Mariana Kiomy Osako

Angiotensin II accelerates osteoporosis by activating osteoclasts

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 (1×10−6 M) significantly increased tartrate‐resistant acid phosphatase (TRAP) ‐positive multinuclear osteoclasts. Of importance, Ang II significantly induced the expression of receptor activator of nf‐κb 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.—Shimizu, H., Nakagami, H., Osako, M. K., Hanayama, R., Kunugiza, Y., Kizawa, T., Tomita, T., Yoshikawa, H., Ogihara, T., Morishita, R. Angiotensin II accelerates osteoporosis by activating osteoclasts. FASEB J. 22, 2465–2475 (2008)

Estrogen inhibits vascular calcification via vascular RANKL system: Common mechanism of osteoporosis and vascular calcification

Rationale Arterial calcification and osteoporosis are associated in postmenopausal women. RANK (the receptor activator of nuclear factor &kgr;B), RANKL (RANK ligand), and osteoprotegerin are key proteins in bone metabolism and have been found at the site of aortic calcification. The role of these proteins in vasculature, as well as the contribution of estrogen to vascular calcification, is poorly understood. Objective To clarify the mechanism of RANKL system to vascular calcification in the context of estrogen deficiency. Methods and Results RANKL induced the calcification inducer bone morphogenetic protein-2 by human aortic endothelial cells (HAECs) and decreased the calcification inhibitor matrix Gla protein (MGP) in human aortic smooth muscle cells (HASMCs), as quantified by real-time PCR and Western blot analysis. RANKL also induced bone-related gene mRNA expression and calcium deposition (Alizarin red staining) followed by the osteogenic differentiation of HASMCs. Estrogen inhibited RANKL signaling in HAECs and HASMCs mainly through estrogen receptor &agr;. Apolipoprotein E–deficient mice fed with Western high-fat diet for 3 months presented atherosclerotic calcification (Oil red and Alizarin red staining) and osteoporosis (microcomputed tomographic analysis) after ovariectomy and increased expression of RANKL, RANK, and osteopontin in atherosclerotic lesion, as detected by in situ hybridization. Estrogen replacement inhibited osteoporosis and the bone morphogenetic protein osteogenic pathway in aorta by decreasing phosphorylation of smad-1/5/8 and increasing MGP mRNA expression. Conclusions RANKL contributes to vascular calcification by regulating bone morphogenetic protein-2 and MGP expression, as well as bone-related proteins, and is counteracted by estrogen in a receptor-dependent manner.Rationale: Arterial calcification and osteoporosis are associated in postmenopausal women. RANK (the receptor activator of nuclear factor κB), RANKL (RANK ligand), and osteoprotegerin are key proteins in bone metabolism and have been found at the site of aortic calcification. The role of these proteins in vasculature, as well as the contribution of estrogen to vascular calcification, is poorly understood. Objective: To clarify the mechanism of RANKL system to vascular calcification in the context of estrogen deficiency. Methods and Results: RANKL induced the calcification inducer bone morphogenetic protein-2 by human aortic endothelial cells (HAECs) and decreased the calcification inhibitor matrix Gla protein (MGP) in human aortic smooth muscle cells (HASMCs), as quantified by real-time PCR and Western blot analysis. RANKL also induced bone-related gene mRNA expression and calcium deposition (Alizarin red staining) followed by the osteogenic differentiation of HASMCs. Estrogen inhibited RANKL signaling in HAECs and HASMCs mainly through estrogen receptor α. Apolipoprotein E–deficient mice fed with Western high-fat diet for 3 months presented atherosclerotic calcification (Oil red and Alizarin red staining) and osteoporosis (microcomputed tomographic analysis) after ovariectomy and increased expression of RANKL, RANK, and osteopontin in atherosclerotic lesion, as detected by in situ hybridization. Estrogen replacement inhibited osteoporosis and the bone morphogenetic protein osteogenic pathway in aorta by decreasing phosphorylation of smad-1/5/8 and increasing MGP mRNA expression. Conclusions: RANKL contributes to vascular calcification by regulating bone morphogenetic protein-2 and MGP expression, as well as bone-related proteins, and is counteracted by estrogen in a receptor-dependent manner. # Novelty and Significance {#article-title-42}

Prevention of osteoporosis by angiotensin-converting enzyme inhibitor in spontaneous hypertensive rats

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.

Vascular protective effects of ezetimibe in ApoE-deficient mice

One of the major risk factors for ischemic disease is hyperlipidemia, which is mainly regulated by endogenous cholesterol synthesis in the liver and dietary absorption in the small intestine. In this study, we evaluated the vascular protective effects of a potent cholesterol absorption inhibitor, ezetimibe. ApoE-deficient mice were fed a chow or high-fat diet with or without ezetimibe (5mg/(kgday)) for 3 months. Co-treatment with ezetimibe significantly reduced plasma cholesterol (by 76%; from 1592 to 381mg/dL) and LDL cholesterol (by 78%; from 1515 to 319mg/dL), and increased HDL cholesterol (by 187%; from 16 to 46mg/dL) in high-fat diet mice. Consistently, a marked inhibitory effect of ezetimibe on the development of lipid-rich plaque was observed, as assessed by oil red O staining. Of importance, treatment with ezetimibe significantly improved endothelial dysfunction as assessed by the vasodilator response to acetylcholine, accompanied by inhibition of interleukin-6 mRNA and an increase in endothelial nitric oxide synthase (eNOS) mRNA in the aorta. Ezetimibe also suppressed oxidative stress and the ubiquitination-proteasome system in the aorta. Although changes in body weight and several tissue weights were similar in the groups with and without ezetimibe administration, only liver weight was significantly decreased in the ezetimibe-treated group. Interestingly, ezetimibe markedly inhibited lipid accumulation in the liver. Furthermore, ezetimibe increased the mRNA expression of 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) synthase as a counteraction in the liver, but not in the aorta. Overall, ezetimibe significantly prevented atherosclerosis through not only lipid-lowering effects, but also other direct and/or indirect vascular protective actions in ApoE-deficient mice.

Cross-Talk of Receptor Activator of Nuclear Factor-κB Ligand Signaling With Renin–Angiotensin System in Vascular Calcification

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-&kgr;B 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 RANKL activation. In addition, RANKL activated renin–angiotensin II system, especially angiotensin II–converting enzyme and Ang II type 1 receptor. Cross-talk between renin–angiotensin II system and RANKL system might work as a vicious cycle to promote vascular calcification in atherosclerosis. Further studies to inhibit renin–angiotensin II system and RANKL may provide new therapeutic options to prevent and regress vascular calcification.

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.

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

Significance Although a high-serum osteoprotegerin (OPG) level is associated with an unfavorable outcome in ischemic stroke, it is unclear whether OPG is a culprit or an innocent bystander. Here we show that the deletion of OPG and enhanced RANKL/RANK signaling contribute to the reduction of infarct volume with lower brain edema, whereas infarct volume is 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 ischemic brain and were expressed in activated microglia and macrophages. Enhanced RANKL/RANK signaling showed neuroprotective effects with reduced expression in inflammatory cytokines in LPS-stimulated neuron-glia mixed culture. Our findings propose anti-inflammatory roles for RANKL/RANK signaling in ischemic brains. 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.

Effect of an antimicrobial agent on atherosclerotic plaques: assessment of metalloproteinase activity by molecular imaging.

OBJECTIVES Technetium-99m-labeled matrix metalloproteinase inhibitor (MPI) was used for the noninvasive assessment of matrix metalloproteinase (MMP) activity in atherosclerotic plaques after minocycline (MC) intervention. BACKGROUND MMP activity in atherosclerosis contributes to plaque instability. Some antimicrobial agents may attenuate MMP activity. METHODS Atherosclerotic lesions were produced in 38 rabbits with a high cholesterol diet for 4 months; 5 groups of rabbits, in the fourth month, received fluvastatin (FS) (n = 6), low-dose MC (n = 7), high-dose MC (n = 7), a combination of low-dose MC and FS (n = 6), or no intervention (n = 12); 8 unmanipulated rabbits were used as disease controls. Micro-single-photon emission computed tomography imaging was performed in all animals after intravenous MPI administration, followed by pathologic characterization of the aorta. A cell culture study evaluated the effect of MC on MMP production by activated human monocytes. RESULTS MPI uptake was visualized best in untreated atherosclerotic animals (percent injected dose per gram MPI uptake, 0.11 +/- 0.04%). MPI uptake was reduced in the FS (0.06 +/- 0.01%; p < 0.0001), high-dose MC (0.05 +/- 0.01%; p < 0.0001), and MC-FS (0.05 +/- 0.005%; p < 0.0001) groups. Low-dose MC did not resolve MPI uptake significantly (0.08 +/- 0.02; p = 0.167). There was no incremental benefit of the combination of MC and FS. MPI uptake showed a significant correlation with plaque MMP-2, and MMP-9 activity. MMP-9 release from tumor necrosis factor-alpha-activated macrophages was abrogated by incubation with MC. CONCLUSIONS Molecular imaging of MMP activity in atherosclerotic plaque allows for the study of the efficacy of therapeutic interventions. MC administration resulted in substantial reduction in plaque MMP activity and histologically verified plaque stabilization. MC was found to be equally effective as FS.

Decrease in blood pressure and regression of cardiovascular complications by angiotensin II vaccine in mice.

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.

Modification of decoy oligodeoxynucleotides to achieve the stability and therapeutic efficacy.

The decoy oligodeoxynucleotide (ODN) serves as a decoy sequence for a target transcription factor, then inhibiting its binding to the authentic sequence at the promoter, and consequently hinders the gene expression. ODNs should be properly up taken by the cell and tissue, be specific for one nuclear factor, and be stable against intracellular and serum nucleases. Since phosphodiester oligos are easily degradated by nucleases, chemical modification such as phosphorothioation, and structural modification by ligation of the extremities of two single-strand phosphodiester sequence resulting in a dumbbell shaped ODN (Ribbon-type decoy ODN) are performed to increase the stability of ODNs. In combination, phosphorothioation of specific regions in Ribbon-type decoy has further increased its stability, and the introduction of saturated hydrocarbon polymer spacer linking the two double strands also improved the stability and reduced the production cost. The cellular delivery has been optimized by using the biodegradable polymer D,L-lactide-co-glycolide (PLGA) as a carrier to ODN. The nuclear factor-kappa B (NF-κB) is a convergent point of different pathways, with main role in many pathologies, and poses as an ideal target for decoy ODN strategy. Following this we have designed ODN targeting NF-κB, and in this review, we are going to discuss the various modification performed in an attempt to improve the ODN efficacy, and some promising pre-clinical data and clinical trials using NF-κB decoy ODN.