Pretreatment with Clodronate Improved Neurological Function by Preventing Reduction of Posthemorrhagic Cerebral Blood Flow in Experimental Subarachnoid Hemorrhage.

BACKGROUND: Brain perivascular macrophages (PVMs) are potential treatment targets for subarachnoid hemorrhage (SAH), and previous studies revealed that their depletion by clodronate (CLD) improved outcomes after experimental SAH. However, the underlying mechanisms are not well understood. Therefore, we investigated whether reducing PVMs by CLD pretreatment improves SAH prognosis by inhibiting posthemorrhagic impairment of cerebral blood flow (CBF). METHODS: In total, 80 male Sprague-Dawley rats received an intracerebroventricular injection of the vehicle (liposomes) or CLD. Subsequently, the rats were categorized into the prechiasmatic saline injection (sham) and blood injection (SAH) groups after 72 h. We assessed its effects on weak and severe SAH, which were induced by 200- and 300-µL arterial blood injections, respectively. In addition, neurological function at 72 h and CBF changes from before the intervention to 5 min after were assessed in rats after sham/SAH induction as the primary and secondary end points, respectively. RESULTS: CLD significantly reduced PVMs before SAH induction. Although pretreatment with CLD in the weak SAH group provided no additive effects on the primary end point, rats in the severe SAH group showed significant improvement in the rotarod test. In the severe SAH group, CLD inhibited acute reduction of CBF and tended to decrease hypoxia-inducible factor 1α expression. Furthermore, CLD reduced the number of PVMs in rats subjected to sham and SAH surgery, although no effects were observed in oxidative stress and inflammation. CONCLUSIONS: Our study proposes that pretreatment with CLD-targeting PVMs can improve the prognosis of severe SAH through a candidate mechanism of inhibition of posthemorrhagic CBF reduction.

Sirt7 Deficiency Attenuates Neointimal Formation Following Vascular Injury by Modulating Vascular Smooth Muscle Cell Proliferation.

BACKGROUND: Sirt7 is a recently identified sirtuin and has important roles in various pathological conditions, including cancer progression and metabolic disorders. It has previously been reported that Sirt7 is a key molecule in acute myocardial wound healing and pressure overload-induced cardiac hypertrophy. In this study, the role of Sirt7 in neointimal formation after vascular injury is investigated.Methods and Results:Systemic (Sirt7-/-) and smooth muscle cell-specific Sirt7-deficient mice were subjected to femoral artery wire injury. Primary vascular smooth muscle cells (VSMCs) were isolated from the aorta of wild type (WT) and Sirt7-/-mice and their capacity for cell proliferation and migration was compared. Sirt7 expression was increased in vascular tissue at the sites of injury. Sirt7-/-mice demonstrated significant reduction in neointimal formation compared to WT mice. In vitro, Sirt7 deficiency attenuated the proliferation of serum-induced VSMCs. Serum stimulation-induced upregulation of cyclins and cyclin-dependent-kinase 2 (CDK2) was significantly attenuated in VSMCs of Sirt7-/-compared with WT mice. These changes were accompanied by enhanced expression of the microRNA 290-295 cluster, the translational negative regulator of CDK2, in VSMCs of Sirt7-/-mice. It was confirmed that smooth muscle cell-specific Sirt7-deficient mice showed significant reduction in neointima compared with control mice. CONCLUSIONS: Sirt7 deficiency attenuates neointimal formation after vascular injury. Given the predominant role in vascular neointimal formation, Sirt7 is a potentially suitable target for treatment of vascular diseases.

Enhanced oxidative stress contributes to worse prognosis and delayed neurofunctional recovery after striatal intracerebral hemorrhage in 5XFAD mice.

Although Alzheimer's disease (AD) is associated with an increased risk of intracerebral hemorrhage (ICH) caused by hypertension and cerebral amyloid angiopathy, the precise clinical course after hypertensive ICH in AD patients is still unknown. In this study, we investigated how striatal ICH, a frequent site for hypertensive ICH, affected the prognosis of AD. We employed 17- and 18-month-old male 5XFAD (5X) mice and littermate (LT) controls, and striatal ICH was induced by collagenase injection. First, to address the acute effects of ICH on 5X mice, hemorrhagic volume and brain edema were evaluated 3 days after ICH. Next, to address the long-term effects of ICH on 5X mice, morbidity, mortality, neurological function (beam-walking and rotarod tests), and cognitive function (Y-maze and nest-building tests) were monitored. Twenty-eight days later, the animals were euthanized, their brains were isolated, and the cytotoxic alterations were investigated. The results revealed that the acute effects of ICH were not significantly different between 5X and LT mice. In contrast, 5X mice showed significantly higher morbidity and mortality in response to ICH, as well as delayed neurological function recovery, compared to LT mice through 28 days. ICH did not affect cognitive function in either group. Infiltrated macrophages in the perihemorrhagic cortex, gp91phox , p67phox , and COX-2 were significantly increased in 5X mice in response to ICH. We demonstrated that striatal ICH deteriorated prognosis and delayed neurofunctional recovery in 5X mice, which might be associated with enhanced oxidative stress in the presence of AD-like pathology.

