Project description:The ligamentum flavum (LF) is a crucial structure in maintaining spinal stability; however, hypertrophy of the LF is a significant contributor to lumbar spinal canal stenosis (LSCS). However, the mechanisms linking LF hypertrophy to the exacerbation of LSCS remain incompletely understood. In this study, we investigated the cellular proportions and signaling pathways observed in the hypertrophied LF using single-cell RNA sequencing (scRNA-seq). The LF tissues were obtained from three patients undergoing decompressive surgery at the lumbar level. These patients had been diagnosed with LSCS prior to surgery and had an LF thickness exceeding 4 mm. After single-cell dissociation of the LF tissues, scRNA-seq was performed. Fibroblasts accounted for 75% of the total cells, followed by endothelial cells, T cells, macrophages, and B cells. Among heterogeneous types of fibroblasts, we identified that a subset of fibroblasts trans-differentiated into myofibroblasts. Two types of macrophages that exhibited phenotypic plasticity akin to M1 and M2 states were observed. We also identified novel signaling pathways involved in fibroblast and immune cell interaction in the hypertrophied LF, such as GAS and GRN, as well as known signaling pathways, such as TGF-β, PDGF, CXCL, and ANGPTL. Our study highlights the transdifferentiation process from fibroblasts to myofibroblasts in the hypertrophied LF through intrinsic changes in and extrinsic influences of fibroblasts.
Project description:To identify the changes in gene expression profile caused by mechanical stress in the ligamentum flavum, we performed resection of the L3-4 supraspinal muscle and L2-3, L4-5 posterolateral fusion with instrumentation to concentrate the mechanical stress with segmental instability at the L3-4 level. The control group underwent only surgical exposure as a sham operation. Both groups of rabbits were sacrificed at 16 weeks after surgery and total RNA were extracted from ligamentum flavum.
Project description:To identify the changes in gene expression profile caused by mechanical stress in the ligamentum flavum, we performed resection of the L3-4 supraspinal muscle and L2-3, L4-5 posterolateral fusion with instrumentation to concentrate the mechanical stress with segmental instability at the L3-4 level. The control group underwent only surgical exposure as a sham operation. Both groups of rabbits were sacrificed at 1 year after surgery and total RNA were extracted from ligamentum flavum.
Project description:Pericryptal myofibroblasts in the colon and rectum play an important role in regulating the normal colorectal stem cell niche and facilitating tumour progression. Myofibroblasts have previously mostly been distinguished from normal fibroblasts only by the expression of α smooth muscle actin (αSMA). We now identify AOC3, a surface monoamine oxidase, as a new marker of myofibroblasts by showing that it is the target protein of the myofibroblast reacting monoclonal antibody (mAb), PR2D3. The normal and tumour tissue distribution and the cell line reactivity of AOC3 match that expected for myofibroblasts. We have shown that the surface expression of AOC3 is sensitive to digestion by trypsin and collagenase and that anti-AOC3 antibodies can be used for FACS sorting of myofibroblasts obtained by non-enzymatic procedures. Whole genome microarray mRNA expression profiles of myofibroblasts and skin fibroblasts revealed four additional genes that are significantly expressed differentially between these two cell types; NKX2-3 and LRRC17 are expressed in myofibroblasts and SHOX2 and TBX5 in skin fibroblasts. Transforming Growth Factor β (TGFβ) substantially down-regulated AOC3 expression in myofibroblasts but not in skin fibroblasts, in which it dramatically increased the expression of αSMA. A knockdown of NKX2-3 in myofibroblasts caused a decrease of myofibroblast-related gene expression and an increased expression of the fibroblast associated gene, SHOX2, suggesting that NKX2-3 is a key mediator for maintaining myofibroblast characteristics. Our results show that colorectal myofibroblasts, as defined by the expression of AOC3, NKX2-3 and other markers, are a distinctly different cell type from TGFβ activated fibroblasts. colorectal myofibroblast specific markers and expression profiles were sought by comparing four primary myofibroblast cultures to a panel of four dermal and foreskin fibroblast cell lines Four primary myofibroblast cultures established from adult human colon compared to four skin fibroblast cell lines to identify intestinal myofibroblast specific markers
Project description:Pericryptal myofibroblasts in the colon and rectum play an important role in regulating the normal colorectal stem cell niche and facilitating tumour progression. Myofibroblasts have previously mostly been distinguished from normal fibroblasts only by the expression of α smooth muscle actin (αSMA). We now identify AOC3, a surface monoamine oxidase, as a new marker of myofibroblasts by showing that it is the target protein of the myofibroblast reacting monoclonal antibody (mAb), PR2D3. The normal and tumour tissue distribution and the cell line reactivity of AOC3 match that expected for myofibroblasts. We have shown that the surface expression of AOC3 is sensitive to digestion by trypsin and collagenase and that anti-AOC3 antibodies can be used for FACS sorting of myofibroblasts obtained by non-enzymatic procedures. Whole genome microarray mRNA expression profiles of myofibroblasts and skin fibroblasts revealed four additional genes that are significantly expressed differentially between these two cell types; NKX2-3 and LRRC17 are expressed in myofibroblasts and SHOX2 and TBX5 in skin fibroblasts. Transforming Growth Factor β (TGFβ) substantially down-regulated AOC3 expression in myofibroblasts but not in skin fibroblasts, in which it dramatically increased the expression of αSMA. A knockdown of NKX2-3 in myofibroblasts caused a decrease of myofibroblast-related gene expression and an increased expression of the fibroblast associated gene, SHOX2, suggesting that NKX2-3 is a key mediator for maintaining myofibroblast characteristics. Our results show that colorectal myofibroblasts, as defined by the expression of AOC3, NKX2-3 and other markers, are a distinctly different cell type from TGFβ activated fibroblasts. colorectal myofibroblast specific markers and expression profiles were sought by comparing four primary myofibroblast cultures to a panel of four dermal and foreskin fibroblast cell lines