Integrin αVß3 antagonist-c(RGDyk) peptide attenuates the progression of ossification of the posterior longitudinal ligament by inhibiting osteogenesis and angiogenesis.

BACKGROUND: Ossification of the posterior longitudinal ligament (OPLL), an emerging heterotopic ossification disease, causes spinal cord compression, resulting in motor and sensory dysfunction. The etiology of OPLL remains unclear but may involve integrin αVß3 regulating the process of osteogenesis and angiogenesis. In this study, we focused on the role of integrin αVß3 in OPLL and explored the underlying mechanism by which the c(RGDyk) peptide acts as a potent and selective integrin αVß3 inhibitor to inhibit osteogenesis and angiogenesis in OPLL. METHODS: OPLL or control ligament samples were collected in surgery. For OPLL samples, RNA-sequencing results revealed activation of the integrin family, particularly integrin αVß3. Integrin αVß3 expression was detected by qPCR, Western blotting, and immunohistochemical analysis. Fluorescence microscopy was used to observe the targeted inhibition of integrin αVß3 by the c(RGDyk) peptide on ligaments fibroblasts (LFs) derived from patients with OPLL and endothelial cells (ECs). The effect of c(RGDyk) peptide on the ossification of pathogenic LFs was detected using qPCR, Western blotting. Alkaline phosphatase staining or alizarin red staining were used to test the osteogenic capability. The effect of the c(RGDyk) peptide on angiogenesis was determined by EC migration and tube formation assays. The effects of the c(RGDyk) peptide on heterotopic bone formation were evaluated by micro-CT, histological, immunohistochemical, and immunofluorescence analysis in vivo. RESULTS: The results indicated that after being treated with c(RGDyk), the osteogenic differentiation of LFs was significantly decreased. Moreover, the c(RGDyk) peptide inhibited the migration of ECs and thus prevented the nutritional support required for osteogenesis. Furthermore, the c(RGDyk) peptide inhibited ectopic bone formation in mice. Mechanistic analysis revealed that c(RGDyk) peptide could inhibit osteogenesis and angiogenesis in OPLL by targeting integrin αVß3 and regulating the FAK/ERK pathway. CONCLUSIONS: Therefore, the integrin αVß3 appears to be an emerging therapeutic target for OPLL, and the c(RGDyk) peptide has dual inhibitory effects that may be valuable for the new therapeutic strategy of OPLL.

Drug therapy targeting pyrophosphate slows the ossification of spinal ligaments in twy mice.

The lack of an effective drug therapy against ossification of spinal ligament (OSL) warrants investigation into the therapeutic target of this disease. An endogenous inhibitor of biomineralization, pyrophosphate (PPi) is a potential therapy for ectopic ossification; however, exogenous PPi is rapidly hydrolyzed by tissue non-specific alkaline phosphatase (TNAP) present in body fluids. In this study, we examined whether a drug therapy targeting PPi is efficacious for the treatment of OSL using the Enpp1ttw/ttw (twy) mouse model. Twenty male twy mice were randomized into four groups: (i) vehicle (Control); (ii) alkaline phosphatase inhibitor levamisole (5 mg/kg/day sc continuously); (iii) levamisole + exogenous PPi (160 µmol/kg/day sc continuously); and (iv) nuclear retinoic acid receptor-γ (RARγ) agonist (6 µg/kg sc daily). The RARγ agonist, which is a proven inhibitor of ectopic endochondral ossification, was used as a positive control. Treatments commenced when the mice were 5 weeks of age and continued for 4 weeks. Longitudinal micro-computed tomography and postmortem histological analysis were performed. Administration of levamisole alone and in combination with PPi increased serum PPi concentration by 17% and 52%, respectively, compared to that in vehicle-treated mice. The development of OSL in twy mice was suppressed by levamisole + PPi and RARγ agonist treatments, but not by levamisole alone. The levamisole + PPi therapy did not cause osteoporosis, whereas RARγ agonist-treated mice developed osteoporosis. Treatment of twy mice with levamisole in combination with exogenous PPi increased serum PPi level, which slowed the progression of OSL without producing adverse effect on bone. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1256-1261, 2018.

