The balance of nitric oxide (⅐NO) and superoxide anion (O 2 . ) plays an important role in vascular biology. The association of heat shock protein 90 (Hsp90) with endothelial nitric-oxide synthase (eNOS) is a critical step in the mechanisms by which eNOS generates ⅐NO. As eNOS is capable of generating both ⅐NO and O 2 . , we hypothesized that Hsp90 might also mediate eNOS-dependent O 2 . production. To test this hypothesis, bovine coronary endothelial cells (BCEC) were pretreated with geldanamycin (GA, 10 g/ml; 17.8 M) and then stimulated with the calcium ionophore, A23187 (5 M). GA significantly decreased A23187-stimulated eNOS-dependent nitrite production (p < 0.001, n ‫؍‬ 4) and significantly increased A23187-stimulated eNOS-dependent O 2 . production (p < 0.001, n ‫؍‬ 8). A23187 increased phospho-eNOS(Ser-1179) levels by >1.6-fold over vehicle (V)-treated levels. Pretreatment with GA by itself or with A23187 increased phospho-eNOS levels. In unstimulated V-treated BCEC cultures low amounts of Hsp90 were found to associate with eNOS. Pretreatment with GA and/or A23187 increased the association of Hsp90 with eNOS. These data show that Hsp90 is essential for eNOS-dependent ⅐NO production and that inhibition of ATP-dependent conformational changes in Hsp90 uncouples eNOS activity and increases eNOS-dependent O 2 . production.Nitric oxide (⅐NO) and superoxide anion (O 2 . ) play opposing roles in vascular biology. Nitric oxide generation is increased greatly when Hsp90 associates with eNOS 1 in endothelial cell cultures (1, 2). A decrease in the amount of Hsp90 co-precipitating with eNOS is associated with a decrease in ⅐NO production by pulmonary artery endothelial cells exposed to prolonged periods of hypoxia (3). Geldanamycin (GA) is an ansamycin antibiotic that binds to the ATP binding site of Hsp90, thereby inhibiting the ATP/ADP cycle required for the interaction with client proteins such as eNOS (2-4). GA has been used to demonstrate that ⅐NO production in mesentary arteries and rat aortas depends on Hsp90 activity, implying that factors adversely affecting this interaction between Hsp90 and eNOS may be one of the mechanisms for portal hypertension and increased vascular tone (2, 4). Taken together, these reports indicate that Hsp90 is critical for eNOS generation of ⅐NO.Emerging evidence suggests that under pathological conditions eNOS may also generate O 2. (5-

Signals from the intestinal microbiota are important for normal host physiology; alteration of the microbiota (dysbiosis) is associated with multiple disease states. We determined the effect of antibiotic-induced intestinal dysbiosis on circulating cytokine levels and severity of ischemia/reperfusion injury in the heart. Treatment of Dahl S rats with a minimally absorbed antibiotic vancomycin, in the drinking water, decreased circulating leptin levels by 38%, resulted in smaller myocardial infarcts (27% reduction), and improved recovery of postischemic mechanical function (35%) as compared with untreated controls. Vancomycin altered the abundance of intestinal bacteria and fungi, measured by 16S and 18S ribosomal DNA quantity. Pretreatment with leptin (0.12 μg/kg i.v.) 24 h before ischemia/reperfusion abolished cardioprotection produced by vancomycin treatment. Dahl S rats fed the commercially available probiotic product Goodbelly, which contains the leptin-suppressing bacteria Lactobacillus plantarum 299v, also resulted in decreased circulating leptin levels by 41%, smaller myocardial infarcts (29% reduction), and greater recovery of postischemic mechanical function (23%). Pretreatment with leptin (0.12 μg/kg i.v.) abolished cardioprotection produced by Goodbelly. This proof-of-concept study is the first to identify a mechanistic link between changes in intestinal microbiota and myocardial infarction and demonstrates that a probiotic supplement can reduce myocardial infarct size.

