Cyclic tensile strain enhances the expression of Indian hedgehog and progression of ossification of the human cervical posterior longitudinal ligament
ABSTRACT: The initiation and/or progression of ossification of the posterior longitudinal ligament (OPLL) is associated with cyclic tensile strain, but the pathomechanism of OPLL remains unclear. Indian hedgehog (Ihh) and its related signaling are key factors in normal enchondral ossification. However, the relation of OPLL to Ihh is unclear. The purpose of this study is to investigate the contribution of mechanical strain to OPLL and the relation of Ihh to OPLL. Cultured posterior longitudinal ligament cells were subjected to 24 hours of cyclic tensile strain and then analyzed by microarray.
Project description:The pathomechanisms of initiation and progression of ossification of the posterior longitudinal ligament (OPLL) are unclear. Indian hedgehog (Ihh) and related signaling molecules are key factors in normal enchondral ossification. The purpose of this study is to investigate the contribution of mechanical strain to OPLL and the relationship of Ihh with OPLL. Sections of the posterior longitudinal ligament (PLL) were obtained from 49 patients with OPLL and from 7 patients without OPLL. Cultured PLL cells were subjected to 24?hours of cyclic tensile strain. To identify differentially expressed genes associated with cyclic tensile strain, microarray analysis was performed. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified upregulation of various genes, particularly of the Hedgehog signaling pathway; Ihh and related genes had increased expression compared with controls after 24-hour cyclic tensile strain. In immunoblotting analysis, Ihh, Runx2, Sox9, Gli2, Gli3, and smoothened (SMO) had significantly increased expression after 6- or 12-hour cyclic tensile strain. OPLL samples were strongly immunopositive for Ihh, Sox9, Runx2, Gli2, Gli3, and SMO in the ossification front of OPLL. These results suggest that cyclic tensile strain induces abnormal activation of Ihh and related signaling molecules, and this might be important in the ossification process in OPLL.
Project description:Ossification of the posterior longitudinal ligament (OPLL) is formed by heterogeneous ossification of posterior longitudinal ligament. The patho-mechanism of OPLL is still largely unknown. MicroRNAs are small nucleatides that function as regulators of gene expression in almost any biological process. However, few microRNAs are reported to have a role in the pathological process of OPLL. Therefore, we performed high-throughput microRNA sequencing and transcriptome sequencing of primary OPLL and PLL cells in order to decipher the interacting network of microRNAs in OPLL. MRNA and microRNA profiles were done using primary culture cells of human ossification of the posterior longitudinal ligament (OPLL) tissue and normal posterior longitudinal ligament (PLL) tissue.
Project description:We found disease-specific proteins from serum proteomics of ossification of posterior longitudinal ligament, and made knockout mice. We discovered protein peculiar to a disease in serum of the ossification of posterior longitudinal ligament (OPLL), and, as a result of producing knockout mouse, we checked spinal ligament ossification and combination of diabetes and thought that it was a typical mouse and analyzed the sequence of the kidney organization. Overall design: Proteomics analysis carried out OPLL of the wild mouse that the ossification of the ligament was confirmed when glucose and urine glucose showed a mouse to the high value from a knockout
Project description:Ossification of the posterior longitudinal ligament (OPLL) of the spine is characterized by progressive ectopic bone formation in the spinal ligament. To identify the genes related to ectopic ossification of human spinal ligament affected by mechanical stress, analyses using cDNA microarray were carried out using cultured human spinal ligament cells that had been subjected to uniaxial cyclic stretching. cDNA microarrays revealed that ranges of distribution of both up- and down-regulated genes evoked by cyclic stretching were significantly wider in cells from than in non-OPLL cells. Keywords: mechanical-stress response Overall design: Cells were cultured in deformable silicon chamber which were exposed to cyclic stretching (120% extension ratio) for up to 9 hr. After cyclic stretching, total RNA was extracted from each cell monolayer. Target cDNAs transcribed from non-stretched control cells and stretched cells were labeled with Cy3 and Cy5, respectively, and then hybridized with an Agilent Human 1 cDNA Microarray. The microarrays were scanned in both the Cy3 and Cy5 channels with an Affymetrix 428 Array Scanner, and then analyzed using the ImaGene and GeneSight-Lite Software packages.
