ENDOVASCULAR PROCEDURES FOR LOWER LIMB PERIPHERAL ARTERIAL DISEASE IN AN AMBULATORY UNIT.

OBJECTIVES: To analyse the safety and outcomes of endovascular procedures in an ambulatory practice. METHODS: Data were collected from a cohort of patients admitted in an ambulatory unit for an endovascular procedure for lower limb (LL) arterial occlusive disease during a one year period. RESULTS: A total of 168 procedures were carried out in 134 patients. Patients' mean age was 67 (39-91) years and 78% were male. Most patients presented with lower limb ulcer or gangrene (43%) or disabling claudication (40%). Most frequent comorbidities included hypertension (75.4%), dyslipidemia (72.4%) and diabetes mellitus (57.5%). The preferred vascular access for the procedures was the common femoral artery (52%), superficial femoral artery (24%) and humeral artery (21%). Global complication rate was 19% but only one major, non-fatal complication was identified. The most common complication was arterial dissection (8.3%), none compromising blood flow. One-year amputation rate was 6.7%, and one-year mortality was 3.0%. Factors significantly associated with procedure complications were female sex, hypertension and dyslipidemia. CONCLUSION: Ambulatory endovascular procedures for PAD are safe and effective in selected patients. Both the low rate and low severity of complications make them an attractive option in the prospect of diminishing the burden of these patients on the health-care system while improving patient comfort.

Impact of Compliance with a Sepsis Resuscitation Bundle in a Portuguese Emergency Department.

INTRODUCTION: Severe sepsis and septic shock are common conditions with high levels of morbi-mortality surpassing those of coronary heart disease or stroke. The reality of hospital treated sepsis is largely unknown outside of the intensive care unit. We therefore aimed to evaluate the level of compliance with the Surviving Sepsis Campaign 6-hour bundle in a Portuguese emergency department and to relate it to the patient clinical outcomes. MATERIAL AND METHODS: We conducted a retrospective, observational cohort study with 178 severe sepsis/septic shock patients admitted to the intensive and intermediate care unit between January 1st 2012 and December 31st 2012. RESULTS: In the study, period septic shock was diagnosed in 100 patients (56.2%) and severe sepsis in 78 patients (43.8%). Compliance with the sepsis bundle was: (1) 62.9% for lactate measurement; (2) 62.9% for blood cultures before antibiotics; (3) 41.6% for antibiotics in the first 3 hours; (4) 76.4% for fluid administration; (5) 25% for vasopressor administration; (6) 37% for central venous pressure measurement and (7) 39% for central venous oxygen saturation measurement. Full compliance was observed in 22% of the patients. The individual bundle measure - Blood cultures before antibiotics - was significantly associated with a decreased risk of both intensive care unit mortality and 28-day mortality. There was also a trend for an inverse correlation between increased compliance with the full bundle and the intensive care unit and 28-days hospital mortality. DISCUSSION: There was a low compliance with the Surviving Sepsis Campaign 6-hour bundle, a result that replicates the findings in similar international studies. The explanation is complex but it may include the lack of institutional quality monitoring in the emergency department. CONCLUSIONS: The compliance with a sepsis resuscitation bundle starting in the emergency department was positively associated with the outcomes of the septic patients. Nonetheless the bundle was unreliably performed.

