Contribution of reactive oxygen species to cerebral amyloid angiopathy, vasomotor dysfunction, and microhemorrhage in aged Tg2576 mice.

Cerebral amyloid angiopathy (CAA) is characterized by deposition of amyloid ß peptide (Aß) within walls of cerebral arteries and is an important cause of intracerebral hemorrhage, ischemic stroke, and cognitive dysfunction in elderly patients with and without Alzheimer's Disease (AD). NADPH oxidase-derived oxidative stress plays a key role in soluble Aß-induced vessel dysfunction, but the mechanisms by which insoluble Aß in the form of CAA causes cerebrovascular (CV) dysfunction are not clear. Here, we demonstrate evidence that reactive oxygen species (ROS) and, in particular, NADPH oxidase-derived ROS are a key mediator of CAA-induced CV deficits. First, the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, are shown to reduce oxidative stress and improve CV reactivity in aged Tg2576 mice. Second, the observed improvement in CV function is attributed both to a reduction in CAA formation and a decrease in CAA-induced vasomotor impairment. Third, anti-ROS therapy attenuates CAA-related microhemorrhage. A potential mechanism by which ROS contribute to CAA pathogenesis is also identified because apocynin substantially reduces expression levels of ApoE-a factor known to promote CAA formation. In total, these data indicate that ROS are a key contributor to CAA formation, CAA-induced vessel dysfunction, and CAA-related microhemorrhage. Thus, ROS and, in particular, NADPH oxidase-derived ROS are a promising therapeutic target for patients with CAA and AD.

Passive immunotherapy targeting amyloid-ß reduces cerebral amyloid angiopathy and improves vascular reactivity.

Prominent cerebral amyloid angiopathy is often observed in the brains of elderly individuals and is almost universally found in patients with Alzheimer's disease. Cerebral amyloid angiopathy is characterized by accumulation of the shorter amyloid-ß isoform(s) (predominantly amyloid-ß40) in the walls of leptomeningeal and cortical arterioles and is likely a contributory factor to vascular dysfunction leading to stroke and dementia in the elderly. We used transgenic mice with prominent cerebral amyloid angiopathy to investigate the ability of ponezumab, an anti-amyloid-ß40 selective antibody, to attenuate amyloid-ß accrual in cerebral vessels and to acutely restore vascular reactivity. Chronic administration of ponezumab to transgenic mice led to a significant reduction in amyloid and amyloid-ß accumulation both in leptomeningeal and brain vessels when measured by intravital multiphoton imaging and immunohistochemistry. By enriching for cerebral vascular elements, we also measured a significant reduction in the levels of soluble amyloid-ß biochemically. We hypothesized that the reduction in vascular amyloid-ß40 after ponezumab administration may reflect the ability of ponezumab to mobilize an interstitial fluid pool of amyloid-ß40 in brain. Acutely, ponezumab triggered a significant and transient increase in interstitial fluid amyloid-ß40 levels in old plaque-bearing transgenic mice but not in young animals. We also measured a beneficial effect on vascular reactivity following acute administration of ponezumab, even in vessels where there was a severe cerebral amyloid angiopathy burden. Taken together, the beneficial effects ponezumab administration has on reducing the rate of cerebral amyloid angiopathy deposition and restoring cerebral vascular health favours a mechanism that involves rapid removal and/or neutralization of amyloid-ß species that may otherwise be detrimental to normal vessel function.

Cerebral amyloid angiopathy increases susceptibility to infarction after focal cerebral ischemia in Tg2576 mice.

BACKGROUND AND PURPOSE: We and others have shown that soluble amyloid ß-peptide (Aß) and cerebral amyloid angiopathy (CAA) cause significant cerebrovascular dysfunction in mutant amyloid precursor protein (APP) mice, and that these deficits are greater in aged APP mice having CAA compared with young APP mice lacking CAA. Amyloid ß-peptide in young APP mice also increases infarction after focal cerebral ischemia, but the impact of CAA on ischemic brain injury is unknown. METHODS: To determine this, we assessed cerebrovascular reactivity, cerebral blood flow (CBF), and extent of infarction and neurological deficits after transient middle cerebral artery occlusion in aged APP mice having extensive CAA versus young APP mice lacking CAA (and aged-matched littermate controls). RESULTS: We found that aged APP mice have more severe cerebrovascular dysfunction that is CAA dependent, have greater CBF compromise during and immediately after middle cerebral artery occlusion, and develop larger infarctions after middle cerebral artery occlusion. CONCLUSIONS: These data indicate CAA induces a more severe form of cerebrovascular dysfunction than amyloid ß-peptide alone, leading to intra- and postischemic CBF deficits that ultimately exacerbate cerebral infarction. Our results shed mechanistic light on human studies identifying CAA as an independent risk factor for ischemic brain injury.

Cardiac response to hypobaric hypoxia: persistent changes in cardiac mass, function, and energy metabolism after a trek to Mt. Everest Base Camp.

We postulated that changes in cardiac high-energy phosphate metabolism may underlie the myocardial dysfunction caused by hypobaric hypoxia. Healthy volunteers (n=14) were studied immediately before, and within 4 d of return from, a 17-d trek to Mt. Everest Base Camp (5300 m). (31)P magnetic resonance (MR) spectroscopy was used to measure cardiac phosphocreatine (PCr)/ATP, and MR imaging and echocardiography were used to assess cardiac volumes, mass, and function. Immediately after returning from Mt. Everest, total body weight had fallen by 3% (P<0.05), but left ventricular mass, adjusted for changes in body surface area, had disproportionately decreased by 11% (P<0.05). Alterations in diastolic function were also observed, with a reduction in peak left ventricular filling rates and mitral inflow E/A, by 17% (P<0.05) and 24% (P<0.01), respectively, with no change in hydration status. Compared with pretrek, cardiac PCr/ATP ratio had decreased by 18% (P<0.01). Whether the abnormalities were even greater at altitude is unknown, but all had returned to pretrek levels after 6 mo. The alterations in cardiac morphology, function, and energetics are similar to findings in patients with chronic hypoxia. Thus, a decrease in cardiac PCr/ATP may be a universal response to periods of sustained low oxygen availability, underlying hypoxia-induced cardiac dysfunction in healthy human heart and in patients with cardiopulmonary diseases.

Abnormal whole-body energy metabolism in iron-deficient humans despite preserved skeletal muscle oxidative phosphorylation.

Iron deficiency impairs skeletal muscle metabolism. The underlying mechanisms are incompletely characterised, but animal and human experiments suggest the involvement of signalling pathways co-dependent upon oxygen and iron availability, including the pathway associated with hypoxia-inducible factor (HIF). We performed a prospective, case-control, clinical physiology study to explore the effects of iron deficiency on human metabolism, using exercise as a stressor. Thirteen iron-deficient (ID) individuals and thirteen iron-replete (IR) control participants each underwent 31P-magnetic resonance spectroscopy of exercising calf muscle to investigate differences in oxidative phosphorylation, followed by whole-body cardiopulmonary exercise testing. Thereafter, individuals were given an intravenous (IV) infusion, randomised to either iron or saline, and the assessments repeated ~ 1 week later. Neither baseline iron status nor IV iron significantly influenced high-energy phosphate metabolism. During submaximal cardiopulmonary exercise, the rate of decline in blood lactate concentration was diminished in the ID group (P = 0.005). Intravenous iron corrected this abnormality. Furthermore, IV iron increased lactate threshold during maximal cardiopulmonary exercise by ~ 10%, regardless of baseline iron status. These findings demonstrate abnormal whole-body energy metabolism in iron-deficient but otherwise healthy humans. Iron deficiency promotes a more glycolytic phenotype without having a detectable effect on mitochondrial bioenergetics.

IL-6 deficiency protects against angiotensin II induced endothelial dysfunction and hypertrophy.

OBJECTIVE: The goal of this study was to test the hypothesis that IL-6 mediates the increases in superoxide, vascular hypertrophy, and endothelial dysfunction in response to angiotensin II (Ang II). METHODS AND RESULTS: Responses of carotid arteries from control and IL-6-deficient mice were examined after acute (22-hour) incubation with Ang II (10 nmol/L) or chronic infusion of Ang II (1.4 mg/kg/d for 14 days). The hypertrophic response and endothelial dysfunction produced by Ang II infusion was markedly less in carotid arteries from IL-6-deficient mice than that in control mice. IL-6 deficiency also protected against endothelial dysfunction in response to acute (local) Ang II treatment (eg, 100 mumol/L acetylcholine produced 100+/-4 and 98+/-4% relaxation in vehicle-treated and 51+/-4 and 99+/-4% relaxation in Ang II-treated, control, and IL-6-deficient vessels, respectively). Endothelial dysfunction could be reproduced in vessels from IL-6-deficient mice with combined Ang II plus IL-6 (0.1 nmol/L) treatment. Increases in vascular superoxide and IL-6, as well as reductions in endothelial nitric oxide synthase mRNA expression, produced by Ang II were absent in IL-6-deficient mice. CONCLUSIONS: These data demonstrate that IL-6 is essential for Ang II-induced increases in superoxide, endothelial dysfunction, and vascular hypertrophy.