Renal Denervation in the Acute Phase of Ischemic Stroke Provides Brain Protection in Hypertensive Rats.

BACKGROUND AND PURPOSE: Renal denervation (RD) may protect against cardiovascular diseases regardless of blood pressure (BP)-lowering effect. We hypothesized that RD can improve the outcome of acute ischemic stroke (AIS) in hypertensive rats. METHODS: An AIS model of spontaneously hypertensive stroke-prone rats (SHRSPs) was prepared by 90-minute middle cerebral artery occlusion followed by reperfusion. At 30 minutes poststroke, the SHRSPs were subjected to (1) sham operation or (2) RD to determine the beneficial effects of RD posttreatment. In addition, we evaluated the neuroprotective effects of RD posttreatment in Wistar Kyoto rats and RD pretreatment in SHRSPs with AIS. RESULTS: RD significantly ameliorated neurological deficit and infarct volume at 1 and 7 days after middle cerebral artery occlusion. RD immediately and continuously normalized elevated BP during 24 hours. This normalization of BP by RD was associated with the reduction of cerebral blood flow during both middle cerebral artery occlusion and reperfusion periods. Thus, RD-induced BP normalization seems to ameliorate cerebrovascular overperfusion in SHRSPs with AIS. On the contrary, RD did not affect cerebral vascular resistance or cerebral vasoreactivity and did not increase Akt and endothelial NO synthase phosphorylation, thereby indicating no apparent change of cerebrovascular function. RD significantly attenuated cerebral oxidative stress as estimated by dihydroethidium staining and gp91phox expression. The beneficial effects were not seen in Wistar Kyoto rats, whereas RD pretreatment improved neurological function in SHRSPs. CONCLUSIONS: RD posttreatment improved outcome in the hypertensive AIS rat model. Therefore, we suggest that BP normalization by RD may be a promising therapeutic strategy for AIS.

DPP-4 inhibition with linagliptin ameliorates the progression of premature aging in klotho-/- mice.

BACKGROUND: The potential of anti-aging effect of DPP-4 inhibitors is unknown. This study was performed to determine whether linagliptin, a DPP-4 inhibitor, could protect against premature aging in klotho-/- mice. METHODS: Klotho-/- mice exhibit multiple phenotypes resembling human premature aging, including extremely shortened life span, cognitive impairment, hippocampal neurodegeneration, hair loss, muscle atrophy, hypoglycemia, etc. To investigate the effect of linagliptin on these aging-related phenotypes, male klotho-/- mice were divided into two groups: (1) control group fed the standard diet, and (2) linagliptin group fed the standard diet containing linagliptin. Treatment with linagliptin was performed for 4 weeks. The effect of linagliptin on the above mentioned aging-related phenotypes was examined. RESULTS: Body weight of klotho-/- mice was greater in linagliptin group than in control group (11.1 ± 0.3 vs 9.9 ± 0.3 g; P < 0.01), which was associated with greater gastrocnemius muscle weight (P < 0.01) and greater kidney weight (P < 0.05) in linagliptin group. Thus, linagliptin significantly prevented body weight loss in klotho-/- mice. Survival rate of klotho-/- mice was greater in linagliptin group (93%) compared to control group (67%), although the difference did not reach statistical significance (P = 0.08). None of linagliptin-treated klotho-/- mice had alopecia during the treatment (P < 0.05 vs control klotho-/- mice). Latency of klotho-/- mice in passive avoidance test was larger in linagliptin group than in control group (P < 0.05), indicating the amelioration of cognitive impairment by linagliptin. Cerebral blood flow of klotho-/- mice was larger in linagliptin group than in control group (P < 0.01), being associated with greater cerebral phospho-eNOS levels (P < 0.05) in linagliptin group. Neuronal cell number in hippocampal CA1 region was greater in linagliptin group than in control group (P < 0.05). Linagliptin group had greater cerebral phospho-Akt (P < 0.05) and phospho-CREB (P < 0.05) than control group. Thus, linagliptin ameliorated brain aging in klotho-/- mice. The degree of hypoglycemia in klotho-/- mice was less in linagliptin group than in control group, as estimated by the findings of OGTT. CONCLUSIONS: Out work provided the evidence that DPP-4 inhibition with linagliptin slowed the progression of premature aging in klotho-/- mice, and provided a novel insight into the potential role of DPP-4 in the mechanism of premature aging.

