Association between periodontitis and ossification of the posterior longitudinal ligament: a case report.

INTRODUCTION: Ossification of the posterior longitudinal ligament in the spinal ligament compresses the spinal cord, causing various spinal nerve diseases. The ligament tissue is an important connective tissue in the joints, teeth, and spine, which, when torn and damaged, reduces the range of movement. CASE PRESENTATION: We report the treatment of periapical periodontitis and tooth preservation in a 41-year-old Japanese woman previously diagnosed with ossification of the posterior longitudinal ligament. She presented with widespread pain from the paranasal sinuses to the head caused by the onset of periapical periodontitis of the upper front tooth. DISCUSSION: The patient received an oral zinc supplement, which resolved periapical periodontitis over a 2-year follow-up period. CONCLUSION: The findings from this case imply that, when patients with ossification of the posterior longitudinal ligament develop periapical periodontitis, they should be tested for zinc deficiency.

Ubiquitin-dependent proteolysis of CXCL7 leads to posterior longitudinal ligament ossification.

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.

Retrospective Proteomic Analysis of a Novel, Cancer Metastasis-Promoting RGD-Containing Peptide.

Patients who undergo surgical extirpation of a primary liver carcinoma followed by radiotherapy and chemotherapy leading to complete remission are nevertheless known to develop cancerous metastases 3-10 years later. We retrospectively examined the blood sera collected over 8 years from 30 patients who developed bone metastases after the complete remission of liver cancer to identify serum proteins showing differential expression compared to patients without remission. We detected a novel RGD (Arg-Gly-Asp)-containing peptide derived from the C-terminal portion of fibrinogen in the sera of metastatic patients that appeared to control the EMT (epithelial-mesenchymal transition) of cancer cells, in a process associated with miR-199a-3p. The RGD peptide enhanced new blood vessel growth and increased vascular endothelial growth factor levels when introduced into fertilized chicken eggs. The purpose of this study was to enable early detection of metastatic cancer cells using the novel RGD peptide as a biomarker, and thereby develop new drugs for the treatment of metastatic cancer.

Proline/arginine-rich end leucine-rich repeat protein converts stem cells to ligament tissue and Zn(II) influences its nuclear expression.

Our objective was to facilitate ligament tissue reconstruction by characterizing the mechanism of expression of ligament tissue. To accomplish this, we searched for proteins specific to the tissue and introduced them into mesenchymal stem cells. In the two-dimensional phosphorescent gel electrophoresis, the spots in common with the normal human ligament tissue were selected after removing the spots of the normal bone tissue from those of the ossified tissue in the spinal ligament. Proline/arginine-rich end leucine-rich repeat protein (PRELP) was identified in ligament-specific locations by liquid chromatography-tandem mass spectrometry. Transfection of PRELP into mouse mesenchymal stem cells yielded ligament-like connective tissue comprised of parallel fibers. Thus, expression of the PRELP protein could reconstruct the ligament tissue. Since zinc-related proteins were found with high incidence as a result of an array analysis of PRELP's ProtoArray, it was considered that there is a relationship to the zinc metabolism. Tissue induction was mediated by the tumor necrosis factor (TNF)-α via the zinc pathway. PRELP may be a useful gene in syndesmoplasty, provided zinc is present for tissue reconstruction. Chromosome division becomes active with the addition of zinc, and rapid tissue induction takes place in the presence of zinc and TNF-α. Currently, the reconstruction of a ruptured ligament tissue is difficult, but we expect that the PRELP protein expression may facilitate this process. This study describes the discovery of the gene responsible for the differentiation of stem cells into ligament tissue. This important finding may lead to treatments for gonarthrosis, cruciate ligament, and periodontal ligament ruptures, and ossification of the posterior longitudinal ligament.

Advanced glycation end-products downregulating intervertebral disc cell production of proteoglycans in vitro.

OBJECT: The authors sought to clarify the role, if any, of advanced glycation end-products (AGEs) in disc degeneration. METHODS: Intervertebral discs were analyzed for the presence of AGEs and of their receptor (RAGE) by immunohistochemical analysis. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect any RAGE gene expression, and real-time PCR was used to quantify messenger RNA (mRNA) levels of aggrecan and collagen types I and II in nucleus pulposus cells treated with AGEs. Aggrecan protein concentration was determined by enzyme-linked immunosorbent assay. Immunohistochemical analysis revealed that AGEs and RAGE were localized in the nucleus pulposus of the intervertebral disc. Advanced glycation end-products were found to significantly suppress the expression of aggrecan at both mRNA and protein levels in a dose- and time-dependent manner. The levels of collagen types I and II remained unchanged after treatments with AGEs. CONCLUSIONS: These results suggest that the accumulation of AGEs and their interaction with their receptor in the nucleus pulposus might result in the downregulation of aggrecan production responsible for disc degeneration.

Confocal laser microscopy of chondrocytes that received gene transfer using in vitro electroporation.

To develop a gene therapy for osteopathies, this study was conducted to establish a method of transferring the BMP gene, a bone formation factor, to cells and administering the cells with BMP expression to patients with osteopathies. Although virus vectors are frequently used for gene transfer, there has been reported a death case of gene therapy using the adenovirus vector. Therefore, various efforts have been made to prevent such complications. In the present study, we used electroporation by which gene transfer can be efficiently performed without inducing severe complications after electric perforation of the cell membrane. Human bone tissues were initially collected intraoperatively, and BMP-2 and Smad4 genes were cloned and integrated into GFP and DsRed plasmid vectors. Using in vitro electroporation, these plasmid vectors were transferred to the cultured chondrocytes (KTN-1) derived from human herniated intervertebral disk. Confocal laser microscopy revealed that the BMP gene was successfully transferred to the nucleus of chondrocytes in the presence of Smad. Since electroporation facilitated human gene transfer to the target cells, gene therapy using electroporation may facilitate individualized treatment for patients.

Effect of AGEs on human disc herniation: intervertebral disc hernia is also effected by AGEs.

Currently, extracellular matrix MMP has been discussed in relation to the extrusion and spontaneous regression of the herniated mass observed in lumbar disc herniation. However, the question remains as to whether degenerated protein is really the cause of this condition's pathogenesis. We confirmed immunologically by means of electron microscopy that extrusion is caused by the AGEs (advanced glycation end products)-induced cross-linking of collagen, and that spontaneous regression is due to AGE receptors on macrophages. Further, AGEs were found to be already exposed during histogenesis, suggesting a relation to apoptosis. In lumbar disc herniation and aging, glucose-derived AGEs cross-link proteins and cause vascular tissue damage.