Introdução: A sépsis severa e o choque sético são entidades clínicas frequentes com elevada morbi-mortalidade que superam os da doença cardíaca coronária ou do acidente vascular cerebral. A realidade da sépsis tratada no hospital fora das unidades de cuidados intensivos é amplamente desconhecida. Pretende-se neste trabalho estimar o cumprimento da bundle das 6-horas da Surviving Sepsis Campaign num serviço de urgência português e os resultados clínicos dos doentes. Material e Métodos: Estudo retrospetivo, observacional de coorte com 178 pacientes com sépsis severa/choque sético internados na unidade de cuidados intensivos e intermédios entre 1 de Janeiro de 2012 e 31 de Dezembro de 2012. Resultados: Durante o período estudado foi diagnosticado choque sético em 100 pacientes (56,2%) e sépsis severa em 78 pacientes (43,8%). O cumprimento com a bundle foi: (1) 62,9% para a medição de lactatos; (2) 62,9% para a colheita hemoculturas antes da antibioterapia; (3) 41,6% para a administração de antibióticos nas primeiras 3 horas; (4) 76,4% para a administração de fluidos; (5) 25% para a administração de vasopressores; (6) 37% for medição da pressão venosa central; (7) 39% para a medição da saturação venosa central de oxigénio. O cumprimento de todas as medidas foi observado em 22% dos pacientes. A medida 'colheita hemoculturas antes da antibioterapia' esteve significativamente associada a um menor risco de mortalidade na unidade de cuidados intensivos e aos 28 dias. Também se verificou uma tendência para uma correlação inversa entre cumprimento crescente da bundle e a mortalidade na unidade de cuidados intensivos e aos 28 dias. Discussão: A baixa adesão à bundle das 6-horas da Surviving Sepsis Campaign é um resultado que replica o de estudos internacionais semelhantes. A explicação para este fenómeno é complexa mas pode incluir a falta de monitorização da qualidade dos cuidados no serviço de urgência. Conclusões: O cumprimento da bundle de ressuscitação da sépsis desde a chegada do doente ao serviço de urgência está associado positivamente com os resultados clínicos do paciente sético. O cumprimento das medidas não foi no entanto muito elevado.

Arginase II Promotes Macrophage Inflammatory Responses Through Mitochondrial Reactive Oxygen Species, Contributing to Insulin Resistance and Atherogenesis.

BACKGROUND: Macrophage-mediated chronic inflammation is mechanistically linked to insulin resistance and atherosclerosis. Although arginase I is considered antiinflammatory, the role of arginase II (Arg-II) in macrophage function remains elusive. This study characterizes the role of Arg-II in macrophage inflammatory responses and its impact on obesity-linked type II diabetes mellitus and atherosclerosis. METHODS AND RESULTS: In human monocytes, silencing Arg-II decreases the monocytes' adhesion to endothelial cells and their production of proinflammatory mediators stimulated by oxidized low-density lipoprotein or lipopolysaccharides, as evaluated by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Macrophages differentiated from bone marrow cells of Arg-II-deficient (Arg-II(-/-)) mice express lower levels of lipopolysaccharide-induced proinflammatory mediators than do macrophages of wild-type mice. Importantly, reintroducing Arg-II cDNA into Arg-II(-/-) macrophages restores the inflammatory responses, with concomitant enhancement of mitochondrial reactive oxygen species. Scavenging of reactive oxygen species by N-acetylcysteine prevents the Arg-II-mediated inflammatory responses. Moreover, high-fat diet-induced infiltration of macrophages in various organs and expression of proinflammatory cytokines in adipose tissue are blunted in Arg-II(-/-) mice. Accordingly, Arg-II(-/-) mice reveal lower fasting blood glucose and improved glucose tolerance and insulin sensitivity. Furthermore, apolipoprotein E (ApoE)-deficient mice with Arg-II deficiency (ApoE(-/-)Arg-II(-/-)) display reduced lesion size with characteristics of stable plaques, such as decreased macrophage inflammation and necrotic core. In vivo adoptive transfer experiments reveal that fewer donor ApoE(-/-)Arg-II(-/-) than ApoE(-/-)Arg-II(+/+) monocytes infiltrate into the plaque of ApoE(-/-)Arg-II(+/+) mice. Conversely, recipient ApoE(-/-)Arg-II(-/-) mice accumulate fewer donor monocytes than do recipient ApoE(-/-)Arg-II(+/+) animals. CONCLUSIONS: Arg-II promotes macrophage proinflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis. Targeting Arg-II represents a potential therapeutic strategy in type II diabetes mellitus and atherosclerosis. (J Am Heart Assoc. 2012;1:e000992 doi: 10.1161/JAHA.112.000992.).

Period2 gene mutant mice show compromised insulin-mediated endothelial nitric oxide release and altered glucose homeostasis.