Empagliflozin lessened cardiac injury and reduced visceral adipocyte hypertrophy in prediabetic rats with metabolic syndrome.

BACKGROUND: The potential benefit of SGLT2 inhibitors in metabolic syndrome is with prediabetic stage unclear. This work was undertaken to investigate the non-glycemic effect of empagliflozin on metabolic syndrome rats with prediabetes. METHODS: SHR/NDmcr-cp(+/+) rats (SHRcp), a model of metabolic syndrome with prediabetes, were given empagliflozin for 10 weeks to examine the effects on urinary sodium and water balance, visceral and subcutaneous adipocyte, and cardiac injury. Further, the effect of empagliflozin on blood pressure and autonomic nervous system was continuously investigated by using radiotelemetry system. RESULTS: Empagliflozin significantly reduced urinary sodium and water balance of SHRcp only within 1 week of the treatment, but later than 1 week did not alter them throughout the treatment. Empagliflozin significantly reduced body weight of SHRcp, which was mainly attributed to the significant reduction of subcutaneous fat mass. Empagliflozin significantly reduced the size of visceral adipocytes and increased the number of smaller size of adipocytes, which was associated with the attenuation of oxidative stress. Empagliflozin ameliorated cardiac hypertrophy and fibrosis of SHRcp, in association with the attenuation of cardiac oxidative stress and inflammation. However, empagliflozin did not significantly change blood pressure, heart rate, sympathetic activity, or baroreceptor function, as evidenced by radiotelemetry analysis. CONCLUSIONS: Our present work provided the evidence that SGLT2 inhibition reduced visceral adipocytes hypertrophy and ameliorated cardiac injury in prediabetic metabolic syndrome rat, independently of diuretic effect or blood pressure lowering effect. Thus, SGLT2 inhibition seems to be a promising therapeutic strategy for prediabetic metabolic syndrome.

Assessment of the Correlations Between Brain Weight and Brain Edema in Experimental Subarachnoid Hemorrhage.

Because brain edema is correlated with poor outcome in clinical subarachnoid hemorrhage (SAH), appropriate evaluation methods for brain edema are important in experimental SAH studies. Although brain water content (BWC) is widely used to evaluate brain edema in stroke research, the usefulness of brain weight is undetermined. In this study, we examined the role of brain weight in the evaluation of brain edema in experimental SAH. The endovascular perforation model of SAH was used, and rats were assessed by neurological scoring (NS). The brains were quickly removed at 24 h after the operation, and the weights of wet cerebrum (WWC) and dry cerebrum (WDC) were measured to determine the brain water content (BWC). The correlations of those values with each other and to body weight (BW) were then examined to reveal the significance of brain weight. The rats were assigned to sham-operated (n = 8) and SAH (n = 16) groups. There were no significant differences in WWC between the groups (p = 0.61). WWC was correlated with BWC but not with NS in all rats. In addition, WWC was clearly correlated with BW and WDC, which is thought to substitute for the original brain weight. From these results, we suggest that the measurement of brain weight as an evaluation of brain edema is limited and that BW and original brain volume can be confounding factors in evaluation.

DPP-4 inhibition with linagliptin ameliorates cognitive impairment and brain atrophy induced by transient cerebral ischemia in type 2 diabetic mice.

