Dataset Information

Cyclic stretch promotes the ossification of ligamentum flavum by modulating the Indian hedgehog signaling pathway.

ABSTRACT: The Indian hedgehog (IHH) signaling pathway is an important pathway for bone growth and development. The aim of the present study was to examine the role of the IHH signaling pathway in the development of the ossification of ligamentum flavum (OLF) at the cellular and tissue levels. The expression levels and localization of the osteogenic genes Runt-related transcription factor 2 (RUNX2), Osterix, alkaline phosphatase (ALP), osteocalcin (OCN) and IHH were evaluated in OLF tissues by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry. Non-ossified ligamentum flavum (LF) sections were used as control samples. The tissue explant method was used to obtain cultured LF cells. In addition, OLF cells were subjected to cyclic stretch application for 0, 6, 12 or 24 h. The expression levels of osteogenic genes, and the IHH signaling pathway genes IHH, Smoothened (SMO), GLI family zinc finger 1 (GLI1), GLI2 and GLI3 were evaluated with RT-qPCR and western blotting. Osteogenic differentiation was further evaluated by assessing ALP activity and staining. Moreover, the effect of cyclopamine (Cpn), an IHH signaling inhibitor, on osteogenic differentiation was examined. The RT-qPCR and immunohistochemical results indicated that the mRNA and protein expression levels of RUNX2, Osterix, ALP, OCN and IHH were significantly higher in the OLF group compared with the LF group. Furthermore, application of cyclic stretch to OLF cells resulted in greater ALP activity, and significant increases in mRNA and protein expression levels of RUNX2, Osterix, ALP and OCN in a time-d00ependent manner. Cyclic stretch application also led to significant increases in IHH signaling pathway genes, including IHH, SMO, GLI1 and GLI2, while no significant effect was found on GLI3 expression level. In addition, it was found that Cpn significantly reversed the effect of cyclic stretch on the ALP activity, and the expression levels of RUNX2, Osterix, ALP, OCN, GLI1 and GLI2. Collectively, the present results suggested that the IHH signaling pathway may mediate the effect of cyclic stretch on the OLF cells.

SUBMITTER: Gao R

PROVIDER: S-EPMC7339599 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Cyclic stretch promotes the ossification of ligamentum flavum by modulating the Indian hedgehog signaling pathway.

Gao Rui R Shi Changgui C Yang Chengwei C Zhao Yin Y Chen Xiongsheng X Zhou Xuhui X

Molecular medicine reports 20200602 2

The Indian hedgehog (IHH) signaling pathway is an important pathway for bone growth and development. The aim of the present study was to examine the role of the IHH signaling pathway in the development of the ossification of ligamentum flavum (OLF) at the cellular and tissue levels. The expression levels and localization of the osteogenic genes Runt-related transcription factor 2 (RUNX2), Osterix, alkaline phosphatase (ALP), osteocalcin (OCN) and IHH were evaluated in OLF tissues by reverse tran ...[more]

PMID: 32626952

Similar Datasets

Project description:The present study aimed to screen crucial micro (mi)RNAs and long non‑coding (lnc)RNAs involved in the development of ossification of ligamentum flavum (OLF) based on the miRNA‑mRNA and lncRNA‑miRNA‑mRNA competing endogenous (ce)RNA regulatory network analyses, which are rarely reported. The differentially expressed genes (DEGs), differentially expressed lncRNAs (DELs) and differentially expressed miRNAs (DEMs) between 4 OLF and 4 healthy controls were identified using two microarray datasets GSE106253 and GSE106256 collected from the Gene Expression Omnibus database. A protein‑protein interaction (PPI) network was constructed, followed by calculation of topological characteristics and sub‑module analysis in order to obtain hub DEGs. The miRNA‑mRNA and lncRNA‑miRNA networks that were established based on their interaction pairs, obtained from miRwalk and starBase databases, respectively, were integrated to form the ceRNA network. The underlying functions of mRNAs were predicted using the Database for Annotation, Visualization and Integrated Discovery (DAVID). The present study screened 828 DEGs, 119 DELs and 81 DEMs between OLF and controls. PPI network and module analyses identified interleukin (IL)10, adenylate cyclase (ADCY)5, suppressor of cytokine signaling (SOCS)3, G protein subunit gamma (GNG) 4, collagen type II α 1 chain (COL2A1) and collagen type XIII α 1 chain (COL13A1) as hub genes. The miRNA‑mRNA network analysis demonstrated IL10 could be regulated by miR‑210‑3p, while COL13A1 and COL2A1 could be modulated by miR‑329‑3p and miR‑222‑5p, respectively. lncRNA‑miRNA‑mRNA ceRNA network analysis identified that small nucleolar RNA host gene 16‑hsa‑miR‑196a‑5p‑SOCS3, ankyrin repeat and SOCS box containing 16‑AS1‑hsa‑miR‑379‑5p‑GNG4, nuclear enriched abundant transcript 1‑has‑miR‑181b‑5p‑ADCY5, rhophilin 1‑AS1‑hsa‑miR‑299‑3p‑WNT7B interaction axes may be crucial. DAVID analysis predicted IL10, ADCY5, GNG4 and SOCS3 were involved in 'adaptive immune response', 'Chemokine signaling pathway' and 'regulation of apoptosis' processes, while COL2A1, COL13A1 and WNT7B may be ossification related. In conclusion, the identification of these crucial miRNAs and lncRNAs may be conducive for explaining the pathogenesis of OLF and provide certain natural, endogenous and nontoxic drug targets for the treatment of OLF.

Project description:INTRODUCTION: Ossification of the ligamentum flavum (OLF) of the spine is associated with serious neurologic compromise, but the pathomechanism of this process remains unclear. The objective of this study was to investigate the pathomechanism of the ossification process, including the roles of various transcriptional factors in the ossification of human thoracic ligamentum flavum. METHODS: Sections of the thoracic ligamentum flavum were obtained from 31 patients with OLF who underwent posterior thoracic decompression, and from six control patients free of OLF. Cultured ligamentum flavum cells (n = 6, each) were examined with real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis for Sry-type high-mobility group box 9 (Sox9), runt-related transcription factor 2 (Runx2), muscle segment homeobox 2 (Msx2), Osterix, distal-less homeobox 5 (Dlx5), and AP-1. The harvested sections were examined with hematoxylin-eosin, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method, and immunohistochemistry for the transcriptional factors. RESULTS: Compared with the control, the OLF showed disorganization of the elastic fiber bundles and abundant hypertrophic chondrocytes in the ossification front. TUNEL-positive chondrocytes were found near the ossified plaques. The mRNA expression levels of Sox9, Runx2, Msx2, and AP-1 in cultured cells from the ligamentum flavum of OLF patients were significantly different from those of the control. OLF samples were strongly immunoreactive to Sox9, Runx2, and Msx2 at proliferating chondrocytes in the fibrocartilage area. Hypertrophic chondrocytes were positive for Runx2, Osterix, Dlx5, and AP-1. CONCLUSIONS: The ossification process in OLF seems to involve chondrocyte differentiation under the unique expression of transcriptional factors. Accumulation of hypertrophic chondrocytes was evident around the calcified area at the ossification front, and we suggest that the differentiation of these cells seems to be concerned with the ossification process.

Project description:Ligamentum flavum hypertrophy (LFH) is an important cause of spinal canal stenosis and posterior longitudinal ligament ossification. Although a number of studies have focused on the mechanisms responsible for LFH, the cellular mechanisms remain poorly understood. The aim of the present study was to investigate the roles of differentially expressed genes (DEGs) in LFH, elucidate the mechanisms responsible for LFH and provide a potential therapeutic target for further studies. The GSE113212 dataset was downloaded from the Gene Expression Omnibus (GEO) database. The microarray data were analyzed and DEGs were obtained. Bioinformatics methods, such as Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and protein?protein interaction (PPI) network analyses were used to obtain the key genes and signaling pathways. In addition, cells derived from hypertrophied ligamentum flavum were cultured, and the key genes and signaling pathways in ligamentum cells were identified through in vitro cell biology and molecular biology experiments. A total of 2,123 genes were screened as DEGs. Among these DEGs, 1,384 genes were upregulated and 739 genes were downregulated. The KEGG pathway analysis revealed that the DEGs were mainly enriched in the PI3K/AKT signaling pathway, and the PPI network analysis screened A disintegrin and metalloproteinase 10 (ADAM10) as a key gene. In vitro experimental verification revealed that ADAM10 promoted the proliferation of ligamentum flavum cells and led to the hypertrophy of the ligamentum by activating the PI3K/AKT pathway. On the whole, the in vitro experimental results suggested that ADAM10 promoted the proliferation of ligamentum flavum cells by activating the PI3K/AKT pathway, which may represent a pathogenic mechanism of LFH. The findings of the present study may provide a basis and direction for further studies on the cellular mechanisms of LFH and present a potential novel therapeutic target and clinical approach.

Project description:Background and Objectives: Ossification of the ligamentum flavum (OLF) is a relatively common cause of thoracic myelopathy. Surgical treatment is recommended for patients with myelopathy. Generally, open posterior decompression, with or without fusion, is selected to treat OLF. We performed minimally invasive posterior decompression using a microendoscope and investigated the efficacy of this approach in treating limited type of thoracic OLF. Materials and Methods: Microendoscopic posterior decompression was performed for 19 patients (15 men and four women) with thoracic OLF with myelopathy aged between 35 to 81 years (mean age, 61.9 years). Neurological examination and preoperative magnetic resonance imaging (MRI) and computed tomography (CT) were used to identify the location and morphology of OLF. The surgery was performed using a midline approach or a unilateral paramedian approach depending on whether the surgeon used a combination of a tubular retractor and endoscope. The numerical rating scale (NRS) and modified Japanese Orthopedic Association (mJOA) scores were compared pre- and postoperatively. Perioperative complications and the presence of other spine surgeries before and after thoracic OLF surgery were also investigated. Results: Four midline and 15 unilateral paramedian approaches were performed. The average operative time per level was 99 min, with minor blood loss. Nine patients had a history of cervical or lumbar spine surgery before or after thoracic spine surgery. The mean pre- and postoperative NRS scores were 6.6 and 5.3, respectively. The mean recovery rate as per the mJOA score was 33.1% (mean follow-up period, 17.8 months), the recovery rates were significantly different between patients who underwent thoracic spine surgery alone (50.5%) and patients who underwent additional spine surgeries (13.7%). Regarding adverse events, one patient experienced dural tear, another experienced postoperative hematoma, and one other underwent reoperation for adjacent thoracic stenosis. Conclusion: Microendoscopic posterior decompression was applicable in limited type of thoracic OLF surgery including beak-shaped type and multi vertebral levels. However, whole spine evaluation is important to avoid missing other combined stenoses that may affect outcomes.

Dataset Information

Cyclic stretch promotes the ossification of ligamentum flavum by modulating the Indian hedgehog signaling pathway.

Publications

Cyclic stretch promotes the ossification of ligamentum flavum by modulating the Indian hedgehog signaling pathway.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets