Scanning structural mapping at the Life Science X-ray Scattering Beamline.

This work describes the instrumentation and software for microbeam scattering and structural mapping at the Life Science X-ray Scattering (LiX) beamline at NSLS-II. Using a two-stage focusing scheme, an adjustable beam size between a few micrometres and a fraction of a millimetre is produced at the sample position. Scattering data at small and wide angles are collected simultaneously on multiple Pilatus detectors. A recent addition of an in-vacuum Pilatus 900k detector, with the detector modules arranged in a C-shaped configuration, has improved the azimuthal angle coverage in the wide-angle data. As an option, fluorescence data can be collected simultaneously. Fly scans have been implemented to minimize the time interval between scattering patterns and to avoid unnecessary radiation damage to the sample. For weakly scattering samples, an in-vacuum sample environment has been developed here to minimize background scattering. Data processing for these measurements is highly sample-specific. To establish a generalized data process workflow, first the data are reduced to reciprocal coordinates at the time of data collection. The users can then quantify features of their choosing from these intermediate data and construct structural maps. As examples, results from in-vacuum mapping of onion epidermal cell walls and 2D tomographic sectioning of an intact poplar stem are presented.

Daily low-dose folic acid supplementation does not prevent nitroglycerin-induced nitric oxide synthase dysfunction and tolerance: a human in vivo study.

INTRODUCTION: Continuous treatment with nitroglycerin (GTN) causes tolerance and endothelial dysfunction, both of which may involve endothelial nitric oxide synthase (eNOS) dysfunction. eNOS dysfunction may be linked to depletion of tetrahydrobiopterin, and folic acid may be involved in the regeneration of this cofactor. It has been demonstrated that 10 mg/day folic acid supplementation prevents the development of GTN tolerance and GTN-induced endothelial dysfunction. However, the efficacy of daily lower-dose folic acid supplementation for preventing these phenomena has not been investigated. OBJECTIVE: To determine the effect of 1 mg/day folic acid supplementation on responses to sustained GTN therapy. METHODS AND RESULTS: On visit 1, 20 healthy male volunteers were randomly assigned to receive either oral folic acid (1 mg/day) or placebo for one week in a double- blind study. All subjects also received continuous transdermal GTN (0.6 mg/h). On visit 2, forearm blood flow was measured using venous occlusion strain-gauge plethysmography in response to incremental intra-arterial infusions of acetylcholine, N-monomethyl-L-arginine and GTN. Subjects in both groups displayed significantly decreased responses to acetylcholine and N-monomethyl-L-arginine infusions compared with a control group that received no treatment. Responses to GTN were also significantly diminished in both groups (P<0.05 for all). DISCUSSION: The present data demonstrate that daily supplementation with 1 mg folic acid does not prevent the development of GTN-induced eNOS dysfunction or tolerance.

The acute administration of either amiloride or captopril does not prevent endothelial dysfunction induced by ischemia and reperfusion in the human forearm vasculature.

Animal studies have demonstrated the ability of both sodium-hydrogen exchange inhibitors and angiotensin-converting enzyme inhibitors to reduce infarct size and preserve postischemic ventricular function following ischemia and reperfusion (IR) injury. Whether these interventions can also prevent IR-induced impairment of endothelial function in humans has not been investigated. We performed 2 separate double-blind, placebo-controlled, crossover studies. In the first study, 10 healthy volunteers were randomized to receive oral amiloride (10 mg) or a placebo. In a separate study, another group of volunteers (n = 10) was randomized to receive oral captopril (50 mg) or a placebo. At the time of the peak hemodynamic effect of the drug (3 and 1.5 h after administration of amiloride and captopril, respectively), endothelium-dependent, flow-mediated dilatation of the radial artery was measured before and after IR. IR significantly blunted flow-mediated dilatation in all groups (placebo: pre-IR: 6.8% ± 0.7%; post-IR: 2.9% ± 0.9%; P < 0.01; amiloride: pre-IR: 5.9% ± 0.6%; post-IR: 2.1% ± 1.3%; P = 0.01; captopril: pre-IR: 6.0% ± 0.5%; post-IR: 2.0% ± 0.6%; P < 0.01). In humans, neither 10 mg of oral amiloride nor 50 mg of oral captopril was able to provide protection against IR-induced endothelial dysfunction in the peripheral vasculature.

Continuous therapy with transdermal nitroglycerin does not affect biomarkers of vascular inflammation and injury in healthy volunteers.

