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Angiotensin II receptor blockers (ARBs) have a potent ability to inhibit oxidative stress and advanced glycation, in addition to their protective effects originated from blood pressure lowering and angiotensin II type 1 receptor (AT(1))-blockade. To obtain a pharmacological tool to dissect the mechanisms of ARBs' protective benefits in experimental stroke, we synthesized a novel ARB-derivative, R-147176, which is 6,700 times less potent than olmesartan in AT(1)-binding inhibition and therefore has a minimal antihypertensive effect, but retains marked inhibitory effects on oxidative stress and advanced glycation. We evaluated the effect of R-147176 (10-30 mg/kg per day), administered orally or intravenously, on brain infarct volume in transient thread occlusion and photothrombotic models in rats. The antioxidative and antiinflammatory properties were also investigated. R-147176 significantly reduced infarct volume, without influence on blood pressure, in both models. R-147176 significantly reduced the numbers of ED-1-positive cells and of TUNEL-positive cells, and protein carbonyl formation in the damaged brain. This ARB derivative, despite its significantly lower AT1 affinity and virtually no antihypertensive effect, ameliorated ischemic cerebral damage through antioxidative and antiinflammatory properties. These findings suggest potential usefulness of R-147176 as a pharmacological tool to investigate the ARBs' protective effect in experimental stroke and open new therapeutic avenues.

Hypoxia-inducible factor 1 (HIF-1) is regulated by the oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHDs). We recently developed a novel PHD inhibitor, TM6008, that suppresses the activity of PHDs, inducing continuous HIF-1α activation. In this study, we investigated how TM6008 affects cell survival after hypoxic conditions capable of inducing HIF-1α expression and how TM6008 regulates PHDs and genes downstream of HIF-1α. After SHSY-5Y cells had been subjected to hypoxia, TM6008 was added to the cell culture medium under normoxic conditions. Apoptotic cell death was significantly augmented just after the hypoxic conditions, compared with cell death under normoxic conditions. Notably, when TM6008 was added to the media after the cells had been subjected to hypoxia, the expression level of HIF-1α increased and the number of cell deaths decreased, compared with the results for cells cultured in media without TM6008 after hypoxia, during the 7-day incubation period under normoxic conditions. Moreover, the protein expression levels of heme oxygenase 1, erythropoietin, and glucose transporter-3, which were genes downstream of HIF-1α, were elevated in media to which TM6008 had been added, compared with media without TM6008, during the 7-day incubation period under normoxic conditions. However, the protein expression levels of PHD2 and p53 which suppressed cell proliferation were suppressed in the media to which TM6008 had been added. Thus, TM6008, which suppresses the protein expressions of PHD2 and p53, might play an important role in cell survival after hypoxic conditions, with possible applications as a new compound for treatment after ischemic stroke.

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