These results confirm that FPCM significantly promotes islet viability and functionality, enhances engraftment of islet grafts and decreases the critical islet mass needed to reverse hyperglycemia. This promising finding offers a new approach to reducing the number of islet donors per recipient and improving islet transplant outcome.
Assuntos
Colágeno Tipo I/metabolismo , Diabetes Mellitus Experimental/cirurgia , Fibroblastos/transplante , Sobrevivência de Enxerto , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/cirurgia , Alicerces Teciduais , Animais , Apoptose , Glicemia/metabolismo , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
NO (nitric oxide) molecule is produced by various mammalian cell types and plays a significant role in inflammation, infection and wound healing processes. Recently, gNO (gaseous nitric oxide) therapy has been utilized for its potential clinical application as an antimicrobial agent, with special focus on skin infection. In a previous study, we demonstrated that 200 ppm gNO, 8 h/day for three consecutive days significantly reduced the number of bacteria in dermal wounds without compromising the viability and function of skin cells. To increase the feasibility and ease of its clinical use, we propose that different doses of gNO (5 to 10 K ppm) for 8 h and as short as 10 min be used, respectively. To achieve this, we set up in vitro experiments and asked whether (i) different doses of gNO have any toxic effect on immune cells and (ii) gNO has any modulating effect on key ECM (extracellular matrix) components in fibroblasts. To further investigate the effect of gNO, expression of more than 100 key ECM genes have been examined using gene array in human fibroblasts. As immune cells play an important role in wound healing, the effect of gNO on proliferation and viability of human and mouse lymphocytes was also examined. The findings showed that, the 5, 25, 75 and 200 ppm of gNO for 8 h slightly increased the expression of Col 5A3 (collagen type V alpha 3), and gNO at 5 ppm decreased the expression of MMP-1 (matrix metalloproteinase 1), while exposure of fibroblast to 10 K ppm of gNO for 10 min does not show any significant changes in ECM genes. Exposure to gNO resulted in inhibition of lymphocyte proliferation without affecting the cell viability. Taken together, our findings show that skin could be treated with gNO without compromising the role of ECM and immune cells in low concentrations with long time exposure or high concentrations for a shorter exposure time.
Assuntos
Matriz Extracelular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Óxido Nítrico/farmacologia , Pele/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Matriz Extracelular/genética , Fibroblastos/metabolismo , Humanos , Células Jurkat , Linfócitos/citologia , Metaloproteinase 1 da Matriz/genética , CamundongosRESUMO
Matrix metalloproteinases (MMPs) are key elements in extracellular matrix (ECM) degradation and scar remodeling during the wound-healing process. Our previous data revealed that keratinocyte-releasable factors significantly increased the expression of fibroblast MMPs in monolayer-cultured fibroblasts. In this study, we analyzed the differences in the MMP expressions of fibroblasts in a three-dimensional fibroblast-populated collagen gel (3D FPCG) from that in a two-dimensional monolayer-cultured fibroblasts when both co-cultured with keratinocytes. Differential mRNA and protein expression of fibroblasts were examined by microarray, RT-PCR, and western blot. Our results showed that fibroblasts co-cultured with keratinocytes in a 3D FPCG expressed significantly higher MMP1 and MMP3 at the gene and protein levels. Due to the physiological advantages of a 3D FPCG model to a 2D system, we concluded that the 3D FPCG model may provide a better means of understanding the fibroblast-keratinocyte cross-talk during the wound-healing process.
Assuntos
Fibroblastos/enzimologia , Prepúcio do Pênis/citologia , Queratinócitos/enzimologia , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Técnicas de Cocultura , Colágeno/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Indoleamine 2,3-dioxygenase (IDO) can locally suppress T cell-mediated immune responses. It has been shown that defective self-tolerance in early prediabetic female non-obese diabetic (NOD) mice can be attributed to the impaired interferon-gamma (IFN-γ)- induced IDO expression in dendritic cells of these animals. As IFN-γ can induce IDO in both dendritic cells and fibroblasts, we asked the question of whether there exists a similar defect in IFN-γ-induced IDO expression in NOD mice dermal fibroblasts. To this end, we examined the effect of IFN-γ on expression of IDO and its enzymatic activity in NOD dermal fibroblasts. The results showed that fibroblasts from either prediabetic (8 wks of age) female or male, and diabetic female or male (12 and 24 wks of age respectively) NOD mice failed to express IDO in response to IFN-γ treatment. To find underlying mechanisms, we scrutinized the IFN- γ signaling pathway and investigated expression of other IFN-γ-modulated factors including major histocompatibility complex class I (MHC-I) and type I collagen (COL-I). The findings revealed a defect of signal transducer and activator of transcription 1 (STAT1) phosphorylation in NOD cells relative to that of controls. Furthermore, we found an increase in MHC-I and suppression of COL-I expression in fibroblasts from both NOD and control mice following IFN-γ treatment; indicating that the impaired response to IFN-γ in NOD fibroblasts is specific to IDO gene. Finally, we showed that an IFN-γ-independent IDO expression pathway i.e. lipopolysaccharide (LPS)-mediated-c-Jun kinase is operative in NOD mice fibroblast. In conclusion, the findings of this study for the first time indicate that IFN-γ fails to induce IDO expression in NOD dermal fibroblasts; this may partially be due to defective STAT1 phosphorylation in IFN-γ-induced-IDO signaling pathway.