HIF-1α Mediates Isoflurane-Induced Vascular Protection in Subarachnoid Hemorrhage.

OBJECTIVE: Outcome after aneurysmal subarachnoid hemorrhage (SAH) depends critically on delayed cerebral ischemia (DCI) - a process driven primarily by vascular events including cerebral vasospasm, microvessel thrombosis, and microvascular dysfunction. This study sought to determine the impact of postconditioning - the phenomenon whereby endogenous protection against severe injury is enhanced by subsequent exposure to a mild stressor - on SAH-induced DCI. METHODS: Adult male C57BL/6 mice were subjected to sham, SAH, or SAH plus isoflurane postconditioning. Neurological outcome was assessed daily via sensorimotor scoring. Contributors to DCI including cerebral vasospasm, microvessel thrombosis, and microvascular dysfunction were measured 3 days later. Isoflurane-induced changes in hypoxia-inducible factor 1alpha (HIF-1α)-dependent genes were assessed via quantitative polymerase chain reaction. HIF-1α was inhibited pharmacologically via 2-methoxyestradiol (2ME2) or genetically via endothelial cell HIF-1α-null mice (EC-HIF-1α-null). All experiments were performed in a randomized and blinded fashion. RESULTS: Isoflurane postconditioning initiated at clinically relevant time points after SAH significantly reduced cerebral vasospasm, microvessel thrombosis, microvascular dysfunction, and neurological deficits in wild-type (WT) mice. Isoflurane modulated HIF-1α-dependent genes - changes that were abolished in 2ME2-treated WT mice and EC-HIF-1α-null mice. Isoflurane-induced DCI protection was attenuated in 2ME2-treated WT mice and EC-HIF-1α-null mice. INTERPRETATION: Isoflurane postconditioning provides strong HIF-1α-mediated macro- and microvascular protection in SAH, leading to improved neurological outcome. These results implicate cerebral vessels as a key target for the brain protection afforded by isoflurane postconditioning, and HIF-1α as a critical mediator of this vascular protection. They also identify isoflurane postconditioning as a promising novel therapeutic for SAH.

Energy requirements and body composition in stable pediatric intensive care patients receiving ventilatory support.

Energy requirements of pediatric intensive-care patients are unknown due to the difficulty of measuring total energy expenditure in free-living conditions. We investigated energy expenditure and body composition in stable pediatric intensive-care patients receiving long-term ventilatory support. Total energy expenditure and total body water were measured in 10 such patients using the doubly-labeled water method. The patients had significantly lower energy expenditure than healthy children of the same age. Relative to length, fat-free mass deposition was significantly lower, and fat deposition was significantly greater, than in healthy subjects. In general, total energy requirements of these patients are significantly reduced as compared to healthy children, which can be attributed to their lower activity levels and their reduced muscle mass. Although they gain weight similar to healthy children, their weight gain is disproportionately higher in fat. However, one patient with myofibromatosis contradicted this general pattern.

Oral Coenzyme Q10 supplementation does not prevent cardiac alterations during a high altitude trek to everest base cAMP.

Exposure to high altitude is associated with sustained, but reversible, changes in cardiac mass, diastolic function, and high-energy phosphate metabolism. Whilst the underlying mechanisms remain elusive, tissue hypoxia increases generation of reactive oxygen species (ROS), which can stabilize hypoxia-inducible factor (HIF) transcription factors, bringing about transcriptional changes that suppress oxidative phosphorylation and activate autophagy. We therefore investigated whether oral supplementation with an antioxidant, Coenzyme Q10, prevented the cardiac perturbations associated with altitude exposure. Twenty-three volunteers (10 male, 13 female, 46±3 years) were recruited from the 2009 Caudwell Xtreme Everest Research Treks and studied before, and within 48 h of return from, a 17-day trek to Everest Base Camp, with subjects receiving either no intervention (controls) or 300 mg Coenzyme Q10 per day throughout altitude exposure. Cardiac magnetic resonance imaging and echocardiography were used to assess cardiac morphology and function. Following altitude exposure, body mass fell by 3 kg in all subjects (p<0.001), associated with a loss of body fat and a fall in BMI. Post-trek, left ventricular mass had decreased by 11% in controls (p<0.05) and by 16% in Coenzyme Q10-treated subjects (p<0.001), whereas mitral inflow E/A had decreased by 18% in controls (p<0.05) and by 21% in Coenzyme Q10-treated subjects (p<0.05). Coenzyme Q10 supplementation did not, therefore, prevent the loss of left ventricular mass or change in diastolic function that occurred following a trek to Everest Base Camp.

