In microvessels of patients with coronary artery disease (CAD), flow-mediated dilation (FMD) is largely dependent upon the endothelium-derived hyperpolarizing factor HO. The goal of this study is to examine the influence of age and presence or absence of disease on the mechanism of FMD. Human coronary or adipose arterioles (~150 µm diameter) were prepared for videomicroscopy. The effect of inhibiting COX [indomethacin (Indo) or NOS (L-NAME), eliminating HO (polyethylene glycol-catalase (PEG-CAT)] or targeting a reduction in mitochondrial ROS with scavengers/inhibitors [Vitamin E (Vitamin E); phenylboronic acid (PBA)] was determined in children aged 0-18 years; young adults 19-55 years; older adults >55 years without CAD, and similarly aged adults with CAD. Indo eliminated FMD in children and reduced FMD in younger adults. This response was mediated mainly by PGI, as the prostacyclin-synthase-inhibitor trans-2-phenyl cyclopropylamine reduced FMD in children and young adults. L-NAME attenuated dilation in children and younger adults and eliminated FMD in older adults without CAD, but had no effect on vessels from those with CAD, where mitochondria-derived HO was the primary mediator. The magnitude of dilation was reduced in older compared to younger adults independent of CAD. Exogenous treatment with a sub-dilator dose of NO blocked FMD in vessels from subjects with CAD, while prolonged inhibition of NOS in young adults resulted in a phenotype similar to that observed in disease. The mediator of coronary arteriolar FMD evolves throughout life from prostacyclin in youth, to NO in adulthood. With the onset of CAD, NO-inhibitable release of HO emerges as the exclusive mediator of FMD. These findings have implications for use of pharmacological agents, such as nonsteroidal anti-inflammatory agents in children and the role of microvascular endothelium in cardiovascular health.

We examined whether survival of different rat strains administered anthrax lethal toxin is genetically determined. A reproducible test population of first filial generation hybrid rats was bred based on the susceptibility of progenitors to anthrax lethal toxin and to maximize genetic diversity across the strains. These rats were then tested with varying doses of anthrax lethal toxin. We found that all 'sensitive' strains died within 2 h following systemic administration of 240 mg/kg lethal toxin, while one strain survived following a five times higher dose (1.4 mg/kg). The ability of lethal toxin to lyse macrophage cultures derived from the bone marrow of these strains corresponded with in vivo results. We conclude that a rat test population can detect strain differences in response to anthrax lethal toxin. Survival is influenced by the host genome background and is likely due to a single gene with a recessive mode of inheritance.

Hydrogen peroxide (H2O2) has been show to be an endothelial derived factor that derived from the endothelium regulates vascular tone and tissue blood flow. In previous studies, inhibition of the endogenous H2O2 scavenger, catalase, increased the sensitivity of H2O2‐ induced dilation in an endothelium‐dependent manner in human adipose microvessels. Since ROS and their scavengers may be upregulated in disease, the purpose of this study was to determine if catalase modulation of H2O2 dilation is greater in the coronary and peripheral microcirculation of patients with coronary artery disease (CAD).Methods: Arterioles (∼150 μm ID) obtained at surgery from patients with or without CAD (atrial appendage and visceral adipose), were prepared for videomicroscopy. Vasomotor responses to exogenous H2O2 (10–10 to 10–3 M) were evaluated in the presence and absence of the catalase inhibitor 3‐amino‐1,2,4‐triazole (ATZ (4x10–2M)). Results: H2O2 dilated adipose [ED50=7.62x10‐6M, n=7] and coronary arterioles [ED50=8.63x10–6M] from CAD patients. Adipose tissue with CAD were not affected by inhibition of catalase with ATZ [ED50=4.31x10–6M, n=7]. Treatment with ATZ did not enhance dilation to H2O2 in any of the tissues tested. (with CAD [ED50=4.19x10–6M]; without CAD [ED50=1.08x10–5M]). Conclusions: H2O2 signaling is less modulated by endogenous catalase in subjects with than those without CAD.

BackgroundAcetylcholine (ACH) dose‐dependently constricts human coronary atrial arterioles (HCA) but dilates ventricular arterioles in patients with coronary disease. Whether NO mechanisms participate in Ach‐induced dilation in atrial arterioles remains unclear. Since CAD and its risk factors generate ROS that can quench NO, we hypothesized that acetylcholine‐induced dilation can be restored in HCA by restoring redox balance and supporting exogenous NO synthesis.MethodsArterioles (~150 μm) from patients with coronary artery disease were prepared for videomicroscopy. HCA's were pre‐incubated with indomethacin (10‐5), sepiapterin (10‐4), L‐Arginine (10‐3), and Peg‐SOD (300units/ml) for 30 minutes. After preconstruction with endothelin‐1, vasomotor responses to exogenous ACH (10‐9 to 10‐4 M) were evaluated in control or in the presence and absence of N‐nitro‐L‐arginine methyl ester (L‐name; 10‐4)).ResultsACH dilated pretreated compared to control intact arterioles [%max dilation (MD): 80±8% vs. ‐36.4±7%]. Treatment with L‐name abolished dilation (MD:‐0.5±9%).ConclusionsReducing endogenous oxidative stress and providing NOS substrate converts an , ACH‐induced constriction to an NO ‐dependent vasodilation in HCA. There may be a greater sensitivity to ROS in endothelium of atrial vs. ventricular arterioles.

