OPG/RANKL/RANK axis is a critical inflammatory signaling system in ischemic brain in mice.

Osteoprotegerin (OPG) is a soluble secreted protein and a decoy receptor, which inhibits a receptor activator of nuclear factor κB (NF-κB) ligand (RANKL)/the receptor activator of NF-κB (RANK) signaling. Recent clinical studies have shown that a high-serum-OPG level is associated with unfavorable outcome in ischemic stroke, but it is unclear whether OPG is a culprit or an innocent bystander. Here we demonstrate that enhanced RANKL/RANK signaling in OPG(-/-) mice or recombinant RANKL-treated mice contributed to the reduction of infarct volume and brain edema via reduced postischemic inflammation. On the contrary, infarct volume was increased by reduced RANKL/RANK signaling in OPG(-/-) mice and WT mice treated with anti-RANKL neutralizing antibody. OPG, RANKL, and RANK mRNA were increased in the acute stage and were expressed in activated microglia and macrophages. Although enhanced RANKL/RANK signaling had no effects in glutamate, CoCl2, or H2O2-stimulated neuronal culture, enhanced RANKL/RANK signaling showed neuroprotective effects with reduced expression in inflammatory cytokines in LPS-stimulated neuron-glia mixed culture, suggesting that RANKL/RANK signaling can attenuate inflammation through a Toll-like receptor signaling pathway in microglia. Our findings propose that increased OPG could be a causal factor of reducing RANKL/RANK signaling and increasing postischemic inflammation. Thus, the OPG/RANKL/RANK axis plays critical roles in controlling inflammation in ischemic brains.

Therapeutic vaccine against DPP4 improves glucose metabolism in mice.

The increasing prevalence of type 2 diabetes mellitus is associated with a significant economic burden. We developed a dipeptidyl peptidase 4 (DPP4)-targeted immune therapy to increase glucagon-like peptide 1 hormone levels and improve insulin sensitivity for the prevention and treatment of type 2 diabetes mellitus. Immunization with the DPP4 vaccine in C57BL/6J mice successfully increased DPP4 titer, inhibited plasma DPP4 activity, and induced an increase in the plasma glucagon-like peptide 1 level. Moreover, this elevated titer was sustained for 3 mo. In mice fed a high-fat diet, DPP4 vaccination resulted in improved postprandial glucose excursions and insulin sensitivity and, in the diabetic KK-A(y) and db/db mice strains, DPP4 vaccination significantly reduced glucose excursions and increased both plasma insulin and pancreatic insulin content. Importantly, T cells were not activated following challenge with DPP4 itself, which suggests that this vaccine does not induce cell-mediated autoimmunity. Additionally, no significant immune-mediated damage was detected in cells and tissues where DPP4 is expressed. Thus, this DPP4 vaccine may provide a therapeutic alternative for patients with diabetes.

Systemic administration of ribbon-type decoy oligodeoxynucleotide against nuclear factor κB and ets prevents abdominal aortic aneurysm in rat model.

Currently, there is no effective clinical treatment to prevent abdominal aortic aneurysm (AAA). To develop a novel therapeutic approach, we modified decoy oligodeoxynucleotide (ODN) against nuclear factor κB (NFκB) and ets, to a ribbon-shaped circular structure without chemical modification, to increase its resistance to endonuclease for systemic administration. Intraperitoneal administration of ribbon-type decoy ODNs (R-ODNs) was performed in an elastase-induced rat AAA model. Fluorescent isothiocyanate (FITC)-labeled R-ODNs could be detected in macrophages migrating into the aneurysm wall, and NFκB and ets activity were simultaneously inhibited by chimeric R-ODN. Treatment with chimeric R-ODN significantly inhibited aortic dilatation, whereas conventional phosphorothioate decoy ODN failed to prevent aneurysm formation. Significant preservation of elastic fibers was observed with chimeric R-ODN, accompanied by a reduction of secretion of several proteases from macrophages. Activation of matrix metalloproteinase (MMP)-9 and MMP-12, but not MMP-2, was suppressed in the aneurysm wall by chimeric R-ODN, whereas recruitment of macrophages was not inhibited. Treatment with chimeric R-ODN also inhibited the secretion of cathepsin B and K from macrophages. Overall, the present study demonstrated that systemic administration of chimeric R-ODNs prevented aneurysm formation in a rat model. Further modification of the decoy strategy would provide a means of less invasive molecular therapy for human AAA.

Inhibition of experimental abdominal aortic aneurysm progression by nifedipine.