Small-molecule inhibitors of signal transducer and activator of transcription 3 protect against angiotensin II-induced vascular dysfunction and hypertension.

Angiotensin II (Ang II) is known to promote vascular disease and hypertension in part by formation of cytokines, such as interleukin-6. However, the role of signal transducer and activator of transcription 3 (STAT3) in these processes and Ang II/interleukin-6 signaling is unclear. Using 2 models, we tested the hypothesis that STAT3 is essential for Ang II-induced vascular dysfunction and hypertension. Incubation of isolated carotid arteries from C57BL/6J mice with Ang II overnight increased superoxide ≈2-fold and reduced vasodilator responses to the endothelium-dependent agonist acetylcholine by ≈50% versus controls (P<0.05). These effects were prevented by the addition of small-molecular inhibitors of STAT3 activation (S3I-201 or STATTIC). In vivo, administration of Ang II (1.4 mg kg(-1) day(-1)) using osmotic minipumps increased arterial pressure by ≈40 mm Hg at day 14 compared with vehicle-treated mice, and this effect was prevented by S3I-201 treatment (5 mg/kg IP, QOD). After systemic treatment with Ang II, dilator responses to acetylcholine were reduced by ≈30% to 50% in carotid artery and basilar arteries, whereas S3I-201 treatment prevented most of this impairment (P<0.05). In contrast to effects on vascular function and blood pressure, S31-201 did not prevent Ang II-induced hypertrophy in the carotid artery. These findings provide the first evidence that inhibitors of STAT3 activation protect against Ang II-induced oxidative stress, endothelial dysfunction, and hypertension. Because Ang II promotes vascular disease in the presence of multiple cardiovascular risk factors, these results suggest that selective targeting of STAT3 may have substantial therapeutic potential.

Exercise training in dilated cardiomyopathy improves rest and stress cardiac function without changes in cardiac high energy phosphate metabolism.

OBJECTIVE: To determine the effects of short-term exercise training on cardiac function and metabolism during rest and physical exercise in patients with heart failure from dilated cardiomyopathy (DCM). DESIGN: Patients with DCM (n=15, age 58±2 years, NYHA class I-III) were studied before and after 8 weeks of cycle exercise for 20 min, five times per week. MAIN OUTCOME MEASURES: Cardiac volumes, function and high energy phosphate metabolism were measured using cardiac magnetic resonance during rest and 7 min of acute physical exercise (leg-raising). RESULTS: At baseline, average left ventricular ejection fraction (LVEF) was 38±3%, which did not alter during 7 min of exercise. After 8 weeks of home exercise training, there was a 16% improvement in resting LVEF to 44±3% (p<0.01). Training caused a further 20% improvement in LVEF (p<0.05) during acute physical exercise. There was a negative correlation between subjects' baseline level of exercise and change in LVEF (r=-0.67, p<0.05), with sedentary patients having the greatest improvement. Cardiac phosphocreatine (PCr) to ATP ratio did not change during acute physical exercise or after exercise training. CONCLUSIONS: Short-term exercise training improves resting LVEF and LVEF with acute physical exercise with sedentary patients having the greatest improvement. There were no changes in cardiac PCr to ATP, before or after exercise training, suggesting that the improved cardiac function was not caused by improved energetics. Therefore, peripheral factors likely underlie the improved cardiac function in patients with heart failure after short-term exercise.

Two-photon imaging of the mouse eye.

PURPOSE: To image the conventional aqueous outflow pathway and adjacent structures within the intact enucleated mouse eye using a noninvasive microscopy technique. METHODS: Two-photon microscopy (2PM) techniques, including two-photon autofluorescence (2PAF) and second harmonic generation (SHG), were used to obtain images of the trabecular meshwork (TM) region within an intact mouse eye. Cardiac perfusion of fluorescein-conjugated dextran was used to label blood vessels within the eye to serve as an anatomic reference. Eyes were subsequently fixed, paraffin embedded, sectioned, and stained for comparison to the 2PM images. RESULTS: Three-dimensional analyses of multiple 2PM images revealed a well-defined region adjacent to the iris and cornea that is free of SHG signal and consistent with the location of Schlemm's canal. This open region is continuous with smaller tube structures consistent with collector channels. These structures do not label in mice perfused with the vascular probe dextran, supporting the hypothesis that the enclosed spaces are filled with aqueous humor rather than circulating blood. The TM region in the mouse eye was also visible, with a clear SHG signal representing collagen fibers. CONCLUSIONS: These results support the hypothesis that 2PM may be useful for noninvasively imaging the conventional aqueous outflow pathway in mouse models of glaucoma. Studies are ongoing to validate our methodology in live animals.

