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Objective: Wingless/integrase 5a（Wnt5a） pathway is known to regulate the osteogenesis. In this study, we aimed to clarify the role of Wnt5a pathway in aortic valve ectopic calcification. Methods: Human aortic valve interstitial cells（HAVICs） were obtained from calcified aortic valves of patients with calcific aortic valve stenosis（CAVS）. HAVICs were separated to CD34-negative and -positive cells by flow cytometry. We measured real-time PCR, alkaline phosphatase（ALP） activity, Alizarin Red S staining as an index of calcification. Immunohistochemical staining was performed to confirm the distribution of Wnt5a on calcified and normal aortic valves. Results: HAVICs, especially, CD34-negative cells are highly sensitive to tumor necrosis factor-α（ TNF-α, 30 ng/mL）, and which accelerated the Wnt5a gene expression. Wnt5a antagonist, box5, did not down-regulate HAVIC calcification induced by TNF-α. Further, Wnt5a agonist, foxy5, did not accelerated HAVIC calcification induced by TNF-α. There was no significant difference in the TNF-α-induced acceleration of ALP activity between the cells treated with foxy5 and box5. Furthermore, the proportion of Wnt5a positive cells was no difference between calcified and normal valves. Conclusions: We confirmed that Wnt5a pathway does not regulate the TNF-α-induced aortic valve calcification in HAVICs obtained from CAVS patients.

Background Aortic valve stenosis （AVS） is the most common heart valve disease in elderly society. The effective treatment for AVS is surgical aortic valve replacement but with extremely invasive. Although transcatheter aortic valvereplacement also is performed for highly risk patients with minimally invasive, it has to resolve the problem ofbioprosthetic valve durability in future. However, there is no effective medical treatment established for AVS toinhibit acceleration of aortic valve calcification. Objectives We aimed to confirm whether the ascending aorta is the best location for AVS to innovate local and selective medicaltherapy or not. Methods and Results After anesthetized by isoflurane, the wild type rat was stablished on operating table. Opening chest to heart bymedian sternotomy approach, the catheter was inserted into left ventricular from apical for body perfusion. Weinjected saturated solution of amido black 10B （AB） into ascending aorta above sino-tubular junction about 0.5 cmfrom aortic valves without aortic dissection observed, and body perfusion was performed by saline containing heparinin order to prevent embolism happened in vascular. After 30 minutes, the heart with the ascending aorta wasextracted. We used optical microscope to check the existence of AB at around of aortic valves or not. Interestingly,AB was detected at around of aortic valves and valsalva sinus of the aorta and myocardium in all rats. Conclusions These results suggested that AB injected is delivered from the ascending aorta to aortic valves through vasavasorum. These data provide very important evidences for local medical therapy development for AVS patients.

Mesenchymal stem cells（ MSCs） from white adipose tissue（ WAT） have a higher osteogenic differentiation potential than those from brown adipose tissue （BAT）. The purpose of this study was to compare the neuronal differentiation potentials of these cells. In addition, to examine the applicability of these cells for autologous neuronal regeneration, the mouse hind limbs were assessed for neurological function after adipose tissue was harvested from the inguinal and interscapular regions. Adipose-MSCs were isolated from WAT of the inguinal subcutaneous regions and BAT of the interscapular regions in 6-week-old female C57BL/6J mice. The expression of cell-surface markers on the adipose-MSCs was examined by flowcytometry. The markers examined were CD90, Sca1, CD34, and CD45. Neuronal differentiation was assessed by immunocytochemistry using NeuN as a mature neuron marker. MSCs derived from WAT and BAT displayed similar immunophenotypes. Both cell populations differentiated into NeuNpositive cells（ 16.2% ± 1.0% of the WAT-MSCs and 14.7% ± 2.4% of the BAT-MSCs） in vitro. Harvesting the adipose tissue did not affect the neurological properties of the hind limbs. These findings indicate that both WAT-MSCs and BAT-MSCs may be attractive cells for autologous euronalregeneration.

Two sensitive enzymatic fluorometric assays have been developed for adenosine 3’,5’-cyclic monophosphate （cAMP） by Sugiyama et al. （Anal. Biochem. 1990） and for guanosine 3’,5’-cyclic monophosphate （cGMP） by Seya et al. （Anal. Biochem. 1998）. However, to make a new simultaneous comparison of two cyclic nucleotides, distinct measurement methods cause less reliability and longer measurement time. To overcome these problems, we developed a simultaneous measurement method for them. All adenosine nucleotides and GMP were enzymatically degraded using alkaline phosphatase and apyrase. The remaining GDP was converted to GTP by creatine kinase. Cyclic GMP and cAMP, absorbed into a Sep-Pak amino propyl cartridge, were eluted to separate from GTP, and then were simultaneously quantified using improved enzymatic fluorometric assay. The detection limits for cAMP and cGMP were 1 and 5 fmol, respectively. The total measurement time was about 10 h. Using this method, the basal cAMP and cGMP levels in rat aortic smooth muscle cells were confirmed to 4.1 and 0.042 pmol/mg protein, respectively. An adrenergic agonist, isoproterenol （1 μM） increased cAMP approximately 6 fold, while nitric oxide donor, S-nitroso-N-acetylpenicillamine （100 μM） increased cGMP approximately 230 fold. These results suggest that this simultaneous measurement of cGMP and cAMP can provide a more convenient assessment of them in biological samples., 弘前医学. 68, p.52-61. 2017