Period2 (Per2) is an important component of the circadian clock. Mutation of this gene is associated with vascular endothelial dysfunction and altered glucose metabolism. The aim of this study is to further characterize whole body glucose homeostasis and endothelial nitric oxide (NO) production in response to insulin in the mPer2(Brdm1) mice. We show that mPer2(Brdm1) mice exhibit compromised insulin receptor activation and Akt signaling in various tissues including liver, fat, heart, and aortas with a tissue-specific heterogeneous diurnal pattern, and decreased insulin-stimulated NO release in the aortas in both active and inactive phases of the animals. As compared to wild type (WT) mice, the mPer2(Brdm1) mice reveal hyperinsulinemia, hypoglycemia with lower fasting hepatic glycogen content and glycogen synthase level, no difference in glucose tolerance and insulin tolerance. The mPer2(Brdm1) mice do not show increased predisposition to obesity either on normal chow or high fat diet compared to WT controls. Thus, mice with Per2 gene mutation show altered glucose homeostasis and compromised insulin-stimulated NO release, independently of obesity.

Non-genomic vasorelaxant effects of 17ß-estradiol and progesterone in rat aorta are mediated by L-type Ca2+ current inhibition.

AIM: The sex hormones 17ß-estradiol (ßES) and progesterone (PRG) induce rapid non-genomic vasodilator effects which could be protective for the cardiovascular system. The purpose of this study was to analyze the mechanisms underlying their vasodilator effect in rat aortic smooth muscle preparations. METHODS: Endothelium-denuded aorta artery rings were prepared from male Wistar rats and incubated in an organ bath. The contractions of the preparation were recorded through isometric transducers. The effects of the hormones on K(+) current and L-type Ca(2+) current (LTCC) were analyzed by using the whole cell voltage-clamp technique in A7r5 cells. RESULTS: Both ßES and PRG (1-100 µmol/L) concentration-dependently relaxed the endothelium-denuded aortic rings contracted by (-)-Bay K8644 (0.1 µmol/L) or by KCl (60 mmol/L). The IC(50) values of the two hormones were not statistically different. The K(V) channel blocker 4-aminopyridine (2 mmol/L), BK(Ca) channel blocker tetraethylammonium (1 mmol/L) and K(ATP) channel blocker glibenclamide (10 µmol/L) did not significantly modify the relaxant effect of the hormones. On the other hand, the blockage of the intracellular ßES and PRG receptors with estradiol receptor antagonists ICI 182,780 (1 µmol/L) and PRG receptor antagonist mifepristone (30 µmol/L), respectively, did not significantly modify the relaxant action of the hormones. In A7r5 cells, both the hormones (1-100 µmol/L) rapidly and reversibly inhibited the basal and BAY-stimulated LTCC. However, these hormones had no effect on the basal K(+) current. CONCLUSION: The vasorelaxant effects of ßES and PRG are due to the inhibition of LTCC. The K(+) channels are not involved in the effects.

Hyperactive S6K1 mediates oxidative stress and endothelial dysfunction in aging: inhibition by resveratrol.

Mammalian target of rapamycin (mTOR)/S6K1 signalling emerges as a critical regulator of aging. Yet, a role of mTOR/S6K1 in aging-associated vascular endothelial dysfunction remains unknown. In this study, we investigated the role of S6K1 in aging-associated endothelial dysfunction and effects of the polyphenol resveratrol on S6K1 in aging endothelial cells. We show here that senescent endothelial cells displayed higher S6K1 activity, increased superoxide production and decreased bioactive nitric oxide (NO) levels than young endothelial cells, which is contributed by eNOS uncoupling. Silencing S6K1 in senescent cells reduced superoxide generation and enhanced NO production. Conversely, over-expression of a constitutively active S6K1 mutant in young endothelial cells mimicked endothelial dysfunction of the senescent cells through eNOS uncoupling and induced premature cellular senescence. Like the mTOR/S6K1 inhibitor rapamycin, resveratrol inhibited S6K1 signalling, resulting in decreased superoxide generation and enhanced NO levels in the senescent cells. Consistent with the data from cultured cells, an enhanced S6K1 activity, increased superoxide generation, and decreased bioactive NO levels associated with eNOS uncoupling were also detected in aortas of old WKY rats (aged 20-24 months) as compared to the young animals (1-3 months). Treatment of aortas of old rats with resveratrol or rapamycin inhibited S6K1 activity, oxidative stress, and improved endothelial NO production. Our data demonstrate a causal role of the hyperactive S6K1 in eNOS uncoupling leading to endothelial dysfunction and vascular aging. Resveratrol improves endothelial function in aging, at least in part, through inhibition of S6K1. Targeting S6K1 may thus represent a novel therapeutic approach for aging-associated vascular disease.