Complementary techniques for the characterization of thin film Ti/Nb multilayers.

An aberration corrector on the probe-forming lens of a scanning TEM (STEM) equipped with an electron energy-loss spectrometer (EELS) and X-ray energy-dispersive spectrometer (XEDS) has been employed to investigate the compositional variations as a function of length scale in nanoscale Ti/Nb metallic multilayers. The composition profiles of EELS and XEDS were compared with the profiles obtained from the complementary technique of 3D atom probe tomography. At large layer widths (h > or = 7 nm, where h is the layer width) of Ti and Nb, XEDS composition profiles of Ti/Nb metallic multilayers are in good agreement with the EELS results. However, at reduced layer widths (h approximately 2 nm), profiles of EELS and atom probe exhibited similar compositional variations, whereas XEDS results have shown a marked difference. This difference in the composition profiling of the layers has been addressed with reference to the effects of beam broadening and the origin of the signals collected in these techniques. The advantage of using EELS over XEDS for these nanoscaled multilayered materials is demonstrated.

Chiral determination: direct interpretation of convergent-beam electron diffraction patterns using the series expansion of Cowley and Moodie.

Given a small number of structure factors of a known chiral structure of unknown hand, it is shown that the hand can be determined from the sign of the contrast difference of two reflections in a suitably oriented convergent-beam electron diffraction (CBED) pattern. A simple formula for this difference, which takes into account all the significant second-order scattering, is derived using the series expansion of Cowley and Moodie for n-beam diffraction. The reason for the success of a three-beam interpretation is investigated. The method is applied to patterns from thin crystals in which a mirror projection symmetry can be found and its validity is demonstrated by agreement with experiment using samples of known hand. The advantages of recording patterns near major zone axes are discussed as well as some other experimental aspects of chiral determination using CBED.

A combination method of charge density measurement in hard materials using accurate, quantitative electron and X-ray diffraction: the alpha-Al2O3 case.

Current X-ray diffraction techniques intended for "ideally imperfect" specimens provide structure factors only on a relative scale and ever-present multiple scattering in strong low-angle Bragg reflections is difficult to correct. Multiple scattering is implicit in the quantitative convergent beam electron diffraction (QCBED) method, which provides absolutely scaled structure factors. Conventional single crystal X-ray diffraction has proved adequate in softer materials where crystal perfection is limited. In hard materials, the highly perfect nature of the crystals is often a difficulty, due to the inadequacy of the conventional corrections for multiple scattering (extinction corrections). The present study on alpha-Al2O3 exploits the complementarity of synchrotron X-ray measurements for weak and medium intensities and QCBED measurement of the strong low-angle reflections. Two-dimensional near zone axis QCBED data from different crystals at various accelerating voltages, thicknesses, and orientations have been matched using Bloch-wave and multislice methods. The reproducibility of QCBED data is better than 0.5%. The low-angle strong QCBED structure factors were combined with middle and high-angle extinction-free data from synchrotron X-ray diffraction measurements. Static deformation charge density maps for alpha-Al2O3 have been calculated from a multipole expansion model refined using the combined QCBED and X-ray data.

The underglycosylation of plasma alpha 1-antitrypsin in congenital disorders of glycosylation type I is not random.

Conditions under which the glycosylation capacity of cells is limited provide an opportunity for studying the efficiency of site-specific glycosylation and the role of glycosylation in the maturation of glycoproteins. Congenital disorders of glycosylation type 1 (CDG-I) provide such a system. CDG-I is characterized by underglycosylation of glycoproteins due to defects in the assembly or transfer of the common dolichol-pyrophosphate-linked oligosaccharide precursor of asparagine-linked glycans. Human plasma alpha1-antitrypsin is normally fully glycosylated at three asparagine residues (46, 83, and 247), but un-, mono-, di-, and fully glycosylated forms of alpha1-antitrypsin were detected by 2D PAGE in the plasma from patients with CDG-I. The state of glycosylation of the three asparagine residues was analyzed in all the underglycosylated forms of alpha1-antitrypsin by peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was found that asparagine 46 was always glycosylated and that asparagine 83 was never glycosylated in the underglycosylated glycoforms of alpha1-antitrypsin. This showed that the asparagine residues are preferentially glycosylated in the order 46>247>83 in the mature underglycosylated forms of alpha1-antitrypsin found in plasma. It is concluded that the nonoccupancy of glycosylation sites is not random under conditions of decreased glycosylation capacity and that the efficiency of glycosylation site occupancy depends on structural features at each site. The implications of this observation for the intracellular transport and sorting of glycoproteins are discussed.