A case of psoriasis vulgaris with diffuse idiopathic skeletal hyperostosis involved with ossifications of posterior and anterior longitudinal ligament.

Diffuse idiopathic skeletal hyperostosis (DISH) is difficult to distinguish from various forms of inflammatory arthritis, including psoriatic arthritis (PsA), rheumatoid arthritis, and ankylosing spondylitis. A 67-year-old Japanese male had been treated for psoriasis vulgaris for 13 years. Numbness of his right arm and lower limbs and spinal stiffening had developed 7 years prior to his initial evaluation at our facility. He noticed pain mainly while exercising. There were symmetrical marginal syndesmophytes in the spine, from the thoracic vertebrae to the upper lumbar vertebrae, on radiological examinations. We therefore suspected DISH. Furthermore, ossifications of the posterior and anterior longitudinal ligaments were noted in the cervical spine. Laboratory examinations revealed a normal peripheral white blood cell count, serum C-reactive protein, and erythrocyte sedimentation rate, and he was negative for rheumatoid factor. We detected human leukocyte antigen B39 but not B27. All distal interphalangeal joints were swollen but without pain. X-ray imaging showed narrowing of the joint space, and the consolidation of the joint was recognized, but there was no new juxta-articular bone formation. Based on clinical and radiological findings, we concluded that he had DISH and not PsA. DISH was indicated by marked radiological features of the axial skeleton, particularly the thoracic spine, but may also have involved the peripheral joints. DISH is one of the entheseal disorders, and 10% of Japanese middle-aged and elderly men have DISH. Therefore, the differentiation of DISH from PsA is necessary in psoriasis patients with spinal involvement.

A patient with hypophosphatemic rickets and ossification of posterior longitudinal ligament caused by a novel homozygous mutation in ENPP1 gene.

X-linked hypophosphatemic rickets/osteomalacia (XLH), autosomal dominant hypophosphatemic rickets/osteomalacia (ADHR) and autosomal recessive hypophosphatemic rickets/osteomalacia (ARHR1 or ARHR2) are hereditary fibroblast growth factor 23 (FGF23)-related hypophosphatemic rickets showing similar clinical features. We here show a patient with hypophosphatemic rickets and widespread ossification of posterior longitudinal ligament (OPLL). The proband is a 62-year-old female. Her parents are first cousins and showed no signs of rickets or osteomalacia. She showed hypophosphatemic rickets with elevated FGF23 level and had been clinically considered to be suffering from XLH. However, direct sequencing of all coding exons and exon-intron junctions of phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX), FGF23 and dentin matrix protein 1 (DMP1) genes, responsible genes for XLH, ADHR and ARHR1, respectively, showed no mutation. A novel homozygous splice donor site mutation was found at the exon-intron junction of exon 21 of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene responsible for ARHR2 (IVS21+1_3(GTA>CACC)). Subsequent analysis of mRNA revealed that this mutation caused skipping of exon 21 which created a premature stop codon in exon 22. These results indicate that genetic analysis is mandatory for the correct diagnosis of hereditary FGF23-related hypophosphatemic rickets. Because Enpp1 knockout mouse is a model of OPLL, this case also suggests that OPLL is associated with ARHR2.

[Ossification of the common posterior longitudinal ligament of the spine and spinal cord compression in a patient from Senegal]. / Ossification du ligament commun vertébral postérieur et compression médullaire chez un Africain.

INTRODUCTION: Ossification of the posterior longitudinal ligament of the spine is a rare cause medullar compression. OBSERVATION: A 50-year-old man from Senegal was referred with recent-onset mechanical lumbar pain with proximal motor deficiency of the lower limbs and somatosensory disorders. Magnetic resonance imaging revealed layered medullar compression, due to anterior cervical and mixed anterior and posterior thoracic ossification. Corticosteroid treatment led to regression of the pain and neurological disorders within a few days. DISCUSSION: This case report of ossification of the posterior longitudinal ligament of the spine appears to be an idiopathic form corresponding to the "Japanese disease" initially thought to be limited to that population.

Effects of ethane-1-hydroxy-1,1-diphosphonate on ossification of the posterior longitudinal ligament in Zucker fatty rats.