BACKGROUND: It is unclear whether dipeptidylpeptidase-4 (DPP-4) inhibition can counteract the impairment of cognitive function and brain injury caused by transient cerebral ischemia in type 2 diabetes. The present study was undertaken to test our hypothesis that linagliptin, a DPP-4 inhibitor, administration following transient cerebral ischemia can ameliorate cognitive impairment and brain injury in diabetic mice. METHODS: db/db mice, a model of obese type 2 diabetes, were subjected to transient cerebral ischemia by 17 min of bilateral common carotid artery occlusion (BCCAO), and were administered (1) vehicle or (2) linagliptin for 8 weeks or 1 week. For the long-term experiment on 8 weeks of linagliptin treatment, cognitive function, and volume and neuronal cell number of hippocampus and cortex were estimated in each group of mice. For the short-term experiment on 1 week of linagliptin treatment, cerebral IgG extravasation, Iba-1 positive cell number (reactive microglia), oxidative stress, and claudin-5 and gp91phox protein levels were measured in each group of mice. RESULTS: Linagliptin administration almost completely suppressed the circulating DPP-4 activity in db/db mice, but did not significantly reduce blood glucose or ameliorate glucose intolerance in db/db mice. Linagliptin administration following transient cerebral ischemia significantly counteracted cognitive impairment in diabetic mice, as estimated by water maze test and passive avoidance test. Linagliptin administration ameliorated the decrease in cerebral volume and neuronal cell number in hippocampus and cortex of diabetic mice. Linagliptin administration significantly reduced the increase in cerebral IgG extravasation and the increase in reactive microglia caused by transient cerebral ischemia in diabetic mice. Furthermore, linagliptin significantly suppressed the increase in cerebral oxidative stress in transient cerebral ischemia-subjected diabetic mice. Furthermore, linagliptin significantly increased cerebral claudin-5 and significantly decreased gp91phox in diabetic mice subjected to transient cerebral ischemia. CONCLUSIONS: DPP-4 inhibition with linagliptin counteracted cognitive impairment and brain atrophy induced by transient cerebral ischemia in diabetic mice, independently of blood glucose lowering effect. This cerebroprotective effect of linagliptin was associated with the suppression of blood-brain barrier disruption and the attenuation of cerebral oxidative stress. Thus, our present work highlights DPP-4 inhibition as a promising therapeutic strategy for cognitive impairment and cerebral vascular complications in type 2 diabetes.

Apoptosis signal-regulating kinase 1 is a novel target molecule for cognitive impairment induced by chronic cerebral hypoperfusion.

OBJECTIVE: There are currently no specific strategies for the treatment or prevention of vascular dementia. White matter lesions, a common pathology in cerebral small vessel disease, are a major cause of vascular dementia. We investigated whether apoptosis signal-regulating kinase 1 (ASK1) might be a key molecule in cerebral hypoperfusion, associated with blood-brain barrier breakdown and white matter lesions. APPROACH AND RESULTS: A mouse model of cognitive impairment was developed by inducing chronic cerebral hypoperfusion in white matter including the corpus callosum via bilateral common carotid artery stenosis (BCAS) surgery. BCAS-induced white matter lesions caused cognitive decline in C57BL/6J (wild-type) mice but not in ASK1-deficient (ASK1(-/-)) mice. Phosphorylated ASK1 increased in wild-type mouse brains, and phosphorylated p38 and tumor necrosis factor-α expression increased in corpus callosum cerebral endothelial cells after BCAS in wild-type mice but not in ASK1(-/-) mice. BCAS decreased claudin-5 expression and disrupted blood-brain barrier in the corpus callosum of wild-type but not ASK1(-/-) mice. Cerebral nitrotyrosine was increased in wild-type and ASK1(-/-) BCAS mice. Cerebral phosphorylated ASK1 did not increase in wild-type mice treated with NADPH-oxidase inhibitor. A p38 inhibitor and NADPH-oxidase inhibitor mimicked the protective effect of ASK1 deficiency against cognitive impairment. Specific ASK1 inhibitor prevented cognitive decline in BCAS mice. In vitro oxygen-glucose deprivation and tumor necrosis factor-α stimulation caused the disruption of endothelial tight junctions from wild-type mice but not ASK1(-/-) mice. CONCLUSIONS: Oxidative stress-ASK1-p38 cascade plays a role in the pathogenesis of cognitive impairment, through blood-brain barrier breakdown via the disruption of endothelial tight junctions. ASK1 might be a promising therapeutic target for chronic cerebral hypoperfusion-induced cognitive impairment.

Glycemic control with empagliflozin, a novel selective SGLT2 inhibitor, ameliorates cardiovascular injury and cognitive dysfunction in obese and type 2 diabetic mice.