Agents to inhibit the renin-angiotensin system have been reported to suppress the progression of abdominal aortic aneurysm (AAA). However, the effects of calcium channel blockers (CCBs) are still unclear in terms of the inhibition of the progression of AAA. Recently, several effects of CCBs beyond those associated with blood pressure lowering have attracted much interest. In this study, we examined the effects of nifedipine on AAA progression. AAA was induced in rats by transient aortic perfusion with elastase. Then, nifedipine (10 mg/kg/day) and saline (control) were administered to rats by osmotic mini-pump. At 2 and 4 weeks, the size of the AAA, blood pressure and heart rate were measured. Then, to further explore the mechanisms of the progression of AAA, we used human vascular smooth muscle cells (VSMCs). Especially, we focused on NF-kappaB and matrix metalloproteinase-9 (MMP-9). Treatment with nifedipine resulted in a significant inhibition of the progression of AAA such as aneurismal dilation at 14 and 28 days compared to the control (week 2: control, 2.98+/-0.71 mm; nifedipine, 2.37+/-0.64 mm; p<0.05 and week 4: control, 3.28+/-0.98 mm; nifedipine, 2.41+/-0.17 mm; p<0.05). Neither nifedipine nor saline changed blood pressure and heart rate, significantly. Nifedipine (1 microM) significantly suppressed angiotensin II-induced (10(-6) M) NF-kappaB activity in VSMCs by reporter assay (p<0.01). Furthermore, nifedipine (1 microM) inhibited MMP-9 protein expression and activity. Saline did not show such inhibitory effects. Taken together, these results indicated that nifedipine inhibits the progression of experimental AAA possibly through suppression of NF-kappaB and MMP-9 activity, leading to protective effects against AAA beyond those associated with blood pressure lowering.

Improvement of organ damage by a non-depressor dose of imidapril in diabetic spontaneously hypertensive rats.

Many clinical trials have demonstrated that angiotensin converting enzyme inhibitors have protective effects on organ damage, suggesting the importance of inhibition of the renin-angiotensin system. In this study, we investigated the effects of a non-depressor dose of imidapril on organ damage induced by diabetes and hypertension. Diabetes was induced by an intravenous injection of streptozotocin (STZ, 40 mg/kg) in 15-week-old male spontaneously hypertensive rats (SHR). Imidapril (2 mg/kg/day) or vehicle was given orally for 28 days, and then the heart weight, left ventricle mass (LVM), urinary albumin excretion (UAE) and endothelial function were examined, as well as the urinary NOx level and local hepatocyte growth factor (HGF) expression. There were no significant differences between the treated groups in systolic blood pressure and plasma parameters. On the other hand, UAE was significantly suppressed in the imidapril-treated group (450+/-44 mg/day) compared to the vehicle-treated group (963+/-182 mg/day) (p<0.01). Moreover, endothelial function assessed by dilative reaction to acetylcholine as well as cardiac hypertrophy assessed by both heart/body weight ratio and LVM were significantly improved in the imidapril-treated group (p<0.05 and p<0.01, respectively). The urinary NOx concentration and local HGF expression in vessel walls were also significantly increased in the imidapril-treated group (p<0.01). A non-depressor dose of imidapril showed protective effects against organ damage in diabetic SHR, which may be partially due to the increase of HGF and NO.

New concept of vascular calcification and metabolism.

Vascular calcification is recently considered as one of the major complications and an independent risk factor of cardiovascular diseases. Although vascular calcification was commonly regarded as a passive process of mineral adsorption or precipitation, it tends to be an active process associated with the expression of growth factors, matrix proteins, and other bone-related proteins. There are 2 main types of vascular calcification. Intimal calcification is found in atherosclerotic plaques and is associated with the vascular events such as myocardial infarction. Medial calcification is usually associated with age and chronic kidney disease patients, which leads to increased vascular stiffness and reduced vascular compliance. Interestingly, our vascular calcification model using ApoE deficient mice showed intima calcification at sites of atherosclerotic plaques under high fat diet with ovariectomy. Thus, lipid metabolism is one of the therapeutic targets to prevent intima calcification of aorta. Previously we reported that ezetimibe significantly prevented atherosclerosis through lipid-lowering effects in ApoE-deficient mice. Based on these findings, we speculate that ezetimibe might prevent aortic intima calcification, which may give us the benefits to decrease vascular events.

Combination therapy based on the angiotensin receptor blocker olmesartan for vascular protection in spontaneously hypertensive rats.

For hypertension, combination therapies are recommended to achieve a low target blood pressure. In this study, the efficacy of combination therapies for preventing organ damage was investigated in spontaneously hypertensive rats (SHR). Twenty-week-old male SHR were orally administered olmesartan (Olm) (5 mg/kg/day) for the first 4 weeks. Subsequently, rats were randomly divided into 5 groups and administered add-on drugs for another 4 weeks as follows: Olm+Olm (5 mg/kg/day), Olm+azelnidipine (Aze) (30 mg/kg/day), Olm+temocapril (Tem) (10 mg/kg/day), Olm+atenolol (Ate) (5 mg/kg/day), Olm+hydrochlorothiazide (HCTZ) (5 mg/kg/day). Blood pressure and heart rate were measured at weeks 0, 4 and 8 by the tail-cuff method. Heart and kidney weights were determined, and endothelial function was assessed by evaluating the dilator response to acetylcholine. In comparison to untreated control SHR, a significant reduction in systolic blood pressure was observed at weeks 4 and 8 in all groups (p<0.05), while heart rate was significantly reduced at week 8 in only the Olm+Aze and Olm+Ate groups (p<0.05). In all groups, heart but not kidney weight was significantly decreased (p<0.05), and endothelial function was significantly improved (p<0.05) compared to the control SHR. In the Olm+Olm, Olm+Tem and Olm+Aze groups, endothelial function was significantly improved as compared to the other treatment groups (p<0.05). Thus, when using an angiotensin receptor blocker as a first-line therapy, an antihypertensive in the form of an angiotensin-converting enzyme inhibitor, angiotensin receptor blocker, or calcium channel blocker, such as azelnidipine, should be used as a second-line drug to protect against vascular damage.