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We examined the relationship between structural changes of the aorta and pulse wave velocity (PWV), and the effects of antihypertensive treatments on PWV in N S S S S -nitro-L -arginine methyl ester (L-NAME)-treated rats.Twelve-week-old Wistar-Kyoto (WKY) rats were divided into the following groups, all of which received drug treatment in their drinking water: an untreated control group ( n = 36), an L-NAME-treated group (0.7 mg/ml) ( n = 32), an L-NAME and angiotensin converting enzyme (ACE) inhibitor (ACEI)-treated group (imidapril: 0.4 mg/ml) ( n = 8), and an L-NAME and hydralazine-treated group (0.2 mg/ml) ( n = 10). PWV was measured at the same blood pressure (BP) level as in the control group and the wall-to-lumen ratio of the thoracic aorta was evaluated in all groups. In the L-NAME group, PWV increased compared with the value in the control group, at the same time that BP was increasing. After the third day of treatment, PWV was higher in the L-NAME group than in the control group after adjusting BP to the control level, while the wall-to-lumen ratios were equal between the two groups. After the first week of treatment, not only the adjusted PWV, but also the wall-to-lumen ratios were greater in the L-NAME group than in the control group. With administration of antihypertensive agents, both PWV and the thickening of the aortic wall were reduced, but there was no significant difference between the ACEI and hydralazine-treated groups. In conclusion, in a rat model of nitric

Introduction Critical limb ischemia (CLI) is the end-stage of peripheral artery disease caused by atherosclerosis and inflammation. Despite advances in treatment of CLI, substantial number of patients with CLI suffer from severe pain, ulceration, and gangrene. Although the advent of stem cell-based therapy has opened a new avenue for the treatment of various diseases, accumulating evidence suggests current cell-based therapies are safe, but their efficacy is modest in CLI patients. Multilineage-differentiating stress enduring (Muse) cells were first reported by one of the authors and colleagues as endogenous pluripotent-like stem cells residing in the bone marrow, peripheral blood and connective tissue of many organs. Previous studies have demonstrated that systemic administration of exogenous Muse cells improves functional recovery after ischemic organ injury such as myocardial infarction. However, their therapeutic potential in CLI remains unclear. Objective The goal of this study is to elucidate the efficacy and safety of exogenous Muse cell transplantation with animal models of CLI. Methods Muse cells were isolated from human bone marrow-derived mesenchymal stem cells (MSCs) by fluorescence-activated cell sorting with anti-SSEA-3 antibody. To establish an animal model of CLI, 12–14-week-old male BALB/c mice were anesthetized and subjected to left femoral artery and vein resection. At 24 hours after establishment of hindlimb ischemia, mice were randomly divided into 5 groups and treated as follows: Group 1, intravenous injection of PBS as control group; group 2, intravenous injection of 3 × 104 SSEA3-negative MSCs (non-Muse MSCs); group 3, intravenous injection of 3 × 104 Muse cells; group 4, intramuscular injection of 3 × 104 Muse cells into ischemic limb; group 5, intramuscular injection of 2 × 105 mouse bone marrow-derived mononuclear cells (BM-MNCs) into ischemic limb (n=5 for each group). Hindlimb blood flow was evaluated by laser Doppler flowmetry for 14 days and expressed as the ratio of ischemic to non-ischemic hindlimb. Results We found that intramuscular injection of xenogeneic Muse cells without immunosuppression significantly improved the blood flow in the ischemic hindlimb compared to control at day 7 and 14 (0.67 vs. 0.52, p=0.0002 and 0.74 vs. 0.53, p<0.0001, respectively) similar to intramuscular injection of allogenic BM-MNCs. In addition, we also found that intravenous injection of xenogeneic Muse cells without immunosuppression significantly improved the blood flow in the ischemic hindlimb compared to control at day 7 and 14 (0.72, p<0.0001 and 0.67, p=0.004, respectively). In contrast, intravenous injection of xenogeneic non-Muse MSCs did not improve blood flow in the ischemic hindlimb. Conclusion Our result suggests that Muse cell-based regenerative therapy could be a novel, less invasive, cost-effective, and subsequently more effective treatment for CLI patients. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research (KAKENHI)

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Introduction Klotho-deficient mice have extensive vascular calcification. However, the mechanism involved has not been elucidated. We analyzed the endothelial cells of Klotho-deficient mice (Klotho-ECs) and found a novel functional role for the Klotho-protein in the maintenance of vascular homeostasis. Methods and Results Primary-cultured endothelial cells from the aorta of wild-type- (WT) and Klotho- deficient mice were loaded with fura-2 and stimulated with VEGF. In WT-ECs, intracellular Ca(2+) concentration ([Ca(2+)]i) elevated to the peak level, and it decreased to the basal level in +5 minutes. Whereas, in Klotho-ECs, after similar peak level to WT-ECs, the elevation of [Ca(2+)]i was sustained at ~35% of the peak level for more than 10 minutes. In Ca(2+) free medium, this sustained elevation was abolished. Immunostaining revealed that, in Klotho-ECs, VEGF-induced co-internalization of the Ca(2+) channel; TRPC-1, with VEGF-receptor-2 (VEGFR-2) was abolished, thereby, at 30 minutes after VEGF-stimulation, expression levels of TRPC-1 in the plasmamembrane was 2.2-fold higher than in WT-ECs. Klotho-protein constitutively bound to VEGFR-2 and TRPC-1, strengthened their association, and promoted their co-internalization. VEGF-induced activation of Rac-1 and reorganization of F-actin, which are involved in internalization, were impaired in Klotho-ECs, and restored by the replacement of Klotho-protein. VEGF-induced phosphorylation of VEGFR-2 and its downstream signaling molecules, including Src, Akt, PLCγ, and Caveolin1, was attenuated. As the biological consequence of sustained increase in [Ca(2+)]i, activation of calpain and caspase-3 followed by apoptosis was promoted in Klotho-ECs in vivo and vitro. Also, expression of VE-cadherin in the plasmamembrane was reduced and intimal hyperpermeability was observed in the aorta of Klotho-deficient mice, which presumably lead to exudation of the Ca(2+)/phosphor -rich serum into the vessel walls, followed by extensive vascular calcification. Conclusion Thus, Klotho-protein regulates ligand-induced Ca(2+) influx and subsequent biological functions involved in vascular calcification.