BACKGROUND: There has been uncertainty regarding the benefit of glycemic control with antidiabetic agents in prevention of diabetic macrovascular disease. Further development of novel antidiabetic agents is essential for overcoming the burden of diabetic macrovascular disease. The renal sodium glucose co-transporter 2 (SGLT2) inhibitor is a novel antihyperglycemic agent for treatment of type 2 diabetes. This work was performed to determine whether empagliflozin, a novel SGLT2 inhibitor, can ameliorate cardiovascular injury and cognitive decline in db/db mouse, a model of obesity and type 2 diabetes. METHODS: (1) Short-term experiment: The first experiment was performed to examine the effect of 7 days of empagliflozin treatment on urinary glucose excretion and urinary electrolyte excretion in db/db mice. (2) Long-term experiment: The second experiment was undertaken to examine the effect of 10 weeks of empagliflozin treatment on cardiovascular injury, vascular dysfunction, cognitive decline, and renal injury in db/db mice. RESULTS: (1) Short-term experiment: Empagliflozin administration significantly increased urinary glucose excretion, urine volume, and urinary sodium excretion in db/db mice on day 1, but did not increase these parameters from day 2. However, blood glucose levels in db/db mice were continuously decreased by empagliflozin throughout 7 days of the treatment. (2) Long-term experiment: Empagliflozin treatment caused sustained decrease in blood glucose in db/db mice throughout 10 weeks of the treatment and significantly slowed the progression of type 2 diabetes. Empagliflozin significantly ameliorated cardiac interstitial fibrosis, pericoronary arterial fibrosis, coronary arterial thickening, cardiac macrophage infiltration, and the impairment of vascular dilating function in db/db mice, and these beneficial effects of empagliflozin were associated with attenuation of oxidative stress in cardiovascular tissue of db/db mice. Furthermore, empagliflozin significantly prevented the impairment of cognitive function in db/db mice, which was associated with the attenuation of cerebral oxidative stress and the increase in cerebral brain-derived neurotrophic factor. Empagliflozin ameliorated albuminuria, and glomerular injury in db/db mice. CONCLUSIONS: Glycemic control with empagliflozin significantly ameliorated cardiovascular injury and remodeling, vascular dysfunction, and cognitive decline in obese and type 2 diabetic mice. Thus, empagliflozin seems to be potentially a promising therapeutic agent for diabetic macrovascular disease and cognitive decline.

DPP-4 inhibitor linagliptin ameliorates cardiovascular injury in salt-sensitive hypertensive rats independently of blood glucose and blood pressure.

BACKGROUND: It remains to be elucidated whether dipeptidylpeptidase-4 (DPP-4) inhibitor can ameliorate cardiovascular injury in salt-sensitive hypertension. The present study was undertaken to test our hypothesis that linagliptin, a DPP-4 inhibitor, administration initiated after onset of hypertension and cardiac hypertrophy can ameliorate cardiovascular injury in Dahl salt-sensitive hypertensive rats (DS rats). METHODS: High-salt loaded DS rats with established hypertension and cardiac hypertrophy were divided into two groups, and were orally given (1) vehicle or (2) linagliptin (3 mg/kg/day) once a day for 4 weeks, and cardiovascular protective effects of linagliptin in DS rats were evaluated. RESULTS: Linagliptin did not significantly affect blood pressure and blood glucose levels in DS rats. Linagliptin significantly lessened cardiac hypertrophy in DS rats, as estimated by cardiac weight and echocardiographic parameters. Linagliptin significantly ameliorated cardiac fibrosis, cardiac macrophage infiltration, and coronary arterial remodeling in DS rats. Furthermore, linagliptin significantly mitigated the impairment of vascular function in DS rats, as shown by the improvement of acetylcholine-induced or sodium nitroprusside-induced vascular relaxation by linagliptin. These cardiovascular protective effects of linagliptin were associated with the attenuation of oxidative stress, NADPH oxidase subunits, p67phox and p22 phox, and angiotensin-converting enzyme (ACE). CONCLUSIONS: Our results provided the experimental evidence that linagliptin treatment initiated after the appearance of hypertension and cardiac hypertrophy protected against cardiovascular injury induced by salt-sensitive hypertension, independently of blood pressure and blood glucose. These beneficial effects of linagliptin seem to be attributed to the reduction of oxidative stress and ACE.

Rosuvastatin ameliorates early brain injury after subarachnoid hemorrhage via suppression of superoxide formation and nuclear factor-kappa B activation in rats.

BACKGROUND: Statins, or 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, have been suggested to possess pleiotropic effects, including antioxidant and anti-inflammatory properties. We investigated the protective effects of pretreatment with rosuvastatin, a relatively hydrophilic statin, on early brain injury (EBI) after a subarachnoid hemorrhage (SAH), using the endovascular perforation SAH model. METHODS: Eighty-six male Sprague-Dawley rats were randomly divided into 3 groups: (1) sham operation, (2) SAH+vehicle, and (3) SAH+10 mg/kg rosuvastatin. Rosuvastatin or vehicle was orally administered to rats once daily from 7 days before to 1 day after the SAH operation. After SAH, we examined the effects of rosuvastatin on the neurologic score, brain water content, neuronal cell death estimated by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate nick end labeling staining, blood-brain barrier disruption by immunoglobulin G (IgG) extravasation, oxidative stress, and proinflammatory molecules. RESULTS: Compared with the vehicle group, rosuvastatin significantly improved the neurologic score and reduced the brain water content, neuronal cell death, and IgG extravasation. Rosuvastatin inhibited brain superoxide production, nuclear factor-kappa B (NF-κB) activation, and the increase in activated microglial cells after SAH. The increased expressions of tumor necrosis factor-alpha, endothelial matrix metalloproteinase-9, and neuronal cyclooxygenase-2 induced by SAH were prevented by rosuvastatin pretreatment. CONCLUSIONS: The present study demonstrates that rosuvastatin pretreatment ameliorates EBI after SAH through the attenuation of oxidative stress and NF-κB-mediated inflammation.