Abstract-Hypoxia causes localized pulmonary arterial (PA) constriction to divert blood flow to optimally ventilated regions of the lung. is an essential mechanism to balance perfusion with ventilation and is unique to the pulmonary circulation. This action regulates one of the most important physiological parameters in mammals, arterial oxygen tension. The acute response is biphasic in isolated perfused vessels, whereas sustained hypoxia in vivo can lead to structural remodeling and matrix deposition in pulmonary arteries, which results in increased arterial tone. 1 The biochemical mechanisms that have been hypothesized to underlie this response are varied, 1-3 except for the consensus that HPV is a multifactorial manifestation, with "elements of energy, oxygen and lipid metabolism" (page 1192). 3 Recently, specific candidates that mediate HPV have been carefully examined (eg, Archer et al 2 ). Nitric oxide (NO), cytochrome P450 metabolites, leukotrienes, or direct effects of oxygen on ion channels in vascular smooth muscle cells have been proposed as modulators/mediators of acute hypoxic responses of the pulmonary vasculature, but none appears to account for all the features of hypoxic vasoconstriction. 2 Our overriding goal was to understand adaptation of pulmonary arteries (PAs) to chronic hypoxia. Arachidonic acid (AA) metabolites modulate vasoactivity, and oxygen is a substrate in eicosanoid synthesis, which makes these products ideal candidates to contribute to HPV. 5-hydroxyeicosatetraenoic acid (HETE), the major metabolites of the 5-lipoxygenase pathway, is increased in macrophages exposed to hypoxia. 4 However, hypoxic pulmonary vasoconstriction is not affected by the 5-lipoxygenase inhibitor MK886 in perfused rabbit lungs. 5 To examine the effects of subacute hypoxia on pulmonary metabolism of AA and the reactivity of PAs to these metabolites, we used an animal model in which rabbit kits Original

Rationale: A strong association has emerged between the gut microbiome and atherosclerotic disease. Our recent data suggest L. plantarum 299v (Lp299v) supplementation reduces infarct size in male rats. Limited human data are available on the impact of Lp299v on the vasculature. Objective: To determine whether oral Lp299v supplementation improves vascular endothelial function and reduces systemic inflammation in humans with stable coronary artery disease (CAD). Methods and Results: Twenty men with stable CAD consumed a drink containing Lp299v (20 billion CFU) once daily for six weeks. Following a 4-week washout, subjects were given an option of additionally participating in a 10-day study of oral liquid Vancomycin (250 mg 4x daily). Vascular endothelial function was measured by brachial artery flow-mediated dilation (FMD). Before and following Lp299v, plasma short-chain fatty acids, trimethylamine oxide (TMAO), and adipokine levels were measured. Additional plasma samples underwent unbiased metabolomic analyses using liquid chromatography/mass spectroscopy (UHPLC/MS). 16S rDNA sequencing was used to determine changes of the stool microbiome. Arterioles from CAD patients were obtained and endothelium-dependent vasodilation was measured by video-microscopy following intra-luminal incubation with plasma from Lp299v study subjects. Lp299v supplementation improved brachial FMD (P=0.008) without significant changes in plasma cholesterol profiles, fasting glucose, or body mass index. Vancomycin did not impact FMD. Lp299v supplementation decreased circulating levels of IL-8 (P=0.01), IL-12 (P=0.02), and leptin (P=0.0007) but did not significantly change plasma TMAO concentrations (P=0.27). Plasma propionate (P=0.004) increased while acetate levels decreased (P=0.03). Post-Lp299v plasma improved endothelium-dependent vasodilation in resistance arteries from patients with CAD (P=0.02).16S rRNA analysis the showed Lactobacillus genus was enriched in post-probiotic stool samples without other changes. Conclusions: Lp299v improved vascular endothelial function and decreased systemic inflammation in men with CAD, independent of changes in traditional risk factors and TMAO. Circulating gut-derived metabolites likely account for these improvements and merit further study. Clinical Trial: NCT01952834

Intestinal microbiota determine severity of myocardial infarction in rats. We determined whether low molecular weight metabolites derived from intestinal microbiota and transported to the systemic circulation are linked to severity of myocardial infarction. Plasma from rats treated for seven days with the non-absorbed antibiotic vancomycin or a mixture of streptomycin, neomycin, polymyxin B and bacitracin was analyzed using mass spectrometry-based metabolite profiling platforms. Antibiotic-induced changes in the abundance of individual groups of intestinal microbiota dramatically altered the host’s metabolism. Hierarchical clustering of dissimilarities separated the levels of 284 identified metabolites from treated vs. untreated rats; 193 were altered by the antibiotic treatments with a tendency towards decreased metabolite levels. Catabolism of the aromatic amino acids phenylalanine, tryptophan and tyrosine was the most affected pathway comprising 33 affected metabolites. Both antibiotic treatments decreased the severity of an induced myocardial infarction in vivo by 27% and 29%, respectively. We then determined whether microbial metabolites of the amino acids phenylalanine, tryptophan and tyrosine were linked to decreased severity of myocardial infarction. Vancomycin-treated rats were administered amino acid metabolites prior to ischemia/reperfusion studies. Oral or intravenous pretreatment of rats with these amino acid metabolites abolished the decrease in infarct size conferred by vancomycin. Inhibition of JAK-2 (AG-490, 10 μM), Src kinase (PP1, 20 μM), Akt/PI3 kinase (Wortmannin, 100 nM), p44/42 MAPK (PD98059, 10 μM), p38 MAPK (SB203580, 10 μM), or KATP channels (glibenclamide, 3 μM) abolished cardioprotection by vancomycin, indicating microbial metabolites are interacting with cell surface receptors to transduce their signals through Src kinase, cell survival pathways and KATP channels. These inhibitors have no effect on myocardial infarct size in untreated rats. This study links gut microbiota metabolites to severity of myocardial infarction and may provide future opportunities for novel diagnostic tests and interventions for the prevention of cardiovascular disease.

