Chronic Graft Versus Host Disease (cGVHD) can present in 5-15% of cases with fibrotic skin involvement. This variant is defined as sclerotic-cGVHD and it has been suggested as a useful model to study the immunopathogenesis of systemic sclerosis (SSc) fibrosis. We analysed the histopathological features of cGVHD and scl-GVHD focusing on amount of extracellular matrix and vessel lumen ratio as well as immunohistochemical features peculiar of SSc like decreased expression of the lipid raft protein caveolin-1. Additionally, we aimed to identify the genes that may bridge the immune activation and the fibrotic response in SSc by comparative analysis of the transcriptome of skin biopsies from SSc, cGVHD and scl-GVHD. Thirteen patients with diagnosis of cGVHD, 93 with SSc and 3 healthy volunteers were enrolled in the University Hospital of Verona, Italy. Skin biopsies, serum samples and clinical data were collected for 5 Patients with cGVHD, 9 with scl-GVHD, 8 with SSc and 3 from healthy controls, only sera and clinical data were collected from 80 SSc patients. Biopsies were split in two halves, one stored in RNA later and the other one fixed in parafolmadehide for paraffin embedding. We selected 84 genes as intrinsic scleroderma signature from a metanalysis of microarray data from skin biopsies of 36 SSc patients. Real time PCR of all the genes was performed in cGVHD and scl-GVHD skin biopsies. Results were validated by Immunofluorescence followed by confocal laser scanning microscopy on skin biopsies and ELISA on serum. In vitro studies on epithelial cells were performed for functional validation. Correlation of the ELISA results and clinical parameters was performed using SPSS 18 software. Scl-GVHD skin biopsies showed a thickened dermis with increased ECM and reduction of total vessel lumen similar to SSc, whereas cGVHD did not. Moreover the expression of caveolin-1 was markedly decreased in scl-GVHD compared to healthy skin or cGVHD, similarly to SSc. RT-PCR of the 86 genes of the intrinsic SSc signature showed that only 4 genes had a specific and concordant pattern of expression in scl-GVHD and SSc, among these Secreted Frizzled Receptor 4. Double-immunofluorescence followed by confocal laser scanning microscopy of scl-GVHD and SSc skin biopsies showed that the source of increased expression of SFRP4 were dermal fibroblasts, epithelial cells in the germinal layer of the epidermis and Melanocytes. Further characterization of SFRP4 positive cells in the germinal layer indicated that these cells were vimentin positive and caveolin-1 negative. In vitro studies on epithelial cells indicated that TGF-β induced epithelial to Mesenchymal transition was indeed associated with decrease by 35% in caveolin-1 expression and increase of SFRP4 expression of 52% at mRNA level and 68% at protein level. Additionally, The serum levels of SFRP4 correlated inversely with Diffusion lung capacity (r=0.234 p=0.001) and positively with mRSS in patients with normal lung function.
Epithelial cells undergoing Epithelial Mesenchymal Transition in Systemic Sclerosis lack caveolin-1 and modulate WNT signaling in the dermis by secreting SFRP4
TINAZZI, Ilaria
2012
Abstract
Chronic Graft Versus Host Disease (cGVHD) can present in 5-15% of cases with fibrotic skin involvement. This variant is defined as sclerotic-cGVHD and it has been suggested as a useful model to study the immunopathogenesis of systemic sclerosis (SSc) fibrosis. We analysed the histopathological features of cGVHD and scl-GVHD focusing on amount of extracellular matrix and vessel lumen ratio as well as immunohistochemical features peculiar of SSc like decreased expression of the lipid raft protein caveolin-1. Additionally, we aimed to identify the genes that may bridge the immune activation and the fibrotic response in SSc by comparative analysis of the transcriptome of skin biopsies from SSc, cGVHD and scl-GVHD. Thirteen patients with diagnosis of cGVHD, 93 with SSc and 3 healthy volunteers were enrolled in the University Hospital of Verona, Italy. Skin biopsies, serum samples and clinical data were collected for 5 Patients with cGVHD, 9 with scl-GVHD, 8 with SSc and 3 from healthy controls, only sera and clinical data were collected from 80 SSc patients. Biopsies were split in two halves, one stored in RNA later and the other one fixed in parafolmadehide for paraffin embedding. We selected 84 genes as intrinsic scleroderma signature from a metanalysis of microarray data from skin biopsies of 36 SSc patients. Real time PCR of all the genes was performed in cGVHD and scl-GVHD skin biopsies. Results were validated by Immunofluorescence followed by confocal laser scanning microscopy on skin biopsies and ELISA on serum. In vitro studies on epithelial cells were performed for functional validation. Correlation of the ELISA results and clinical parameters was performed using SPSS 18 software. Scl-GVHD skin biopsies showed a thickened dermis with increased ECM and reduction of total vessel lumen similar to SSc, whereas cGVHD did not. Moreover the expression of caveolin-1 was markedly decreased in scl-GVHD compared to healthy skin or cGVHD, similarly to SSc. RT-PCR of the 86 genes of the intrinsic SSc signature showed that only 4 genes had a specific and concordant pattern of expression in scl-GVHD and SSc, among these Secreted Frizzled Receptor 4. Double-immunofluorescence followed by confocal laser scanning microscopy of scl-GVHD and SSc skin biopsies showed that the source of increased expression of SFRP4 were dermal fibroblasts, epithelial cells in the germinal layer of the epidermis and Melanocytes. Further characterization of SFRP4 positive cells in the germinal layer indicated that these cells were vimentin positive and caveolin-1 negative. In vitro studies on epithelial cells indicated that TGF-β induced epithelial to Mesenchymal transition was indeed associated with decrease by 35% in caveolin-1 expression and increase of SFRP4 expression of 52% at mRNA level and 68% at protein level. Additionally, The serum levels of SFRP4 correlated inversely with Diffusion lung capacity (r=0.234 p=0.001) and positively with mRSS in patients with normal lung function.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14242/182287
URN:NBN:IT:UNIVR-182287