Continuous exposure to nitroglycerin (GTN) results in development of tolerance and is associated with increased free radical production and abnormal endothelial function. Elevated plasma biomarkers of inflammation have been shown to be associated with endothelial dysfunction in most cardiovascular conditions. It remains unclear whether exposure to GTN is also associated with increased biomarkers of endothelial and vascular injury or vascular inflammation. In an investigator-blind study, a total of 28 healthy volunteers were randomized to continuous therapy with GTN (0.6 mg/h 24 h/day for 7 days) or no therapy. Venous blood was collected on day 0 and day 7. Plasma levels of markers such as asymmetric dimethyl-arginine (ADMA), human soluble P-selectin, interleukin-6, tumor necrosis factor-alpha, intercellular adhesion molecule-1, and oxidized low-density lipoproteins were measured. The levels of blood markers on day 0 were similar in the control and GTN-treated groups. After 7 days of GTN exposure, there were no significant changes in the different markers of vascular inflammation and injury either in the GTN or control group (all p > 0.5). The present study documents that prolonged continuous therapy with transdermal GTN therapy is not associated with changes in markers of vascular inflammation and injury.

Once daily therapy with isosorbide-5-mononitrate causes endothelial dysfunction in humans: evidence of a free-radical-mediated mechanism.

OBJECTIVES: The aim of the study was to determine if isosorbide-5-mononitrate (IS-5-MN) 120 mg, taken once daily for 7 days, is associated with evidence of endothelial dysfunction and whether this effect is determined by increased free radical production. BACKGROUND: Tolerance to nitroglycerin is associated with increased free radical production and abnormal endothelial function. To date, no data is available concerning the effect of IS-5-MN, administered in clinically employed dosages, on endothelial function in humans. METHODS: A total of 19 healthy volunteers were randomized in a double-blind fashion to therapy with IS-5-MN (120 mg once daily) or placebo. After 7 days of treatment, forearm blood flow responses to acetylcholine (Ach; 7.5, 15, and 30 microg/min) and N-monomethyl-L-arginine (L-NMMA; 1, 2, and 4 mumol/min) were measured. In a separate study, after 7 days of therapy with IS-5-MN 120 mg once daily, the responses to Ach were assessed during intra-arterial coinfusion of vitamin C (24 mg/min) or saline. RESULTS: As compared with placebo, IS-5-MN caused significant blunting of the responses to both Ach (peak responses: placebo 127 +/- 31%; IS-5-MN 52 +/- 24%) and L-NMMA (peak responses: placebo 41 +/- 5%; IS-5-MN 22 +/- 8%). Vitamin C completely restored the forearm blood flow responses to Ach (peak responses: vitamin C 180 +/- 33%; saline 107 +/- 17%). CONCLUSIONS: We document for the first time that IS-5-MN impairs endothelial function in humans in vivo. Suggesting a role of oxygen free radicals, nitrate-induced abnormalities in endothelium-dependent vasomotor responses were reversed by the antioxidant vitamin C.

Nitroglycerin attenuates human endothelial progenitor cell differentiation, function, and survival.

Endothelial progenitor cells (EPCs) participate in angiogenesis and the response to chronic ischemia. Risk factors and cardiovascular disease attenuate EPC number, function, and survival. Continuous therapy with nitroglycerin (glyceryl trinitrate; GTN) is associated with increased vascular oxidative stress, leading to nitrate tolerance and endothelial dysfunction. Thus, GTN therapy may also affect EPCs. The purpose of this study was to determine whether continuous exposure to GTN in vivo or during ex vivo expansion affects the circulating number and functional characteristics of human EPCs. To determine the effects of continuous in vivo GTN exposure, EPCs isolated from 28 healthy males before and after receiving 0.6 mg/h GTN (n=17) or no treatment (n=11) for 1 week were expanded for 6 days and compared. To determine the effects of continuous ex vivo GTN exposure, EPCs isolated before randomization were expanded for 6 days in medium supplemented with 100 nM, 300 nM, or 1 microM GTN. EPCs expanded without GTN served as controls (n=10). In vivo, GTN exposure significantly increased the percentage of circulating cells expressing the EPC marker CD34 and increased the susceptibility of expanded EPCs to apoptosis but had no impact on the phenotypic differentiation or migration of EPCs. Ex vivo, GTN exposure increased apoptosis while decreasing phenotypic differentiation, migration, and mitochondrial dehydrogenase activity of EPCs, compared with EPCs expanded in the absence of GTN. Taken together, these results suggest that continuous GTN therapy might impair EPC-mediated processes, an effect that could be detrimental in the setting of ischemic cardiovascular disease.