Recent clinical studies suggest that several antihypertensive drugs, especially angiotensin-converting enzyme inhibitors, reduced bone fractures. To clarify the relationship between hypertension and osteoporosis, we focused on the role of angiotensin II (Ang II) on bone metabolism. In bone marrow-derived mononuclear cells, Ang II (1x10(-6) M) significantly increased tartrate-resistant acid phosphatase (TRAP) -positive multinuclear osteoclasts. Of importance, Ang II significantly induced the expression of receptor activator of NF-kappaB ligand (RANKL) in osteoblasts, leading to the activation of osteoclasts, whereas these effects were completely blocked by an Ang II type 1 receptor blockade (olmesartan) and mitogen-activated protein kinase kinase inhibitors. In a rat ovariectomy model of estrogen deficiency, administration of Ang II (200 ng/kg/min) accelerated the increase in TRAP activity, accompanied by a significant decrease in bone density and an increase in urinary deoxypyridinoline. In hypertensive rats, treatment with olmesartan attenuated the ovariectomy-induced decrease in bone density and increase in TRAP activity and urinary deoxypyridinoline. Furthermore, in wild-type mice ovariectomy with five-sixths nephrectomy decreased bone volume by microcomputed tomography, whereas these change was not detect in Ang II type 1a receptor-deficient mice. Overall, Ang II accelerates osteoporosis by activating osteoclasts via RANKL induction. Blockade of Ang II might become a novel therapeutic approach to prevent osteoporosis in hypertensive patients.

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, CoCl 2 , or H 2 O 2 -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.cerebral ischemia | neuroprotection | immune cells

Key Words: RANKL Ⅲ vascular calcification Ⅲ BMP-2 Ⅲ MGP Ⅲ estrogen V ascular calcification is one of the major complications of cardiovascular disease and an independent risk factor for myocardial infarction and cardiac death, 1 among other outcomes. Arterial calcification is also associated with osteoporosis, especially in postmenopausal women. 2 Recent evidence strongly suggests the biological linkages on both disease mechanisms based on the presence of bone-related proteins 3 and bone-related cells at the site of calcification. 4 Accordingly, a triad of key proteins in bone metabolism has emerged as new players in cardiovascular diseases: RANK (the receptor activator of nuclear factor B), the RANK ligand (RANKL), and osteoprotegerin (OPG).In bone, RANKL and OPG are expressed in osteoblasts, and the receptor RANK is expressed in osteoclast cells. The major role of RANKL in bone is the stimulation of osteoclast activity 5 and inhibition of osteoclast apoptosis. 6 RANKL binds not only to RANK but also to OPG,7,8 which is secreted as a soluble protein that lacks transmembrane and cytoplasmic domains. Because of this peculiarity of a decoy receptor, the major biological action described for OPG has been through the binding to RANKL and consequent inhibition of RANK stimulation, then decreasing the osteoclast differentiation and activity in bone. 7,8 OPG-deficient mice develop severe osteoporosis resulting from marked increase in osteoclast activation, and interestingly two-thirds of the animals have profound vascular medial calcification. 9 Similar to OPG-deficient mouse phenotype, postmenopausal women also have high incidence of osteoporosis and vascular calcification, 10 which suggests that RANK-RANKL system may work not only in osteoporosis but also in arterial calcification.

Vaccines have been recently developed to treat various diseases such as cancer, rheumatoid arthritis and Alzheimer’s disease in addition to infectious diseases. However, before use in the clinical setting, vaccines targeting self-antigens must be demonstrated to be effective and safe, evoking an adequate humoral immune response from B cells while avoiding T cell activation in response to self. Although the vaccine targeting angiotensin II (Ang II) is efficient in rodents and humans, little is known regarding the immunological activation and safety of the vaccine. In this study, we evaluated the efficiency and safety of an Ang II peptide vaccine in mice. Immunization with Ang II conjugated to keyhole limpet hemocyanin (KLH) successfully induced the production of anti-Ang II antibody, which blocked Ang II signaling in human aortic smooth muscle cells. However, Ang II itself did not activate T cells, as assessed by the proliferation and lymphokine production of T cells in immunized mice, whereas KLH activated T cells. In an Ang II-infused model, the non-immunized mice showed high blood pressure (BP), whereas the immunized mice (Ang II-KLH) showed a significant decrease in systolic BP, accompanied by significant reductions in cardiac hypertrophy and fibrosis. Importantly, anti-Ang II antibody titer was not elevated even after the administration of large amounts of Ang II, indicating that Ang II itself boosted antibody production, most likely due to less activation of T cells. In addition, no accumulation of inflammatory cells was observed in immunized mice, because endogenous Ang II would not activate T cells after immunization with Ang II-KLH. Taken together, these data indicate that vaccines targeting Ang II might be effective to decrease high BP and prevent cardiovascular complications without severe side effects.