Project description:Ossification of the posterior longitudinal ligament (OPLL) is formed by heterogeneous ossification of posterior longitudinal ligament. The patho-mechanism of OPLL is still largely unknown. Recently, disorders of metabolism are thought to be the center of many diseases such as OPLL. Advanced glycation end product (AGE) are accumulated in many extracellular matrixes such as ligament fibers, and it can functions as cellular signal through its receptor (RAGE), contributing to various events such as atherosclerosis or oxidative stress. However, its role in OPLL formation is not yet known. Therefore, we performed high-through-put RNA sequencing on primary posterior longitudinal ligament cells treated with different doses of AGEs (1µM, 5µM and negative control), with or without BMP2 (1µM). mRNA profiles of Primary human posterior longitudinal ligament cells stimulated with various stimuli (Control, 1µM AGE-BSA, 5µM AGE-BSA, 1µM AGE-BSA with BMP2, 5µM AGE-BSA with BMP2) were generated by deep sequencing on Ion Proton
Project description:<h4>Introduction</h4>Ossification of the posterior longitudinal ligament (OPLL) is a significantly critical pathology that can eventually cause serious myelopathy. Ossification commences in the vertebral posterior longitudinal ligaments, and intensifies and spreads with the progression of the disease, resulting in osseous projections and compression of the spinal cord. However, the paucity of histological studies the underlying mechanisms of calcification and ossification processes remain obscure. The pathological process could be simulated in the ossifying process of the ligament in mutant spinal hyperostotic mouse (twy/twy). The aim of this study is to observe that enlargement of the nucleus pulposus followed by herniation, disruption and regenerative proliferation of annulus fibrosus cartilaginous tissues participated in the initiation of ossification of the posterior longitudinal ligament of twy/twy mice.<h4>Materials and methods</h4>The mutant twy/twy mice (6 to 22-week-old) were used in the present study. The vertebral column was analyzed histologically and immunohistochemically.<h4>Results</h4>We observed that the enlargement of the nucleus pulposus followed by herniation, disruption and regenerative proliferation of annulus fibrosus cartilaginous tissues participated in the initiation of ossification of posterior longitudinal ligament of twy/twy mice. In this regards, the cells of the protruded hyperplastic annulus fibrosus invaded the longitudinal ligaments and induced neovascularization and metaplasia of primitive mesenchymal cells to osteoblasts in the spinal ligaments of twy/twy mice.<h4>Conclusion</h4>Since genetic mechanisms could play a role in human OPLL, the age-related enlargement of the nucleus pulposus in the twy/twy mouse may primarily occur as a result of overproduction of mucopolysaccharide matrix material induced by certain genetic abnormalities.
Project description:In order to investigate the function and mechanism of Ossification of Posterior Longitudinal Ligament (OPLL) primary ligament cell derived exosomes, we take advantages of high throughput sequencing technology to fully reveal the small RNA content of both OPLL and normal posterior longitudinal ligament (PLL) cell derived exosomes. Overall design: We collect the posterior longitudinal ligament tissues of OPLL and PLL patients during surgery, PLL samples were collected from myelopathy patients who underwent ACCF operation. The collected samples were washed with PBS and subjected to primary culture immediately. The samples were cut into small pieces and attached to culture flasks for primary culture, 10%FBS high-glucose DMEM were used as culture medium for primary ligament cell culture. When cells reach 80% confluent, the cell were lysed with trypsin for passage. After 2 passages, the primary cells were ready to perform exosome collection. For exosome collection, we use 1% exosome free FBS/DMEM for culture, the medium were collected at a 3-days interval, and the collected culture medium of both OPLL and PLL primary cell were subsequently subjected to ultracentrifugation. After the ultarcentrifugation, the pellet were washed with PBS and ultarcentrifugated again before use.
Project description:This study describes the discovery of the gene responsible for differentiation of stem cells into ligament tissue. This important finding may lead to the development of treatments for gonarthrosis, rupture of the cruciate ligament and periodontal ligament, and ossification of the posterior longitudinal ligament. This study describes the discovery of the gene (A) responsible for differentiation of stem cells into ligament tissue. The transfection of this gene into mouse mesenchymal stem cells resulted in the formation of ligament-like connective tissue composed of parallel fibres. We performed microarray analysis of four samples: stem cells (sample1), ligament-like tissue from stem cells transfected with A (sample 4), ligament tissue from A-transgenic mice (sample 2) , and ligament tissue from wild type mice (sample 3).
Project description:Ossification of the posterior longitudinal ligament (OPLL) is a genetic disorder which involves pathological heterotopic ossification of the spinal ligaments. Although studies have identified several genes that correlated with OPLL, the underlying regulation network is far from clear. Through small RNA sequencing, we compared the microRNA expressions of primary posterior longitudinal ligament cells form OPLL patients with normal patients (PLL) and identified 218 dysregulated miRNAs (FDR?<?0.01). Furthermore, assessing the miRNA profiling data of multiple cell types, we found these dysregulated miRNAs were mostly OPLL specific. In order to decipher the regulation network of these OPLL specific miRNAs, we integrated mRNA expression profiling data with miRNA sequencing data. Through computational approaches, we showed the pivotal roles of these OPLL specific miRNAs in heterotopic ossification of longitudinal ligament by discovering highly correlated miRNA/mRNA pairs that associated with skeletal system development, collagen fibril organization, and extracellular matrix organization. The results of which provide strong evidence that the miRNA regulatory networks we established may indeed play vital roles in OPLL onset and progression. To date, this is the first systematic analysis of the micronome in OPLL, and thus may provide valuable resources in finding novel treatment and diagnostic targets of OPLL.