An angiotensin II receptor blocker-calcium channel blocker combination prevents cardiovascular events in elderly high-risk hypertensive patients with chronic kidney disease better than high-dose angiotensin II receptor blockade alone.

The OSCAR study was a multicenter, prospective randomized open-label blinded end-point study of 1164 Japanese elderly hypertensive patients comparing the efficacy of angiotensin II receptor blocker (ARB) uptitration to an ARB plus calcium channel blocker (CCB) combination. In this prospective study, we performed prespecified subgroup analysis according to baseline estimated glomerular filtration rate (eGFR) with chronic kidney disease (CKD) defined as an eGFR <60 ml/min per 1.73 m(2). Blood pressure was lower in the combined therapy than in the high-dose ARB cohort in both groups with and without CKD. In patients with CKD, significantly more primary events (a composite of cardiovascular events and noncardiovascular death) occurred in the high-dose ARB group than in the combination group (30 vs. 16, respectively, hazard ratio 2.25). Significantly more cerebrovascular and more heart failure events occurred in the high-dose ARB group than in the combination group. In patients without CKD, however, the incidence of primary events was similar between the two treatments. The treatment-by-subgroup interaction was significant. Allocation to the high-dose ARB was a significant independent prognostic factor for primary events in patients with CKD. Thus, the ARB plus CCB combination conferred greater benefit in prevention of cardiovascular events in patients with CKD compared with high-dose ARB alone. Our findings provide new insight into the antihypertensive strategy for elderly hypertensive patients with CKD.

p-Cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by activation of NADPH oxidase.

The accumulation of p-cresyl sulfate (PCS), a uremic toxin, is associated with the mortality rate of chronic kidney disease patients; however, the biological functions and the mechanism of its action remain largely unknown. Here we determine whether PCS enhances the production of reactive oxygen species (ROS) in renal tubular cells resulting in cytotoxicity. PCS exhibited pro-oxidant properties in human tubular epithelial cells by enhancing NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) activity. PCS also upregulated mRNA levels of inflammatory cytokines and active TGF-ß1 protein secretion associated with renal fibrosis. Knockdown of p22(phox) or Nox4 expression suppressed the effect of PCS, underlining the importance of NADPH oxidase activation on its mechanism of action. PCS also reduced cell viability by increasing ROS production. The toxicity of PCS was largely suppressed in the presence of probenecid, an organic acid transport inhibitor. Administration of PCS for 4 weeks caused significant renal tubular damage in 5/6-nephrectomized rats by enhancing oxidative stress. Thus, the renal toxicity of PCS is attributed to its intracellular accumulation, leading to both increased NADPH oxidase activity and ROS production, which, in turn, triggers induction of inflammatory cytokines involved in renal fibrosis. This mechanism is similar to that for the renal toxicity of indoxyl sulfate.

Voltage-dependent N-type Ca2+ channels in endothelial cells contribute to oxidative stress-related endothelial dysfunction induced by angiotensin II in mice.

N-type voltage-dependent Ca(2+)channels (VDCCs), expressed predominantly in the nervous system, play pivotal roles in sympathetic regulation of the circulatory system. Although N-type VDCCs are also reportedly expressed in the vasculature, their pathophysiological role is obscure. We demonstrated that oxidative stress-related endothelial dysfunction induced by angiotensin (Ang) II is suppressed in mice lacking the N-type VDCC α1B subunit (Cav 2.2). Impairment of endothelium-dependent relaxation of the thoracic aorta observed following Ang II treatment in wild-type (WT) mice was significantly attenuated in the Ang II-treated Cav 2.2-deficient mice, despite the comparable increase of the blood pressure in the two groups of mice. The thoracic aorta of the Cav 2.2-deficient mice showed a smaller positive area of oxidative stress markers as compared to the WT mice. The Ang II-induced endothelial dysfunction was also suppressed by cilnidipine, an L/N-type VDCC blocker, but not by amlodipine, an L-type VDCC blocker; however, this unique effect of cilnidipine was completely abolished in the Cav 2.2-deficient mice. Furthermore, selective inhibition of N-type VDCCs by ω-conotoxin GVIA dramatically suppressed the production of reactive oxygen species (ROS) as well as agonist-induced Ca(2+) influx in the vascular endothelial cells. These results suggest that N-type VDCCs expressed in the vascular endothelial cells contribute to ROS production and endothelial dysfunction observed in Ang II-treated hypertensive mice.