PURPOSE: Using Zucker fatty rats as an animal model, we evaluate the effectiveness of ethane-1-hydroxy-1,1-diphosphonate on ossification of the posterior longitudinal ligament by histopathologically investigating the prodromal, early, and advanced stages of ossification of the spinal ligaments. METHODS: 73 Zucker fatty rats were allocated to the ethane-1-hydroxy-1,1-diphosphonate group (n=33) and the control group (n=40). The former group was fed ethane-1-hydroxy-1,1-diphosphonate daily. The feed was given starting 2 months after birth and continued until the rats were killed at 3 to 18 months later. Chemical analysis of the blood, radiographic tests, and histopathological examination were then conducted for both groups. RESULTS: The results showed that ossification of the spinal ligaments involved excessive cartilage cell proliferation around areas affected by enthesitis; enlargement of the fibrocartilage tissue layer; ligament thickening; calcification of the matrix around the cartilage cells; and ossification of the spinal ligaments through enchondral ossification. Radiographic examinations showed that osteoproliferation in vertebral bodies in rats receiving ethane-1-hydroxy-1,1-diphosphonate was generally suppressed compared with controls, whereas histopathological examinations found no clear difference in cartilage cell proliferation in areas affected by enthesitis between the two groups, indicating the absence of calcification or osteo-proliferation in areas affected by enthesitis for the rats receiving ethane-1-hydroxy-1,1-diphosphonate. CONCLUSION: Ethane-1-hydroxy-1,1-diphosphonate is effective in suppressing progressive ligament ossification.

Management of cerebrospinal fluid leakage complicating anterior procedures through thoracotomy: report of three cases.

Postoperative cerebrospinal fluid (CSF) leakage is a serious complication accompanying an anterior procedure through thoracotomy, and it is difficult to cure. In this report, we present three patients with CSF leakage in the thoracic spine complicating anterior decompression and fusion for ossification of posterior longitudinal ligament who were treated by surgical or nonsurgical methods. As a surgical method, direct closure by fixing substitute dura mater with fibrin adhesive sealant or cyanoacrylate adhesive was performed in two patients. This technique was effective but required another thoracotomy. As a nonsurgical method, intrapleural administration of OK-432 through chest drainage tubes was also effective to reduce intrapleural effusions in one patient, but with this method, care must be taken for neurotoxic reactions. Both techniques seem to be useful and effective for postoperative intrapleural CSF leakages complicating anterior procedures through thoracotomy.

Calcitonin simultaneously regulates both periosteal hyperostosis and trabecular osteopenia in the spinal hyperostotic mouse (twy/twy) in vivo.

The twy (tiptoe-walking-Yoshimura) mouse, established in Japan in 1978 by brother-sister mating of ICR strain mice, is a valuable mutant as a model of ossification of the posterior longitudinal ligament (OPLL). OPLL causes severe myelopathy and has been thought to be very similar to ankylosing spinal hyperostosis (ASH) and diffuse idiopathic skeletal hyperostosis (DISH). In the twy mouse, both an increase in vertebral cortical membranous bone formation and a decrease in trabecular bone mass due to accelerated bone resorption occur simultaneously. This process is attributed to an inherited autosomal recessive single gene (twy). Calcitonin's suppression of bone resorption has been well established in the past, whereas the effects of this hormone on bone formation remain to be defined. Of particular interest is the simultaneous action of calcitonin on the abnormally accelerated bone formation and resorption. Thirty twy mice and 14 ICR mice were divided into seven groups, and changes induced by calcitonin on vertebral cortical appositional rate and on trabecular bone mass were investigated histomorphometrically. Results were (1) osteoclastic activity on trabecular surface was clearly suppressed by chicken calcitonin injected subcutaneously for 4 weeks; (2) no significant difference between the lumbar vertebral periosteal bone formation of calcitonin (CA) and vehicle-administrated twy mice groups. However, on the periosteal surface of the cervical vertebrae of the 6-week-old twy mice, the abnormally accelerated bone formation was suppressed by CA administration. This was also true for the elderly twy mice, although the effect was less pronounced. In conclusion, CA suppressed the abnormally hyperactivated periosteal bone formation. Results also suggested a possible therapeutic value of CA for OPLL.