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 glucagonlike 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.

A recent analysis of clinical studies suggests that angiotensin-converting enzyme (ACE) inhibitors might reduce bone fractures. In this study, we examined whether an ACE inhibitor might attenuate osteoporosis in a hypertensive rat model. In spontaneous hypertensive rats (SHRs), estrogen deficiency induced by ovariectomy (OVX) resulted in a significant increase in osteoclast activation as assessed by the tartrate-resistant acid phosphatase (TRAP) activity in the tibia, accompanied by a significant decrease in bone density evaluated by dual-energy X-ray absorptiometry and an increase in urinary deoxypyridinoline. Treatment with an ACE inhibitor, imidapril, attenuated OVX-induced decrease in bone density and increase in TRAP activity and urinary deoxypyridinoline. As ACE inhibitors possess the effects of blockade of the renin-angiotensin system (RAS) and activation of the bradykinin-nitric oxide pathway, we examined the contribution of both pathways in an OVX-induced osteoporosis model. Administration of nitro-L-arginine methylester (L-NAME) did not alter TRAP activity, urinary deoxypyridinoline or bone density, whereas the administration of a subpressor dose of angiotensin II accelerated the increase in TRAP activity in the tibia, accompanied by a significant decrease in bone density and an increase in urinary deoxypyridinoline. Thus, ACE inhibitors prevented osteoporosis, probably because of the inhibition of RAS, but not of nitric oxide. Overall, ACE inhibitors attenuated osteoporosis in a hypertensive rat model through the blockade of RAS.

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V ascular calcification is highly correlated with cardiovascular mortality and associated with hypertension.1 Intima arterial calcification occurs in conjunction with atherosclerosis, and media calcification is usually present with systemic mineral imbalance caused by chronic renal disease. Although both types are pathologically distinct, there is an association between the molecular mechanism regulating vascular calcification in intima and media. We and others have described the involvement of receptor activator of nuclear factor κB ligand (RANKL) system with bone morphogenetic protein (BMP)-2 and BMP-4 axis in the development of intima and media calcification, respectively. 2,3RANKL system consists of a triad of proteins: the membrane-bound RANK, its ligand RANKL, and the decoy receptor osteoprotegerin (OPG). This system occurs in immune system, 4,5 mammary glands development, 6 and has been studied extensively in bone metabolism, where osteoblasts express RANKL and OPG, which regulate osteoclast differentiation via RANK activation. 7,8 The first insight about the importance of this system in vasculature came from studies in OPG −/− mice that show early onset of osteoporosis and severe media calcification. 9 The unopposed RANKL signaling present in OPG −/− mice was then related to the development of vascular calcification. Recently, we have described that RANKL plays an important role in aortic calcification under estrogen deficiency by acting on 2 important molecules of the calcification process: the inducer of calcification, BMP-2, and the inhibitor, matrix Gla protein. Objective-Vascular calcification is accelerated by hypertension and also contributes to hypertension; however, it is an enigma why hypertension and vascular calcification are a vicious spiral. The present study elucidates the cross-talk between renin-angiotensin II system and receptor activator of nuclear factor-κB ligand (RANKL) system in vascular calcification. Approach and Results-Angiotensin (Ang) II (10 −7 mol/L) significantly increased calcium deposition as assessed by Alizarin Red staining, associated with a significant increase in the expression of RANKL, RANK, and bone-related genes, such as cbfa1 and msx2, in human aortic vascular smooth muscle cells. Infusion of Ang II (100 ng/kg per minute) in ovariectomized ApoE −/− mice under high-fat diet significantly increased the expression of RANKL system and calcification in vivo, whereas administration of Ang II receptor blocker (olmesartan, 3 mg/kg per day) decreased the calcification and bone markers' expression. In addition, male OPG −/− mice showed a significant increase in vascular calcification followed by Ang II infusion as compared with wild type. Conversely, RANKL significantly increased Ang II type 1 receptor and angiotensin II-converting enzyme expression in vascular smooth muscle cells via extracellular signal-regulated protein kinase phosphorylation.Conclusions-The present study demonstrated that Ang II significantly induced vascular calcification in vitro and in vivo through...