Human aortic valve interstitial cells obtained from patients with aortic valve stenosis are vascular endothelial growth factor receptor 2 positive and contribute to ectopic calcification.

Since aortic valve stenosis (AVS) is the most frequent and serious valvular heart disease in the elderly, and is accompanied by irreversible valve calcification, medicinal prevention of AVS is important. Although we recently demonstrated that human aortic valve interstitial cells (HAVICs) obtained from patients with AVS were highly sensitive to ectopic calcification stimulation, the cell types contributing to calcification are unknown. We aimed to immunocytochemically characterize HAVICs and identify their contribution to valve calcification. HAVICs were isolated from patients with AVS and cultured on non-coated dishes. Immunocytochemical features and HAVIC differentiation were analyzed in passage 1 (P1). The immunohistochemical features of the calcified aortic valve were analyzed. Most cultured P1 HAVICs were CD73-, CD90-, and CD105-positive, and CD45-and CD34-negative. HAVICs were vascular endothelial growth factor receptor 2 (VEGFR2)-positive; however, approximately half were α-smooth muscle actin (SMA)-positive, colonized, and easily differentiated into osteoblastic cells. Calcified aortic valve immunohistochemistry showed that all cells were positive for VEGFR2 and partly α-SMA. Further, VEGFR2-positive cells were more sensitive to tumor necrosis factor-α-induced ectopic calcification with or without α-SMA positivity. We conclude that HAVICs obtained from patients with AVS are VEGFR2-positive undifferentiated mesenchymal cells and may contribute to aortic valve ectopic calcification.

Effect of teriparatide on ligamentum flavum mesenchymal stem cells isolated from patients with ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament (OPLL) within the spinal canal sometimes leads to severe myelopathy. Teriparatide (TPD) is a recombinant human parathyroid hormone (PTH) (1-34), which promotes osteogenesis of mesenchymal stem cells (MSCs) via PTH 1 receptor (PTH1R). Although ligamentum flavum (LF)-MSCs from patients with OPLL have a high osteogenic potency, the effect of TPD on them remains unknown. In this study, we determined PTH1R expression in LF-MSCs from patients with OPLL and investigated whether TPD promotes osteogenic differentiation in them. First, LF-MSCs were isolated from patients with OPLL and cervical spondylotic myelopathy (CSM) (controls). Cultured LF-MSCs were treated with different concentrations of TPD on days 0, 7, and 14. On day 21, osteogenic gene expression was quantified. Mineralization was measured based on optical density after Alizarin Red S staining. LF-MSCs from both groups expressed PTH1R at the same level. TPD did not enhance osteogenic gene expression and mineralization in LF-MSCs from both groups. TPD did not promote the osteogenic differentiation of LF-MSCs from patients with OPLL. Thus, it may be safe for patients with OPLL. However, further confirmation of our results with in vivo studies is necessary.

Menaquinone-4 Accelerates Calcification of Human Aortic Valve Interstitial Cells in High-Phosphate Medium through PXR.

Recently, we confirmed that in human aortic valve interstitial cells (HAVICs) isolated from patients with aortic valve stenosis (AVS), calcification is induced in high inorganic phosphate (high-Pi) medium by warfarin (WFN). Because WFN is known as a vitamin K antagonist, reducing the formation of blood clots by vitamin K cycle, we hypothesized that vitamin K regulates WFN-induced HAVIC calcification. Here, we sought to determine whether WFN-induced HAVIC calcification in high-Pi medium is inhibited by menaquinone-4 (MK-4), the most common form of vitamin K2 in animals. HAVICs obtained from patients with AVS were cultured in α-modified Eagle's medium containing 10% FBS, and when the cells reached 80%-90% confluency, they were further cultured in the presence or absence of MK-4 and WFN for 7 days in high-Pi medium (3.2 mM Pi). Intriguingly, in high-Pi medium, MK-4 dose-dependently accelerated WFN-induced HAVIC calcification and also accelerated the calcification when used alone (at 10 nM). Furthermore, MK-4 enhanced alkaline phosphatase (ALP) activity in HAVICs, and 7 days of MK-4 treatment markedly upregulated the gene expression of the calcification marker bone morphogenetic protein 2 (BMP2). Notably, MK-4-induced calcification was potently suppressed by two pregnane X receptor (PXR) inhibitors, ketoconazole and coumestrol; conversely, PXR activity was weakly increased, but in a statistically significant and dose-dependent manner, by MK-4. Lastly, in physiologic-Pi medium, MK-4 increased BMP2 gene expression and accelerated excess BMP2 (30 ng/ml)-induced HAVIC calcification. These results suggest that MK-4, namely vitamin K2, accelerates calcification of HAVICs from patients with AVS like WFN via PXR-BMP2-ALP pathway. SIGNIFICANCE STATEMENT: For aortic valve stenosis (AVS) induced by irreversible valve calcification, the most effective treatment is surgical aortic or transcatheter aortic valve replacement, but ∼20% of patients are deemed unsuitable because of its invasiveness. For effective drug treatment strategies for AVS, the mechanisms underlying aortic valve calcification must be elucidated. Here, we show that menaquinone-4 accelerates warfarin-induced calcification of AVS-patient human aortic valve interstitial cells in high inorganic phosphate medium; this effect is mediated by pregnane X receptor-bone morphogenetic protein 2-alkaline phosphatase signaling, which could be targeted for novel drug development.

Neurotrophic Factor Secretion and Neural Differentiation Potential of Multilineage-differentiating Stress-enduring (Muse) Cells Derived from Mouse Adipose Tissue.

Multilineage-differentiating stress-enduring (Muse) cells are endogenous pluripotent stem cells that can be isolated based on stage-specific embryonic antigen-3 (SSEA-3), a pluripotent stem cell-surface marker. However, their capacities for survival, neurotrophic factor secretion, and neuronal and glial differentiation are unclear in rodents. Here we analyzed mouse adipose tissue-derived Muse cells in vitro. We collected mesenchymal stem cells (MSCs) from C57BL/6 J mouse adipose tissue and separated SSEA-3+, namely Muse cells, and SSEA-3-, non-Muse cells, to assess self-renewability; pluripotency marker expression (Nanog, Oct3/4, Sox2, and SSEA-3); spontaneous differentiation into endodermal, mesodermal, and ectodermal lineages; and neural differentiation capabilities under cytokine induction. Neurally differentiated Muse and non-Muse cell functions were assessed by calcium imaging. Antioxidant ability was measured to assess survival under oxidative stress. Brain-derived neurotrophic factor (BDNF), vascular endothelial cell growth factor (VEGF), and hepatocyte growth factor (HGF) secretion were analyzed in enzyme-linked immunosorbent assays. SSEA-3+ Muse cells (6.3 ± 1.9% of mouse adipose-MSCs), but not non-Muse cells, exhibited self-renewability, spontaneous differentiation into the three germ layers, and differentiation into cells positive for Tuj-1 (27 ± 0.9%), O4 (17 ± 3.4%), or GFAP (23 ± 1.3%) under cytokine induction. Neurally differentiated Muse cells responded to KCl depolarization with greater increases in cytoplasmic Ca2+ levels than non-Muse cells. Cell survival under oxidative stress was significantly higher in Muse cells (50 ± 2.7%) versus non-Muse cells (22 ± 2.8%). Muse cells secreted significantly more BDNF, VEGF, and HGF (273 ± 12, 1479 ± 7.5, and 6591 ± 1216 pg/mL, respectively) than non-Muse cells (133 ± 4.0, 1165 ± 20, and 2383 ± 540 pg/mL, respectively). Mouse Muse cells were isolated and characterized for the first time. Muse cells showed greater pluripotency-like characteristics, survival, neurotrophic factor secretion, and neuronal and glial-differentiation capacities than non-Muse cells, indicating that they may have better neural-regeneration potential.

Warfarin calcifies human aortic valve interstitial cells at high-phosphate conditions via pregnane X receptor.

Warfarin, a vitamin K antagonist, is the most common anticoagulant used to prevent thromboembolisms associated with atrial fibrillation or following valvular surgery. Although several studies have revealed that long-term warfarin use accelerates aortic valve calcification and the development of aortic stenosis (AS), the detailed mechanism for this phenomenon remains unclear. Therefore, our aim was twofold: to establish the conditions for warfarin-induced calcification of human aortic valve interstitial cells (HAVICs) using high-inorganic phosphate (Pi) conditions and to investigate the underlying mechanism. We prepared and cultured HAVICs from aortic valves affected by calcific aortic valve stenosis (AS group) and aortic valves affected by aortic regurgitation but without any signs of calcification (non-AS group). Under Pi concentrations of 3.2 mM, warfarin significantly increased the calcification and alkaline phosphatase (ALP) activity of AS but not non-AS group HAVICs. Furthermore, gene expression of bone morphogenetic protein 2 (BMP2), a calcigenic marker, was significantly increased following 7 days of warfarin treatment. Warfarin-induced calcification of AS group HAVICs at 3.2 mM Pi was significantly inhibited by dorsomorphin, a Smad inhibitor, and the pregnane X receptor (PXR) inhibitors, ketoconazole and coumestrol, but was unaffected by SN-50, an NF-κB inhibitor. Warfarin was also able to increase BMP2 gene expression at a physiological Pi concentration (1.0 mM). Furthermore, excess BMP2 (30 ng/mL) facilitated warfarin-induced ALP upregulation and HAVIC calcification, an effect which was significantly reduced in the presence of coumestrol. Together, our results suggest that warfarin accelerates calcification of HAVICs from AS patients via the PXR-BMP2-ALP pathway.

Facilitation of Chemotaxis Activity of Mesenchymal Stem Cells via Stromal Cell-Derived Factor-1 and Its Receptor May Promote Ectopic Ossification of Human Spinal Ligaments.

Mesenchymal stem cells (MSCs) have been used to elucidate the pathogenesis of numerous diseases. Our recent study showed that MSCs may conduce to the ossification of spinal ligaments. Stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) regulate MSC migration. Moreover, their expression is elevated in sites of damage and remodeling in pathologic states. We explored the possible role of the SDF-1/CXCR4 axis in the chemotactic behavior of MSCs in the ossification of spinal ligaments. Specimens of thoracic vertebra ossified ligamentum flavum (OLF) and non-OLF plaques were received from patients in whom we had performed spine surgery. Paraffin-embedded tissue sections were prepared for immunohistochemical staining. Cultured MSCs from the ligamentum flavum were prepared for in vitro analyses. We observed SDF-1 and CXCR4 localization immunohistochemically in the perivascular area and collagenous matrix of ligaments and in chondrocytes near the ossification front of OLF. And then, immunohistochemical staining showed a close relationship between MSCs and the SDF-1/CXCR4 axis. In the in vitro analyses, expression of the SDF-1/CXCR4 and the migratory capacity of MSCs in OLF were remarkably higher compared with non-OLF MSCs. Furthermore, the migration of MSCs was upregulated by SDF-1 and downregulated by treatment with AMD3100 (C28H54N88HCl), a specific antagonist for CXCR4. All in vitro test data showed a significant difference in MSCs from OLF compared with non-OLF MSCs. Our results reveal that the SDF-1/CXCR4 axis may contribute to an MSC-mediated increase in the ossification process, indicating that the SDF-1/CXCR4 axis may become a potential target for a novel therapeutic strategy for ossification of spinal ligaments.

Matrix Gla protein negatively regulates calcification of human aortic valve interstitial cells isolated from calcified aortic valves.

Calcified aortic valve stenosis (CAS) is a common heart valve disease in elderly people, and is mostly accompanied by ectopic valve calcification. We recently demonstrated that tumor necrosis factor-α (TNF-α) induces calcification of human aortic valve interstitial cells (HAVICs) obtained from CAS patients. In this study, we investigated the role of matrix Gla protein (MGP), a known calcification inhibitor that antagonizes bone morphogenetic protein 2 (BMP2) in TNF-α-induced calcification of HAVICs. HAVICs isolated from aortic valves were cultured, and calcification was significantly induced with 30 ng/mL TNF-α. Gene expression of the calcigenic marker, BMP2, was significantly increased in response to TNF-α, while the gene and protein expression of MGP was strongly decreased. To confirm the role of MGP, MGP-knockdown HAVICs and HAVICs overexpressing MGP were generated. In HAVICs, in which MGP expression was inhibited by small interfering RNA, calcification and BMP2 gene expression were induced following long-term culture for 32 days in the absence of TNF-α. In contrast, HAVICs overexpressing MGP had significantly decreased TNF-α-induced calcification. These results suggest that MGP acts as a negative regulator of HAVIC calcification, and as such, may be helpful in the development of new therapies for ectopic calcification of the aortic valve.

Ketamine suppresses the proliferation of rat C6 glioma cells.

The present study investigated the effects of N-methyl-D-aspartate receptor (NMDAR) antagonist ketamine, on the growth of gliomas. To analyze the effects of ketamine treatment, rat C6 glioma cells arising from astrocytes, and RNB cells representing non-malignant astrocytes, were examined. In ketamine-treated C6 cells, the gene expression changes associated with cell proliferation following ketamine treatment were evaluated using a cDNA microarray. A cell proliferation assay was performed to analyze the dose-dependent proliferation of C6 glioma and RNB cells following culture (72 h) with ketamine treatment (0-100 µM). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed following cell incubation with/without ketamine, to confirm if the ketamine-induced cell death of C6 glioma and RNB cells were due to apoptosis. In addition, cell proliferation and TUNEL assays were performed following cell incubations with a selective NMDAR antagonist, D-2-amino-5-phosphonovaleric acid (D-AP5). Analysis of the cDNA microarray indicated that the growth of C6 glioma cells were suppressed by the effects of ketamine. Furthermore, results of the proliferation assay confirmed that ketamine treatment inhibited C6 cell proliferation, most notably at a dose of 30 µM (n=7, 66.4%; P<0.001). The TUNEL assay results revealed that ketamine induced an apoptotic effect on C6 glioma cells, with a significant effect on the rate of death observed at all tested concentrations (3, 10, 30 and 100 µM). Results of the aforementioned proliferation and TUNEL assay experiments were reproduced when ketamine was replaced with a selective NMDAR antagonist, D-AP5. However, the NMDARantagonist-induced effects were not observed in RNB cell cultures. Although it would be premature to apply the results from the present study to human cases, these results indicated that ketamine is an anesthetic candidate providing potential benefit for glioma resection.

Suppression of osteogenic differentiation in mesenchymal stem cells from patients with ossification of the posterior longitudinal ligament by a histamine-2-receptor antagonist.

Mesenchymal stem cells (MSCs) in ossification of the posterior longitudinal ligament (OPLL) patients have a high propensity toward osteogenesis. Histamine receptor H2 (H2R) antagonists (H2 blockers) like famotidine decrease ossification in patients, by an unclear mechanism. To confirm that MSCs express H2R and to clarify how H2 blockers suppress osteogenic differentiation, we used spinal-ligament MSCs from patients with OPLL or with cervical spondylotic myelopathy (CSM) (control). The MSCs were treated with 10, 30, or 100nM famotidine for 7 or 21 days. Flow cytometry revealed that cells from both groups expressed MSC surface markers CD44, CD90, and CD105 (> 97.5%) but not CD34 or CD45 (< 2.5%). Immunoblotting showed that the MSCs from both groups expressed H2R, but those from OPLL patients expressed it at higher levels. Real-time qPCR indicated the H2R expression was significantly suppressed by 30nM famotidine for 7 days or by 30 or 100nM for 21 days. However, histidine decarboxylase, a key enzyme in histamine production, did not change significantly after famotidine addition. Famotidine treatment at 100nM for 21 days significantly suppressed mRNA expression of the osteogenic markers osteocalcin (OCN), bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (RUNX2) only in OPLL-derived MSCs. Immunoblots showed that famotidine suppressed BMP2 and OCN in the OPLL group and H2R and RUNX2 in both groups. These results suggest famotidine inhibits osteogenic differentiation in OPLL-derived MSCs by acting as an H2R antagonist, but also by decreasing H2R expression, and support the clinical use of famotidine to treat OPLL.

High Osteogenic Potential of Adipose- and Muscle-derived Mesenchymal Stem Cells in Spinal-Ossification Model Mice.

STUDY DESIGN: Basic experiments in a mouse model of ossification of the posterior longitudinal ligament (OPLL). OBJECTIVE: To assess the osteogenic potential of mesenchymal stem cells (MSCs) obtained from muscle and adipose tissue in Tiptoe-walking (ttw) mice, in which cervical OPLL compresses the spinal cord and causes motor and sensory dysfunction. SUMMARY OF BACKGROUND DATA: In humans, MSCs have been implicated in the pathogenesis of cervical OPLL. Cervical OPLL in ttw mice causes chronic compression of the spinal cord. Few studies have compared the MSC osteogenic potential with behavioral changes in an OPLL animal model. METHODS: We compared the osteogenic potential and behavioral characteristics of MSCs from ttw mice (4 to 20 weeks old) with those from control wild-type mice (without hyperostosis). Ligament ossification was monitored by micro-computed tomography and pathology; tissues were double stained with fluorescent antibodies against markers for MSCs (CD45 and CD105), at 8 weeks. The Basso Mouse Scale was used to assess motor function, and heat and mechanical tests to assess sensory function. The osteogenic potential of adipose and muscle MSCs was assessed by Alizarin Red S absorbance, staining for osteogenic mineralization, and real-time quantitative polymerase chain reaction for osteogenesis-related genes. RESULTS: Spinal-ligament ossification began in ttw mice at 8 weeks of age, and the ossified area increased with age. Immunofluorescence staining identified MSCs in the ossification area. The ttw mice became hyposensitive at 8 weeks of age, and Basso Mouse Scale scores showed motor-function deficits starting at 12 weeks of age. Alizarin Red S staining for mineralization showed a higher osteogenic potential in the adipose- and muscle-derived MSCs from ttw mice than from wild-type mice at 4, 8, and 20 weeks of age. Real-time quantitative polymerase chain reaction showed that ttw MSCs strongly expressed osteogenesis-related genes. CONCLUSION: MSCs derived from muscle and adipose tissue in ttw mice had a high osteogenic potential. LEVEL OF EVIDENCE: N/A.

1-Methyl-2-undecyl-4(1H)-quinolone, a derivative of quinolone alkaloid evocarpine, attenuates high phosphate-induced calcification of human aortic valve interstitial cells by inhibiting phosphate cotransporter PiT-1.

An abnormally high serum phosphate level induces calcific aortic stenosis (CAS), which is characterized by ectopic valve calcification and stenosis of the orifice area. Inhibition of ectopic calcification is a critical function of any internal medical therapy for CAS disease. The aim of the present study was to investigate the inhibitory effects of several derivatives of evocarpine, methanolic extracts from the fruits of Evodia rutaecarpa Bentham (Japanese name: Go-Shu-Yu) on the high phosphate-induced calcification of human aortic valve interstitial cells (HAVICs) obtained from patients with CAS. High phosphate (3.2 mM) concentrations significantly increased the calcification of HAVICs after 7 days of culture. This calcification was completely inhibited in the presence of sodium phosphonoformate (PFA), a selective inhibitor of the type III sodium-dependent phosphate cotransporter (PiT-1). PiT-1 contributes to phosphate uptake, resulting in calcification. 1-Methyl-2-undecyl-4(1H)-quinolone (MUQ; 30-300 nM), but not evocarpine or its derivatives dihydroevocarpine and 1-methyl-2-nonyl-4(1H)-quinolone, inhibited the high phosphate-induced HAVICs calcification in a concentration-dependent manner. Although all of the evocarpine derivatives attenuated alkaline phosphatase activity, only MUQ also decreased PiT-1 gene expression with cellular PiT-1 protein diminution. These results suggest that MUQ mitigated high phosphate-induced HAVICs calcification by inhibiting PiT-1 gene expression.

Hyaluronic acid induces the release of growth factors from platelet-rich plasma.

BACKGROUND/OBJECTIVE: Platelet-rich plasma (PRP) and hyaluronic acid (HA) injection are both therapeutic options for osteoarthritis and chronic tendinopathy. Although several comparative studies on the two have been published, the effects of mixing PRP and HA are not fully understood. The purpose of this study is to investigate the influence of HA on platelets in PRP by measuring releasing growth factors. METHODS: PRP was produced from nine healthy adult volunteers (mean age, 32.8 ± 2.9 years; range, 29-37) with a commercial separation system. HA of weight-average molecular weight of 50-120 kDa was used. PRP group (PRP 1 mL + phosphate buffered saline 0.2 mL) and PRP + HA group (PRP 1 mL + HA 0.2 mL) were incubated at 37°C for 2 hours. The amounts of transforming growth factor ß1 (TGF-ß1) and platelet-derived growth factor (PDGF-AA) released from the PRP and PRP + HA samples were measured on Day 0, Day 3, and Day 5. In addition, the same growth factors on Day 5 were measured for PRP + high HA group (PRP 1 mL + HA 0.6 mL) with five donors. After collecting all of the samples on Day 5, the remaining gels were observed with Giemsa stain. Statistical analyses were performed using paired t tests to compare the PRP and HA groups at each time point, and a one-way analysis of variance (one-way ANOVA) with Tukey post hoc tests was used to compare the PRP, PRP + HA, and PRP + high HA groups. RESULTS: The TGF-ß1 concentrations in the PRP and PRP + HA were 24.3 ± 7.2 µg/mL and 22.4 ± 1.8 µg/mL (p = 0.689) on Day 0, 17.2 ± 13.9 µg/mL and 25.4 ± 7.1 µg/mL (p = 0.331) on Day 3, and 12.7 ± 10.5 µg/mL and 33.7 ± 8.3 µg/mL (p = 0.034) on Day 5. The TGF-ß1 concentrations on Day 5 were 24.1 ± 5.2 µg/mL (PRP group), 28.3 ± 2.4 µg/mL (PRP + HA), and 31.9 ± 4.8 µg/mL (PRP + high HA; one-way ANOVA: p = 0.003; post hoc PRP vs. PRP + HA: p = 0.016). The PDGF-AA concentrations in the PRP and PRP + HA groups were 2.30 ± 1.21 µg/mL and 2.32 ± 0.79 µg/mL (p = 0.931) on Day 0, 2.03 ± 0.53 µg/mL and 2.13 ± 0.73 µg/mL (p = 0.500) on Day 3, and 1.51 ± 0.40 µg/mL and 2.00 ± 0.52 µg/mL (p = 0.003) on Day 5. The PDGF-AA concentrations were 1.48 ± 0.46 µg/mL (PRP group), 1.94 ± 0.57 µg/mL (PRP + HA), and 2.69 ± 0.70 µg/mL (PRP + high HA; one-way ANOVA: p = 0.0002; PRP vs. PRP + high HA: p = 0.002; PRP + HA vs. PRP + high HA: p = 0.011) on Day 5. The PRP showed larger coagulated masses than the PRP + HA. The high concentration HA group had the smallest coagulated mass of all of the group. CONCLUSION: The levels of growth factors released by PRP on Day 5 were increased by the addition of HA. A mixture of PRP and HA may be a more effective therapy than PRP or HA alone for osteoarthritis and tendinopathy.

Decreased DNA methylation in the promoter region of the WNT5A and GDNF genes may promote the osteogenicity of mesenchymal stem cells from patients with ossified spinal ligaments.

Mesenchymal stem cells (MSCs) isolated from spinal ligaments with ectopic ossification have a propensity toward the osteogenic lineage. To explore epigenetic control of the osteogenic features of MSCs, we treated MSCs obtained from the spinal ligaments of ossification of yellow ligament (OYL) patients and non-OYL patients with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5AdC). We compared the non-OYL groups (untreated and treated with 5AdC) with the OYL groups (untreated and treated with 5AdC) by genome-wide microarray analysis. Next, we used methylated DNA immunoprecipitation combined with quantitative real-time PCR to assess gene methylation. Ninety-eight genes showed expression significantly increased by 5AdC treatment in MSCs from non-OYL patients but not from OYL patients. In contrast, only two genes, GDNF and WNT5A, showed significantly higher expression in OYL MSCs compared with non-OYL MSCs without 5AdC treatment. Both genes were hypermethylated in non-OYL MSCs but not in OYL MSCs. Small interfering RNA targeted to each gene decreased expression of the target gene and also several osteogenic genes. Both small interfering RNAs also suppressed the activity of alkaline phosphatase, a typical marker of osteogenesis. These results suggest that the osteogenic features of MSCs from OYL patients are promoted by unmethylated WNT5A and GDNF genes.

Identification of a novel COL2A1 mutation (c.1744G>A) in a Japanese family: a case report.

INTRODUCTION: Mutations in the gene encoding the type II collagen gene (COL2A1) have been found to affect the entire skeletal system. Recently, inheritable skeletal dysplasia caused by novel COL2A1 mutations has been linked to an inherited disease of the hip joint that neither involves the entire skeletal system nor is characterized by the presence of concomitant disorders, such as spinal or ocular abnormalities. CASE PRESENTATION: A 27-year-old Japanese woman previously diagnosed with avasucular necrosis (AVN) of the femoral head on the basis of radiological findings was referred to the study site for surgical management of a painful hip joint. She had no history of disease but suffered from bilateral hip joint lesions. Analysis of her pedigree revealed that bilateral hip joint lesions affected more than three generations of her family. Based on these findings, haplotype analysis of her and her family members was performed by examining select candidate genes from the critical interval for epiphyseal dysplasia of the femoral head on 12q13 and sequencing the promoter and exonic regions of COL2A1. CONCLUSION: A novel COL2A1 mutation (c.1744G>A) was identified within one Japanese family.

8-Methyltryptanthrin-induced differentiation of P19CL6 embryonal carcinoma cells into spontaneously beating cardiomyocyte-like cells.

Enhancement of cardiac differentiation is critical to stem cell transplantation therapy for severe ischemic heart disease. The aim of this study was to investigate whether several derivatives of tryptanthrin (1), extracted from the medicinal plant Polygonum tinctorium, induce the differentiation of P19CL6 mouse embryonal carcinoma cells into beating cardiomyocyte-like cells. P19CL6 cells were cultured in α-MEM supplemented with 10% FBS including a test compound or vehicle. Drug-induced differentiation was assessed by measuring the number of beating and nonbeating aggregates and the area of beating aggregates, and the expression of genes involved in cardiac differentiation was evaluated by real-time PCR. A 1 µM concentration of 8-methyltryptanthrin (2) induced the differentiation of P19CL6 cells into cardiomyocyte-like cells to a significantly greater degree than 1% dimethyl sulfoxide (DMSO), a conventional differentiation inducer of P19CL6 cells. Furthermore, 2 strongly increased both the number and the area of spontaneously beating aggregates in comparison with DMSO. Two distinct genes of the calcium channel family, Cav1.2 and Cav3.1, underlying cardiac automaticity were significantly expressed in the presence of 2. Gap junction genes GJA1 and GJA5 contributing to the synchronized contraction of the myocardium were also induced significantly by 2. These results suggest that 2 successfully differentiated P19CL6 cells into spontaneously beating cardiomyocyte-like cells by activating the gene expression of pacemaker channels and gap junctions.

Recent advances in research on human aortic valve calcification.

Aortic valve calcification can aggravate aortic stenoses, and it is a significant cause of sudden cardiac death. The increasing number of patients with age-related calcification is a problem in developed nations. However, the only treatment option currently available is highly invasive cardiac valve replacement. Therefore, clarification of the etiology of calcification is urgently needed to develop drug therapies and prevention methods. Recent studies have revealed that calcification is not a simple sedimentation of a mineral through a physicochemical phenomenon; various factors dynamically contribute to the mechanism. Further, we are finally beginning to understand the cellular origins of calcification, which had been unclear for a long time. Based on these findings that help to clarify potential drug targets, we expect to establish drug therapies that reduce the stress on patients. In this paper, I introduce the latest findings on cells that are most likely to contribute to calcification and on calcification-related factors that may lead to the development of drug therapies.

Osteogenic lineage commitment of mesenchymal stem cells from patients with ossification of the posterior longitudinal ligament.

Ectopic bone formation is thought to be responsible for ossification of the posterior longitudinal ligament of the spine (OPLL). Mesenchymal stem cells (MSCs) were isolated from spinal ligaments and shown to play a key role in the process of ectopic ossification. The purpose of this study was to explore the capacity of these MSCs to undergo lineage commitment and to assess the gene expression changes between these committed and uncommitted MSCs between OPLL and non-OPLL patients. Spinal ligament-derived cells were obtained from OPLL patients or patients with cervical spondylotic myelopathy (non-ossified) for comparison (n=8 in each group). MSCs from the two patient cohorts were evaluated for changes in colony forming ability; osteogenic, adipogenic and chondrogenic differentiation potential; and changes in gene expression following induction with lineage-specific conditions. We show that the osteogenic differentiation potential was significantly higher in MSCs from OPLL patients than in those from non-OPLL patients. In addition, alkaline phosphatase activity and several osteogenic-related genes expressions (bone morphogenetic protein 2, runt-related transcription factor 2 and alkaline phosphatase) were significantly higher in the OPLL group than in the non-OPLL group. However, single cell cloning efficiency, adipogenic and chondrogenic differentiation, and the expression of adipogenic and chondrogenic-related genes were equivalent between MSCs harvested from OPLL and non-OPLL patient samples. These findings suggest an increase in the osteogenic differentiation potential of MSCs from OPLL patients and that this propensity toward the osteogenic lineage may be a causal factor in the ossification in these ligaments.

CD34-negative mesenchymal stem-like cells may act as the cellular origin of human aortic valve calcification.

Although various osteogenic inducers contribute to the calcification of human aortic valve interstitial cells, the cellular origin of calcification remains unclear. We immunohistochemically investigated the cellular origin of valve calcification using enzymatically isolated cells from both calcified and non-calcified human aortic valve specimens. CD73-, 90-, and 105-positive and CD45-negative mesenchymal stem-like cells (MSLCs) were isolated from both types of valve specimens using fluorescence-activated cell sorting. MSLCs were further sorted into CD34-negative and -positive cells. Compared with CD34-positive cells, CD34-negative MSLCs were significantly more sensitive to high inorganic phosphate (3.2 mM), calcifying easily in response. Furthermore, immunohistochemical staining showed that significantly higher numbers (~7-9-fold) of CD34-negative compared with CD34-positive MSLCs were localized in calcified aortic valve specimens obtained from calcified aortic stenosis patients. These results suggest that CD34-negative MSLCs are responsible for calcification of the aortic valve.

Immunohistochemical localization of mesenchymal stem cells in ossified human spinal ligaments.

Mesenchymal stem cells (MSCs) have been isolated from various tissues and used for elucidating the pathogenesis of numerous diseases. In our previous in vitro study, we showed the existence of MSCs in human spinal ligaments and hypothesized that these MSCs contributed to the pathogenesis of ossification of spinal ligaments. The purpose of this study was to use immunohistochemical techniques to analyze the localization of MSCs in ossified human spinal ligaments in situ. Ossified (OLF) or non-ossified ligamentum flavum (non-OLF) samples from the thoracic vertebra were obtained from patients who had undergone posterior spinal surgery. Serial sections were prepared from paraffin-embedded samples, and double immunofluorescence staining was performed using antibodies against markers for MSCs (CD73, CD90 and CD105), endothelial cells (CD31), pericytes (α-smooth muscle actin), and chondrocytes (S100). Immunolocalization of MSCs was observed in the perivascular area and collagenous matrix in spinal ligaments. Markers for MSCs and pericytes were co-expressed in the perivascular area. Compared with non-OLF, OLF had a large amount of neovascularization in the fragmented ligament matrix, and a high accumulation of MSCs around blood vessels. The prevalence of MSCs in OLF within collagenous matrix was significantly higher than that in non-OLF. Chondrocytes near the ossification front in OLF also presented expression of MSC markers. MSCs may contribute to the ectopic ossification process of OLF through endochondral ossification.

Post injury changes in the properties of mesenchymal stem cells derived from human anterior cruciate ligaments.

PURPOSE: The anterior cruciate ligament (ACL) rarely heals spontaneously after rupture. Mesenchymal stem cells (MSCs) contribute to healing in various tissues, therefore, they may also have a key role in healing after ACL rupture. The purpose of this study was to investigate the properties of MSCs in ruptured ACLs. METHODS: Human ACL samples were harvested from patients undergoing primary ACL reconstruction, and samples were classified by the number of days post rupture (phase I<21 days; phase II 21­56 days; phase III 57­139 days phase IV≥140 days). We evaluated the characteristics of MSCs, such as colony-forming capacity, differentiation potential and cell-surface markers. RESULTS: There was a tendency for high colony-forming capacity during phases I and II, which tended to decrease in phase III. Chondrogenic, adipogenic and osteogenic differentiation potential was maintained until phase II but decreased in phase III. Most surface-epitope expression was consistent from phase I to III: positive for CD44, CD73, CD90 and CD105; negative for CD11b, CD19, CD34, CD45 and human leukocyte antigen-D-related (HLA-DR). The presence of these surface markers proved the existence of MSCs in ruptured ACL tissue. CONCLUSIONS: Our results suggest that colony-forming and differentiation potential decrease over time. It is important to consider changes in properties of MSCs and use ACL tissue in the acute phase of rupture when biological manipulation is required.