Elucidation of the inhibitory effect of (+)-hopeaphenol on polyinosinic-polycytidylic acid-induced innate immunity activation in human cerebral microvascular endothelial cells.

Drug development for regulating the innate immune system is important for the prevention and treatment of autoinflammatory and autoimmune diseases. In this context, we investigated the effect of resveratrol derivatives on the inflammatory reactions in the brain. Resveratrol, which can be found in Vitis plants in the form of oligomers, exhibits neuroprotective effects; however, its regulatory effects on innate immunity are still unclear. We examined the effects of (+)-hopeaphenol, a resveratrol tetramer, and its derivatives on the polyinosinic-polycytidylic acid (poly IC)-induced production of interferon (IFN)-ß and C-X-C motif chemokine 10 (CXCL10) in the cultured human cerebral microvascular endothelial cell line hCMEC/D3. (+)-Hopeaphenol (1-10 µM) inhibited the poly IC-induced production of not only CXCL10 but also retinoic acid-inducible gene-I in a dose-dependent manner and significantly reduced the poly IC-induced IFN-ß gene expression and protein release from hCMEC/D3 cells by inhibiting the phosphorylation of p65 but not that of the interferon regulatory transcription factor IRF3. A docking study indicated a high affinity of (+)-hopeaphenol for p65. These results suggest that (+)-hopeaphenol can regulate the innate immune system by inhibiting the poly IC/IFN-ß/CXCL10 signaling axis via suppression of the phosphorylation of the transcription factor NF-ĸB.

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.

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

In the original publication of the article, part of Fig. 1 was published incorrectly.

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.

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.

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.

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.

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.

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.

Cytosolic Ca2+-induced apoptosis in rat cardiomyocytes via mitochondrial NO-cGMP-protein kinase G pathway.

Previously, we showed that in adult rat cardiomyocytes, nitric oxide (NO) donors stimulate mitochondrial cGMP production, followed by cytochrome c release, independently of the mitochondrial permeable transition pore. We investigated whether mitochondrial cGMP-induced cytochrome c release from cardiac mitochondria is Ca(2+)-sensitive. Mitochondria and primary cultured cardiomyocytes were prepared from left ventricles of male Wistar rats. The cytosolic Ca(2+) concentration was adjusted with Ca(2+)-EGTA buffers. Cytochrome c released from mitochondria was measured by Western blotting. Cardiomyocyte apoptosis was assessed by Annexin V staining. Cytochrome c release from cardiac mitochondria was evoked by buffered Ca(2+) (1 µM); this was inhibited by NO-cGMP pathway inhibitors such as N(G)-monomethyl-l-arginine monoacetate (inhibitor of NO synthase), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (NO scavenger), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, NO-sensitive guanylyl cyclase inhibitor) and voltage-dependent anion channel (VDAC) inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, but not by cyclosporin A (mitochondrial permeable transition pore inhibitor). Furthermore, this release was significantly and dose dependently inhibited by 0.3-3 µM KT5823 (protein kinase G inhibitor). At the cellular level, intracellular perfusion of cardiomyocytes with buffered Ca(2+) (1 µM) also induced apoptosis, which was inhibited in the presence of ODQ. A membrane-permeable cGMP analog, 8-Br-cGMP, but not cGMP itself, mimicked buffered Ca(2+) actions in both cardiac mitochondria and cardiomyocytes. We further confirmed an increase in protein kinase G activity by adding cGMP in mitochondrial protein fraction. Our results suggest that mitochondrial NO-cGMP pathway-induced cytochrome c release from cardiac mitochondria, triggered by increased cytosolic Ca(2+), occurs through VDAC via the stimulation of an undiscovered mitochondrial protein kinase G.

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.

Mesenchymal stem cell isolation and characterization from human spinal ligaments.

Mesenchymal stem cells (MSCs) have a fibroblast-like morphology, multilineage potential, long-term viability and capacity for self-renewal. While several articles describe isolating MSCs from various human tissues, there are no reports of isolating MSCs from human spinal ligaments, and their localization in situ. If MSCs are found in human spinal ligaments, they could be used to investigate hypertrophy or ossification of spinal ligaments. To isolate and characterize MSCs from human spinal ligaments, spinal ligaments were harvested aseptically from eight patients during surgery for lumbar spinal canal stenosis and ossification of the posterior longitudinal ligament. After collagenase digestion, nucleated cells were seeded at an appropriate density to avoid colony-to-colony contact. Cells were cultured in osteogenic, adipogenic or chondrogenic media to evaluate their multilineage differentiation potential. Immunophenotypic analysis of cell surface markers was performed by flow cytometry. Spinal ligaments were processed for immunostaining using MSC-related antibodies. Cells from human spinal ligaments could be extensively expanded with limited senescence. They were able to differentiate into osteogenic, adipogenic or chondrogenic cells. Flow cytometry revealed that their phenotypic characteristics met the minimum criteria of MSCs. Immunohistochemistry revealed the localization of CD90-positive cells in the collagenous matrix of the ligament, and in adjacent small blood vessels. We isolated and expanded MSCs from human spinal ligaments and demonstrated localization of MSCs in spinal ligaments. These cells may play an indispensable role in elucidating the pathogenesis of numerous spinal diseases.