Opposing and uncoupling effects of mTOR and S6K1 in the regulation of endothelial tissue factor expression.

Rapamycin has been reported to enhance tissue factor (TF) expression. The present study investigated roles of mammalian target of rapamycin (mTOR) and its downstream S6K1 in this process. We showed here that, consistent with rapamycin, knocking-down mTOR enhanced thrombin-induced TF mRNA and protein levels, whereas silencing S6K1 mitigated up-regulation of TF protein but not TF mRNA level. The enhanced TF protein level upon mTOR-silencing was further augmented by over-expression of a constitutively active S6K1 mutant and reduced by blocking RhoA, p38(mapk) or NF-kappaB. The results reveal an opposing and uncoupling effect of mTOR and S6K1 in regulating TF expression.

Inhibition of S6K1 accounts partially for the anti-inflammatory effects of the arginase inhibitor L-norvaline.

BACKGROUND: Pharmacological inhibition of endothelial arginase-II has been shown to improve endothelial nitric oxide synthase (eNOS) function and reduce atherogenesis in animal models. We investigated whether the endothelial arginase II is involved in inflammatory responses in endothelial cells. METHODS: Human endothelial cells were isolated from umbilical veins and stimulated with TNFalpha (10 ng/ml) for 4 hours. Endothelial expression of the inflammatory molecules i.e. vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin were assessed by immunoblotting. RESULTS: The induction of the expression of endothelial VCAM-1, ICAM-1 and E-selectin by TNFalpha was concentration-dependently reduced by incubation of the endothelial cells with the arginase inhibitor L-norvaline. However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC) had no effects. To confirm the role of arginase-II (the prominent isoform expressed in HUVECs) in the inflammatory responses, adenoviral mediated siRNA silencing of arginase-II knocked down the arginase II protein level, but did not inhibit the up-regulation of the adhesion molecules. Moreover, the inhibitory effect of L-norvaline was not reversed by the NOS inhibitor L-NAME and L-norvaline did not interfere with TNFalpha-induced activation of NF-kappaB, JNK, p38mapk, while it inhibited p70s6k (S6K1) activity. Silencing S6K1 prevented up-regulation of E-selectin, but not that of VCAM-1 or ICAM-1 induced by TNFalpha. CONCLUSION: The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells. The anti-inflammatory properties of L-norvaline are partially attributable to its ability to inhibit S6K1.

The hexosamine biosynthesis inhibitor azaserine prevents endothelial inflammation and dysfunction under hyperglycemic condition through antioxidant effects.