Nanoparticle-mediated delivery of pioglitazone enhances therapeutic neovascularization in a murine model of hindlimb ischemia.

OBJECTIVE: Critical limb ischemia is a severe form of peripheral artery disease (PAD) for which neither surgical revascularization nor endovascular therapy nor current medicinal therapy has sufficient therapeutic effects. Peroxisome proliferator activated receptor-γ agonists present angiogenic activity in vitro; however, systemic administration of peroxisome proliferator-activated receptor-γ agonists is hampered by its side effects, including heart failure. Here, we demonstrate that the nanoparticle (NP)-mediated delivery of the peroxisome proliferator activated receptor-γ agonist pioglitazone enhances its therapeutic efficacy on ischemia-induced neovascularization in a murine model. METHODS AND RESULTS: In a nondiabetic murine model of hindlimb ischemia, a single intramuscular injection of pioglitazone-incorporated NP (1 µg/kg) into ischemic muscles significantly improved the blood flow recovery in the ischemic limbs, significantly increasing the number of CD31-positive capillaries and α-smooth muscle actin-positive arterioles. The therapeutic effects of pioglitazone-incorporated NP were diminished by the peroxisome proliferator activated receptor-γ antagonist GW9662 and were not observed in endothelial NO synthase-deficient mice. Pioglitazone-incorporated NP induced endothelial NO synthase phosphorylation, as demonstrated by Western blot analysis, as well as expression of multiple angiogenic growth factors in vivo, including vascular endothelial growth factor-A, vascular endothelial growth factor-B, and fibroblast growth factor-1, as demonstrated by real-time polymerase chain reaction. Intramuscular injection of pioglitazone (1 µg/kg) was ineffective, and oral administration necessitated a >500 µg/kg per day dose to produce therapeutic effects equivalent to those of pioglitazone-incorporated NP. CONCLUSIONS: NP-mediated drug delivery is a novel modality that may enhance the effectiveness of therapeutic neovascularization, surpassing the effectiveness of current treatments for peripheral artery disease with critical limb ischemia.

Perindopril, a centrally active angiotensin-converting enzyme inhibitor, prevents cognitive impairment in mouse models of Alzheimer's disease.

The purpose of this work was to test whether brain-penetrating angiotensin-converting enzyme (ACE) inhibitors (e.g., perindopril), as opposed to non-brain-penetrating ACE inhibitors (e.g., enalapril and imidapril), may reduce the cognitive decline and brain injury in Alzheimer's disease (AD). We first compared the effect of perindopril, enalapril, and imidapril on cognitive impairment and brain injury in a mouse model of AD induced by intracerebroventricular (i.c.v.) injection of amyloid-ß (Aß)1₋40. Perindopril, with significant inhibition of hippocampal ACE, significantly prevented cognitive impairment in this AD mouse model. This beneficial effect was attributed to the suppression of microglia/astrocyte activation and the attenuation of oxidative stress caused by iNOS induction and extracellular superoxide dismutase down-regulation. In contrast, neither enalapril nor imidapril prevented cognitive impairment and brain injury in this AD mouse. We next examined the protective effects of perindopril on cognitive impairment in PS2APP-transgenic mice overexpressing Aß in the brain. Perindopril, without affecting brain Aß deposition, significantly suppressed the increase in hippocampal ACE activity and improved cognition in PS2APP-transgenic mice, being associated with the suppression of hippocampal astrocyte activation and attenuation of superoxide. Our data demonstrated that the brain-penetrating ACE inhibitor perindopril, as compared to non-brain-penetrating ACE inhibitors, protected against cognitive impairment and brain injury in experimental AD models.

Passive exercise using whole-body periodic acceleration enhances blood supply to ischemic hindlimb.

OBJECTIVE: Whole-body periodic acceleration (WBPA) has been developed as a passive exercise technique to improve endothelial function by increasing shear stress through repetitive movements in spinal axis direction. We investigated the effects of WBPA on blood flow recovery in a mouse model of hindlimb ischemia and in patients with peripheral arterial disease. METHODS AND RESULTS: After unilateral femoral artery excision, mice were assigned to either the WBPA (n=15) or the control (n=13) group. WBPA was applied at 150 cpm for 45 minutes under anesthesia once a day. WBPA significantly increased blood flow recovery after ischemic surgery, as determined by laser Doppler perfusion imaging. Sections of ischemic adductor muscle stained with anti-CD31 antibody showed a significant increase in capillary density in WBPA mice compared with control mice. WBPA increased the phosphorylation of endothelial nitric oxide synthase (eNOS) in skeletal muscle. The proangiogenic effect of WBPA on ischemic limb was blunted in eNOS-deficient mice, suggesting that the stimulatory effects of WBPA on revascularization are eNOS dependent. Quantitative real-time polymerase chain reaction analysis showed significant increases in angiogenic growth factor expression in ischemic hindlimb by WBPA. Facilitated blood flow recovery was observed in a mouse model of diabetes despite there being no changes in glucose tolerance and insulin sensitivity. Furthermore, both a single session and 7-day repeated sessions of WBPA significantly improved blood flow in the lower extremity of patients with peripheral arterial disease. CONCLUSIONS: WBPA increased blood supply to ischemic lower extremities through activation of eNOS signaling and upregulation of proangiogenic growth factor in ischemic skeletal muscle. WBPA is a potentially suitable noninvasive intervention to facilitate therapeutic angiogenesis.

Loss of body weight in old 5xFAD mice and the alteration of gut microbiota composition.

The cause of age-related body weight loss in Alzheimer's disease (AD) is unclear. We compared the differences in food intake, malabsorption, locomotor activity, and gut microbiota composition between 5xFAD mice, a useful model of AD, and wild-type (WT) mice to investigate the mechanisms underlying lower body weight in 5xFAD mice. Fifteen-month-old male 5xFAD mice and age-matched WT mice were divided into four groups: a control diet (CD) or a high-fat diet (HFD). After feeding CD or HFD for eight to nine weeks, 5xFAD mice had a significantly lower body weight than WT mice regardless of diet (p < 0.05). Additionally, the 5xFAD mice did not show a reduction in food intake compared to the WT mice regardless of diet. To evaluate malabsorption, we performed a fecal fat test. There was no obvious fecal fat in both the 5xFAD mice and WT mice. However, 5xFAD mice showed greater locomotor activity than WT mice in the Y-maze test. The comprehensive analysis of gut microbiota composition showed that 15-month-old 5xFAD mice had more Proteobacteria population and fewer Actinobacteria and Bifidobacteriales populations than WT mice. To investigate the effects of fructooligosaccharides (FOS), we administrated FOS to 15-month-old 5xFAD mice. FOS administration decreased Proteobacteria and increased Actinobacteria population, although that did not change Bifidobacteriales population. Moreover, cognitive impairment and body weight of 5xFAD mice were not changed by FOS administration. In conclusion, loss of body weight in 15-month-old 5xFAD mice might be partially derived from excess energy output by hyperactivity. Moreover, 15-month-old 5xFAD mice might have unique alteration of gut microbiota composition and the potential resistance to FOS.

Telmisartan protects against diabetic vascular complications in a mouse model of obesity and type 2 diabetes, partially through peroxisome proliferator activated receptor-γ-dependent activity.

Experimental and clinical data support the notion that peroxisome proliferator-activated receptor γ (PPARγ) activation is associated with anti-atherosclerosis as well as anti-diabetic effect. Telmisartan, an angiotensin receptor blocker (ARB), acts as a partial PPARγ agonist. We hypothesized that telmisartan protects against diabetic vascular complications, through PPARγ activation. We compared the effects of telmisartan, telmisartan combined with GW9662 (a PPARγ antagonist), and losartan with no PPARγ activity on vascular injury in obese type 2 diabetic db/db mice. Compared to losartan, telmisartan significantly ameliorated vascular endothelial dysfunction, downregulation of phospho-eNOS, and coronary arterial remodeling in db/db mice. More vascular protective effects of telmisartan than losartan were associated with greater anti-inflammatory effects of telmisartan, as shown by attenuation of vascular nuclear factor kappa B (NFκB) activation and tumor necrosis factor α. Coadministration of GW9662 with telmisartan abolished the above mentioned greater protective effects of telmisartan against vascular injury than losartan in db/db mice. Thus, PPARγ activity appears to be involved in the vascular protective effects of telmisartan in db/db mice. Moreover, telmisartan, but not losartan, prevented the downregulation of vascular PPARγ in db/db mice and this effect of telmisartan was cancelled by the coadministration of GW9662. Our data provided the first evidence indicating that PPARγ activity of telmisartan contributed to the protective effects of telmisartan against diabetic vascular complication. PPARγ activity of telmisartan was involved in the normalization of vascular PPARγ downregulation in diabetic mice. Thus, telmisartan seems to exert vascular protective effects in hypertensive patients with diabetes.