The grief process in bereaved children is conceptualized as a series of psychological tasks that must be accomplished over time. Early tasks include understanding and self-protection; middle-phase tasks include acceptance and reworking; late tasks pertain to identification and development. This timing model has practical implications for the conduct of psychotherapy at different times during the grief process.

Purpose-To determine the impact of 10 Gy total body irradiation (TBI) or local thorax irradiation, a dose relevant to a radiological terrorist threat, on lipid and liver profile, coronary microvasculature and ventricular function.Materials and methods-WAG/RijCmcr rats received 10 Gy TBI followed by bone marrow transplantation, or 10 Gy local thorax irradiation. Age-matched, non-irradiated rats served as controls. The lipid profile and liver enzymes, coronary vessel morphology, nitric oxide synthase (NOS) isoforms, protease activated receptor (PAR)-1 expression and fibrinogen levels were compared. Two dimensional strain echocardiography assessed global radial and circumferential strain on the heart.Results-TBI resulted in a sustained increase in total and low density lipoprotein (LDL) cholesterol (190±8 vs. 58±6; 82±8 vs. 13±3 mg/dL, respectively). The density of small coronary arterioles was decreased by 32%. Histology revealed complete blockage of some vessels while cardiomyocytes remained normal. TBI resulted in cellular peri-arterial fibrosis whereas control hearts had symmetrical penetrating vessels with less collagen and fibroblasts. TBI resulted in a 32±4% and 28 ±3% decrease in endothelial NOS and inducible NOS protein respectively, and a 21±4% and 35±5% increase in fibrinogen and PAR-1 protein respectively, after 120 days. TBI reduced radial strain (19 Declaration of interest:The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. NIH Public Access Author ManuscriptInt J Radiat Biol. Author manuscript; available in PMC 2010 December 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript ±8 vs. 46±7%) and circumferential strain (-8±3 vs. −15±3%) compared to controls. Thorax-only irradiation produced no changes over the same time frame.Conclusions-TBI with 10 Gy, a dose relevant to radiological terrorist threats, worsened lipid profile, injured coronary microvasculature, altered endothelial physiology and myocardial mechanics. These changes were not manifest with local thorax irradiation. Non-thoracic circulating factors may be promoting radiation-induced injury to the heart.

Reduction of nitrite to nitric oxide during ischemia protects the heart against injury from ischemia/ reperfusion. However the optimal dose of nitrite and the mechanisms underlying nitrite-induced cardioprotection are not known. We determined the ability of nitrite and nitrate to confer protection against myocardial infarction in two rat models of ischemia/reperfusion injury and the role of xanthine oxidoreductase, NADPH oxidase, nitric oxide synthase and K ATP channels in mediating nitrite-induced cardioprotection. In vivo and in vitro rat models of myocardial ischemia/reperfusion injury were used to cause infarction. Hearts (n=6/group) were treated with nitrite or nitrate for 15 min prior to 30 min regional ischemia and 180 min reperfusion. Xanthine oxidoreductase activity was measured after 15 min aerobic perfusion and 30 min ischemia. Nitrite reduced myocardial necrosis and decline in ventricular function following ischemia/reperfusion in the intact and isolated rat heart in a dose or concentration-dependent manner with an optimal dose of 4 mg/kg in vivo and concentration of 10 μM in vitro. Nitrate had no effect on protection. Reduction in infarction by nitrite was abolished by inhibition of flavoprotein reductases and the molybdenum site of xanthine oxidoreductase, and was associated with an increase in activity of xanthine dehydrogenase and xanthine oxidase during ischemia. Inhibition of nitric oxide synthase had no effect on nitrite-induced cardioprotection. Inhibition of NADPH oxidase and K ATP channels abolished nitrite-induced cardioprotection. Nitrite but not nitrate protects against infarction by a mechanism involving xanthine oxidoreductase, NADPH oxidase and K ATP channels.