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Cyclic tensile strain facilitates ossification of the cervical posterior longitudinal ligament via increased Indian hedgehog signaling.

ABSTRACT: 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.

SUBMITTER: Sugita D

PROVIDER: S-EPMC7190672 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Cyclic tensile strain facilitates ossification of the cervical posterior longitudinal ligament via increased Indian hedgehog signaling.

Sugita Daisuke D Nakajima Hideaki H Kokubo Yasuo Y Takeura Naoto N Yayama Takafumi T Matsumine Akihiko A

Scientific reports 20200429 1

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 ...[more]

PMID: 32350355

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Project description:BackgroundA high false-negative rate has been reported for the diagnosis of ossification of the posterior longitudinal ligament (OPLL) using plain radiography. We investigated whether deep learning (DL) can improve the diagnostic performance of radiologists for cervical OPLL using plain radiographs.Materials and methodsThe training set consisted of 915 radiographs from 207 patients diagnosed with OPLL. For the test set, we used 200 lateral cervical radiographs from 100 patients with cervical OPLL and 100 patients without OPLL. An observer performance study was conducted over two reading sessions. In the first session, we compared the diagnostic performance of the DL-model and the six observers. The diagnostic performance was evaluated using the area under the receiver operating characteristic curve (AUC) at the vertebra and patient level. The sensitivity and specificity of the DL model and average observers were calculated in per-patient analysis. Subgroup analysis was performed according to the morphologic classification of OPLL. In the second session, observers evaluated the radiographs by referring to the results of the DL-model.ResultsIn the vertebra-level analysis, the DL-model showed an AUC of 0.854, which was higher than the average AUC of observers (0.826), but the difference was not significant (p = 0.292). In the patient-level analysis, the performance of the DL-model had an AUC of 0.851, and the average AUC of observers was 0.841 (p = 0.739). The patient-level sensitivity and specificity were 91% and 69% in the DL model, and 83% and 68% for the average observers, respectively. Both the DL-model and observers showed decreases in overall performance in the segmental and circumscribed types. With knowledge of the results of the DL-model, the average AUC of observers increased to 0.893 (p = 0.001) at the vertebra level and 0.911 (p < 0.001) at the patient level. In the subgroup analysis, the improvement was largest in segmental-type (AUC difference 0.087; p = 0.002).ConclusionsThe DL-based OPLL detection model can significantly improve the diagnostic performance of radiologists on cervical radiographs.

Project description:Ossification of the posterior longitudinal ligament (OPLL), a spinal ligament, reduces the range of motion in limbs. No treatment is currently available for OPLL, which is why therapies are urgently needed. OPLL occurs in obesity, is more common in men, and has an onset after 40 years of age. The mechanisms underlying OPLL remain unclear. In this study, we performed a serum proteomic analysis in both OPLL patients and healthy subjects to identify factors potentially involved in the development of OPLL, and found reduced levels of a protein that might underlie the pathology of OPLL. We isolated the protein, determined its amino acid sequence, and identified it as chemokine (C-X-C motif) ligand 7 (CXCL7). Based on these proteomics findings, we generated a CXCL7 knockout mouse model to study the molecular mechanisms underlying OPLL. CXCL7-null mice presented with a phenotype of OPLL, showing motor impairment, heterotopic ossification in the posterior ligament tissue, and osteoporosis in vertebrate tissue. To identify the mechanisms of CXCL7 deficiency in OPLL, we searched for single nucleotide polymorphisms and altered DNA exons, but no abnormalities were found. Although miR-340 levels were found to be high in an miRNA array, they were insufficient to reduce CXCL7 levels. Ubiquitin C-terminal hydrolase1 (UCHL1) was found to be overexpressed in CXCL7-null mice and in the sera of patients with OPLL, and was correlated with OPLL severity. Post-translational modifications of proteins with ubiquitin and ubiquitin-like modifiers, orchestrated by a cascade of specialized ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3) enzymes, are thought to control a wide range of cellular processes, and alterations in the ubiquitin-proteasome system have been associated with several degenerative disorders. In addition, the OPLL tissue of CXCL7-null mouse and its primary cells expressed the antibody to ubiquitin (linkage-specific K48). Our data clearly show decreased CXCL7 levels in patients with OPLL, and that OPLL developed in mice lacking CXCL7. Tumor necrosis factor receptor-associated factor (TRAF)6 expression was decreased in CXCL7-null mouse primary cells. Furthermore, K48 polyubiquitination was found in posterior longitudinal ligament ossified tissue and primary cells from CXCL7-null mice. We performed a phosphoproteomics analysis in CXCL7-deficient mice and identified increased phosphorylation of mitogen-activated protein kinase kinase (ME3K)15, ubiquitin protein ligase E3C (UBE3C) and protein kinase C (PKC) alpha, suggesting that ubiquitin-dependent degradation is involved in CXCL7 deficiency. Future studies in the CXCL7-null mouse model are, therefore, warranted to investigate the role of ubiquitination in the onset of OPLL. In conclusion, CXCL7 levels may be useful as a serum marker for the progression of OPLL. This study also suggests that increasing CXCL7 levels in patients can serve as an effective therapeutic strategy for the treatment of OPLL.

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Cyclic tensile strain facilitates ossification of the cervical posterior longitudinal ligament via increased Indian hedgehog signaling.

Publications

Cyclic tensile strain facilitates ossification of the cervical posterior longitudinal ligament via increased Indian hedgehog signaling.

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