Treating to target matrix metalloproteinase 3 normalisation together with disease activity score below 2.6 yields better effects than each alone in rheumatoid arthritis patients: T-4 Study.

OBJECTIVES: To assess whether therapy to achieve both a disease activity score in 28 joints (DAS28) less than 2.6 and matrix metalloproteinase (MMP) 3 normalisation offers better outcomes than either target alone in early rheumatoid arthritis (RA) at 56 weeks: Treating to Twin Targets (T-4) Study. METHODS: 243 early RA patients were randomly allocated to one of four strategy groups: routine care (R group; n=62); DAS28-driven therapy (D group; n=60); MMP-3-driven therapy (M group; n=60); or both DAS28 and MMP-3-driven therapy group (twin; T group; n=61). Medication was started with sulfasalazine (1 g/day) in all intervention groups. Targets were DAS28 less than 2.6 for the D group, MMP-3 normalisation for the M group and both DAS28 less than 2.6 and MMP-3 normalisation for the T group. If the value in question did not fall below the previously measured level, medication was intensified, including methotrexate, other disease-modifying antirheumatic drugs and biological agents. Primary, secondary and outcome measures consisted of the proportions of patients showing clinical remission (DAS28 <2.6), radiographic non-progression (Δmodified total Sharp score ≤0.5), normal physical function (modified health assessment questionnaire score 0), or comprehensive disease remission defined as the combination of clinical remission, radiographic non-progression and normal physical function. RESULTS: Clinical remission at 56 weeks was achieved by more patients in the T group (56%) than in the R group (p<0.0005) or M group (p<0.0005). CONCLUSIONS: Results of the T-4 Study reveal that a twin target strategy can achieve a high clinical remission rate in early RA.

[Bone and calcium update; diagnosis and therapy of bone metabolism disease update. Molecular mechanism in cardiac valve calcification].

When cardiac valve stenosis is accompanied by calcification, symptoms and prognosis become much worse and may cause sudden cardiac death. The prevalence of this disease has increased with the rapidly aging in Japanese society. It has recently been revealed that several genes which relate to physiological ossification and calcification play important roles in this process. To find a suitable target for medical treatment, the molecular mechanism for calcification of cardiac valves should be elucidated in detail. In this review, we summarize the current knowledge on the pathology and molecular mechanism for ectopic calcification of the cardiac valve.

Genetic differences in the osteogenic differentiation potency according to the classification of ossification of the posterior longitudinal ligament of the cervical spine.

STUDY DESIGN: We categorized the four types of ossification of the posterior longitudinal ligament (OPLL) of the cervical spine into two groups. We biochemically investigated the genetic differences in the osteogenic differentiation potency between the two groups. OBJECTIVE: To investigate the genetic differences in the osteogenic differentiation potency according to the OPLL classification. SUMMARY OF BACKGROUND DATA: Clinical studies on OPLL have revealed that the risk of progression of the ossification area is greatest for continuous and mixed type OPLL. However, until now, these four types of OPLL have been studied as a single condition. METHODS: We categorized the four types of OPLL into the OPLL continuous (continuous or mixed type) and OPLL segmental groups (segmental or circumscribed type). Paraspinal ligaments were aseptically obtained from OPLL patients during surgery. The fibroblast-like cells that migrated from the explants were used for experiments. The cells were placed in a 60-mm culture dishes for total ribonucleic acid preparation and 12 well microplates for alkaline phosphatase (ALP) activity staining. After cultures reached confluence, the cells were cultured in osteogenic medium. The messenger ribonucleic acid expression of bone morphogenetic protein-2 (BMP-2), osterix, tumor necrosis factor-α-stimulated gene-6, and ALP was analyzed by quantitative real time-polymerase chain reaction. Osteogenic differentiation of fibroblast-like cells was determined by histochemically detecting ALP production. RESULTS: After osteogenic induction, BMP-2 expression increased in the OPLL continuous and segmental groups. Osterix expression increased in the OPLL continuous group only. Tumor necrosis factor-α-stimulated gene-6 expression was suppressed in the OPLL continuous and segmental groups. ALP expression as well as ALP activity staining was higher in the OPLL continuous group than in the OPLL segmental group. CONCLUSION.: The study revealed genetic differences in the osteogenic differentiation potency between the OPLL continuous and segmental groups. We propose to distinguish OPLL continuous group from segmental group in biochemical studies on OPLL.