Hexosamine biosynthetic pathway (HBP) accounts for some cardiovascular adverse effects of hyperglycemia. We investigated whether the HBP inhibitor azaserine protects against hyperglycemia-induced endothelial damage dependently of HBP. Human endothelial cells isolated from umbilical veins were exposed either to a high (30.5 mmol/l) or low concentration of glucose (5.5 mmol/l) for 4 days, followed by a stimulation with TNF-alpha (1 ng/ml, 24 h). The blockade of the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase inhibited HBP flux and oxidative stress (generation of superoxide and peroxynitrite) under the hyperglycemic condition and prevented the synergistic stimulation of VCAM-1 and ICAM-1 expression by hyperglycemia and TNF-alpha. In the cells cultured under a low-glucose condition when no increased HBP flux occurred, azaserine enhanced the manganese-superoxide dismutase (MnSOD) protein level and also inhibited the oxidative stress and the expression of VCAM-1 and ICAM-1 in response to TNF-alpha. Moreover, the polyphenol resveratrol inhibited the oxidative stress and adhesion molecule expression and did not decrease the HBP flux under the hyperglycemia condition. In addition, in isolated rat aortas exposed to hyperglycemic buffer for 5 h when no significant HBP flux occurred, azaserine upregulated the MnSOD protein level and prevented decreased endothelium-dependent relaxations to acetylcholine. In conclusion, hyperglycemia independently increases oxidative stress and HBP flux, amplifies endothelial inflammation, and impairs endothelial function mainly through oxidative stress and not the HBP pathway. Azaserine protects against hyperglycemic endothelial damage through its antioxidant effect independently of inhibiting HBP pathway.

O-linked beta-N-acetylglucosamine during hyperglycemia exerts both anti-inflammatory and pro-oxidative properties in the endothelial system.

Elevated cellular levels of protein O-linked beta-N-acetylglucosamine (O-GlcNAc) through hexosamine biosynthesis pathway (HBP) are suggested to contribute to cardiovascular adverse effects under chronic hyperglycemic condition associated with oxidative stress and inflammation. Conversely, enhancing O-GlcNAc levels have also been demonstrated being protective against myocardial ischemia/reperfusion injury. We recently demonstrated that hyperglycemia increases oxidative stress and HBP flux in endothelial cells and enhances endothelial expression of vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in response to tumor necrosis factor-alpha (TNFalpha) through oxidative stress rather than HBP pathway. Here we present further complementary data showing that enhancing O-GlcNAc levels by glucosamine does not mimic hyperglycemia's effect on TNFalpha-induced endothelial VCAM-1 and ICAM-1 expression. Glucosamine however inhibits ICAM-1 (not VCAM-1) expression and induces superoxide generation in the cells. The results further suggest that increased O-GlcNAc levels do not mediate the enhancing effect of hyperglycemia on the endothelial inflammatory responses to TNFalpha. In contrast, it exerts certain anti-inflammatory effects accompanied by pro-oxidative properties. Further work should delineate the exact role of HPB pathway in different aspects of cardiovascular functions, especially those of diabetic cardiovascular complications.

Mutation of the circadian clock gene Per2 alters vascular endothelial function.

BACKGROUND: The circadian clock regulates biological processes including cardiovascular function and metabolism. In the present study, we investigated the role of the circadian clock gene Period2 (Per2) in endothelial function in a mouse model. METHODS AND RESULTS: Compared with the wild-type littermates, mice with Per2 mutation exhibited impaired endothelium-dependent relaxations to acetylcholine in aortic rings suspended in organ chambers. During transition from the inactive to active phase, this response was further increased in the wild-type mice but further decreased in the Per2 mutants. The endothelial dysfunction in the Per2 mutants was also observed with ionomycin, which was improved by the cyclooxygenase inhibitor indomethacin. No changes in the expression of endothelial acetylcholine-M3 receptor or endothelial nitric oxide synthase protein but increased cyclooxygenase-1 (not cyclooxygenase-2) protein levels were observed in the aortas of the Per2 mutants. Compared with Per2 mutants, a greater endothelium-dependent relaxation to ATP was observed in the wild-type mice, which was reduced by indomethacin. In quiescent aortic rings, ATP caused greater endothelium-dependent contractions in the Per2 mutants than in the wild-type mice, contractions that were abolished by indomethacin. The endothelial dysfunction in the Per2 mutant mice is not associated with hypertension or dyslipidemia. CONCLUSIONS: Mutation in the Per2 gene in mice is associated with aortic endothelial dysfunction involving decreased production of NO and vasodilatory prostaglandin(s) and increased release of cyclooxygenase-1-derived vasoconstrictor(s). The results suggest an important role of the Per2 gene in maintenance of normal cardiovascular functions.