Light chain amyloidosis (AL), a plasma cell dyscrasia, is often fatal in patients with heart failure yet the mechanism of injury is unknown. We hypothesize that AL light chains (LC) acutely cause endothelial dysfunction in coronary arterioles. The aims are to measure endothelium-dependent and independent dilation and reactive oxygen species (ROS) in human coronary arterioles exposed to LC from AL subjects. Urine LC was isolated from 3 AL subjects by dialysis and lyophilization. Separately, 15 coronary arterioles were isolated from discarded atrial tissue from 12 non-AL subjects without significant vascular disease or diabetes undergoing open heart surgery (8 males, 70 ± 11 years). Arterioles were pressure-mounted and vessel diameter measured by videomicroscopy. Post-preconstriction with endothelin-1, vessels were exposed to vehicle and sequential doses of bradykinin (endothelium-mediated,10 −10 to 10 −6 M) then papaverine (endothelium-independent, 10 −4 M) were given and dilation was measured. Dilator response was again measured after 20 μg/mL of LC. Coronary arterioles were separately exposed to vehicle and LC and mitochondrial superoxide (MitoSox Red) and hydrogen peroxide (dichlorofluoroscein) were measured by fluorescence microscopy. see figure. Brief exposure to AL amyloid light chains impairs endothelium-dependent coronary vasodilation. This is associated with increased ROS production. This is the first human study demonstrating endothelial dysfunction in coronary microvasculature caused by AL LC. The vascular dysfunction and oxidative stress may be an important mechanism in the pathophysiology of this fatal disease.

BackgroundFlow‐induced dilation (FID) is an important physiological stimulus in regulating tissue blood flow. FID is typically mediated by endogenous vasodilators including nitric oxide (NO) and prostacyclin. In humans with coronary artery disease (CAD), FID occurs but is not mediated by NO; rather hydrogen peroxide (H2O2) is responsible. In the coronary circulation H2O2 derives from the mitochondria, but it is not known if this is a generalized process that also involves adipose arterioles. We examined the hypothesis that the mitochondrial electron transport chain critically contributes to FID in human adipose arterioles.MethodsVisceral adipose arterioles (~150 μm ID) obtained at surgery from patients with or without CAD were cannulated and pressurized (60 mmHg) for videomicroscopy. After constriction with endothelin‐1, vasomotor responses to flow were evaluated in the presence and absence of the rotenone (1microM), a mitochondrial complex I inhibitor. At the end of each experiment, papaverine (0.1 mM) was added to obtain the maximal arteriolar diameter and assess non‐specific effects of rotenone.ResultsFID was reduced by rotenone in adipose arterioles from patients with CAD (%max dilation: 33±10 vs. 73±10%). In contrast, rotenone had no effect on FID arterioles from patients without CAD (%max dilation: 61±9 vs. 65±6%) or on the dilation to papaverine in either group.ConclusionsWe speculate that in patients with but not in those without CAD FID requires an intact proximal mitochondrial electron transport. This is a general response seen in multiple vascular beds.

BACKGROUND/AIMS Over 90% of drug candidates fail in clinical trials, incurring high costs to the pharmaceutical industry. To address this problem, PhysioGenix has developed a novel combinatorial breeding strategy, the PharmGenix™ panel, to capture greater genetic diversity within the rat genome and allow for preclinical drug screening. Tacrine, which causes hepatotoxicity in a small percentage of the human population, was tested to demonstrate the utility of this panel. METHODS A single dose of tacrine (35 mg/kg) was administered to each of six hybrid PharmGenix™ strains, CD‐IGS and CDF strains. Rats were euthanized twenty‐four hours later and serum analyzed for alanine transaminase (ALT) and aspartate transaminase (AST) levels, indicators of hepatotoxicity. RESULTS Tacrine did not significantly elevate ALT levels in CD‐IGS or CDF, however, the PharmGenix™ panel showed significant ALT elevations in five of the six strains, ranging from 111% to 142% of control values. CD‐IGS and CDF exhibited increases in AST levels of 82% and 174%, respectively, while the PharmGenix ™ rats showed much higher AST levels, ranging between 621% and 1069% of control values in four of the six strains. CONCLUSIONS The ability to detect significant AST and ALT elevations in the PharmGenix™ panel, but not in CD‐IGS or CDF, suggests a genetic component underlies the development of tacrine toxicity and may have alerted the pharmaceutical industry to toxicity currently seen in a small percentage of the human population. Clinical Pharmacology & Therapeutics (2005) 79, P82–P82; doi: