Smoking and environmental tobacco smoke (ETS) exposure are major risk factors for cardiovascular disease (CVD), although the exact components and pathophysiological mechanisms responsible for this association remain unclear. Polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (BaP), are ubiquitous environmental contaminants that form during organic material combustion and are thus found in cigarette smoke, vehicle exhaust particles, and air pollution. We hypothesize that PAHs are key agents responsible for mediating the cigarette smoke effects in the cardiovascular system, including increased oxidative stress, inflammation, and arterial stiffness.<p> Arterial stiffness is a powerful, independent predictor of cardiovascular risk and is regulated, in part, by vasoactive mediators derived from the endothelium. The first objective of this project was to determine whether pulse wave dP/dt collected from radiotelemetry-implanted rats is a reliable indicator of changes in arterial stiffness following administration of vasoactive drugs or acute ETS exposure. Anaesthetized rats were administered a single dose of saline (vehicle control), acetylcholine, norepinephrine, and N(G)-nitro-L-arginine methyl ester (L-NAME) via the tail vein, allowing a washout period between injections. Acetylcholine decreased and norepinephrine increased dP/dt compared to saline vehicle. Injection of the nitric oxide (NO) synthase inhibitor L-NAME decreased plasma nitrate/nitrite (NOx), but transiently increased dP/dt. For the ETS experiment, rats were exposed for one hour to sham, low dose ETS, or high dose ETS. Exposure to ETS did not significantly alter dP/dt or plasma endothelin-1 (ET-1) levels, but increased plasma NOx levels at the high ETS exposure and increased plasma nitrotyrosine levels in both ETS groups. In conclusion, acute changes in NO production via acetylcholine or L-NAME alter the arterial pulse wave dP/dt consistently with the predicted changes in arterial stiffness. Although acute ETS appears to biologically inactivate NO, a concomitant increase in NO production at the high ETS exposure may explain why ETS did not acutely alter dP/dt.<p> The second objective of this project was to compare the effects of subchronic ETS and BaP exposure on circadian blood pressure patterns, arterial stiffness, and possible sources of oxidative stress in radiotelemetry-implanted rats. Pulse wave dP/dt was used as an indicator of arterial stiffness, and was compared to both structural (wall thickness) and functional (NO production and bioactivity, ET-1 levels) features of the arterial wall. In addition, histology of lung, heart, and liver were examined as well as pulmonary and hepatic detoxifying enzyme activity (cytochrome P450 specifically CYP1A1). Daily ETS exposure for 28 days altered the circadian pattern of heart rate and blood pressure in rats, with a loss in the normal dipping pattern of blood pressure during sleep. Subchronic ETS exposure also increased dP/dt in the absence of any structural modifications in the arterial wall. Although NO production and ET-1 levels were not altered by ETS, there was increased biological inactivation of NO via peroxynitrite production (as indicated by increased plasma nitrotyrosine levels). Thus, vascular stiffness and failure of blood pressure to dip precede structural changes in rats exposed to ETS for 28 days. Exposure to ETS also caused increased number of lung neutrophils as well as increased CYP1A1 activity in lung microsomes.<p> Since ETS-induced increases in arterial stiffness occurred as early as day 7, radiotelemetry-implanted rats were exposed daily to intranasal BaP for 7 days. Similar to ETS, BaP exposure altered circadian blood pressure patterns and reduced blood pressure dipping during sleep. Thus, in support of part of our hypothesis, the PAH component of cigarette smoke may be responsible for the ETS-induced increase in blood pressure and the loss of dipping pattern during sleep. Increased neutrophil recruitment was observed in the lungs of both ETS- and BaP-exposed rats, suggesting that lung inflammatory reactions may be involved in the disruption of circadian blood pressure rhythms. Unlike ETS however, BaP exposure did not significantly alter pulse wave dP/dt, endothelial function, or lung CYP1A1 activity. Thus, contrary to our hypothesis, the reduction in NO bioactivity and increased arterial stiffness caused by ETS cannot be explained by BaP at the dose and length of the exposure in the current study. Production of reactive metabolites in the lung following ETS exposure may be responsible, at least in part, for the increases in oxidative stress in the vasculature, leading to reduced NO bioactivity and increased arterial stiffness. Oxidative stress caused by BaP exposure may have been insufficient to reduce NO bioactivity in the peripheral vasculature. Therefore arterial stiffness was not increased and factors other than NO may be responsible for the increase in blood pressure observed with ETS and BaP exposure.

Abnormal leukocyte adhesion (i.e. leukostasis) to retinal vascular endothelial cells occurs in early diabetes. The processes of leukostasis have been clearly demonstrated in the vascular endothelium of patients with diabetes. In non-proliferative DR, clinical outcomes are manifested by excessive permeability from inflammatory progression leading to inner blood retinal barrier disruption, endothelial cell damage and widespread capillary nonperfusion. Diabetes promotes vascular leakage in DR by upregulation of adhesion molecules. Moreover, many of the pathological changes in NPDR are related to abnormalities in retinal blood flow. Studies have shown that specific circulating markers of inflammatory activity and endothelial dysfunction are associated with clinical signs of diabetic retinopathy. However, few have found an association between circulating levels of inflammatory and endothelial dysfunctional markers and abnormal retinal hemodynamics in patients with non-proliferative DR. The specific aims of this thesis are as follows: (Chapter 3)To correlate baseline levels of inflammatory and endothelial dysfunction markers and 1) baseline retinal arteriolar hemodynamics and 2) any disturbance in retinal hemodynamics over 6-month time in terms of vessel diameter, blood velocity, maximum-to-minimum velocity ratio and volumetric flow. In Chapter 4: To correlate circulating levels of inflammatory and endothelial dysfunction markers and 1) baseline vascular reactivity and 2) any disturbance in vascular reactivity after 6-month time in terms of vessel diameter, blood velocity, maximum-to-minimum velocity ratio and volumetric flow in patients with increasing non-proliferative diabetic retinopathy (NPDR) severity. Methods for Chapter 3: Diabetes subjects were stratified into either mild-to-moderate (Group 2) or moderate-to-severe (Group 3) NPDR based on their retinopathy status. Age-matched non-diabetics were recruited as controls (Group 1). Forearm blood sample was collected to determine baseline levels of inflammatory and endothelial dysfunctional markers. At visit 1, baseline retinal hemodynamics was acquired using Canon Laser Blood Flowmeter. Patients returned for a visit 2 (6 month follow-up visit) and retinal hemodynamics was reassessed. Baseline levels of inflammatory and endothelial dysfunctional markers compared between groups and correlated with both baseline and change in retinal hemodynamic parameters over 6-month time. For Chapter 4: Diabetes subjects were stratified into either mild-to-moderate NPDR or moderate-to-severe NPDR based on their retinopathy status. Age-matched non-diabetics were recruited as controls. At visit 1, forearm blood sample was collected to determine levels of inflammatory and endothelial dysfunctional markers and baseline vascular reactivity response was acquired. Retinal blood flow data was acquired while subjects breathed air. Retinal blood flow measurements were then acquired after exposure to isocapnic hyperoxic stimuli. At visit 2 (6 month follow-up), retinal vascular reactivity was reassessed. Baseline levels of inflammatory and endothelial dysfunctional markers compared between groups and correlated with both magnitude of baseline and change in vascular reactivity in terms of retinal hemodynamics. Results of Chapter 3: Maximum-to-minimum velocity ratio (max: min) was found to be significantly elevated in the group 3 compared to group 1 at baseline (0.72 vs. 0.49, after Bonferroni correction P<0.01). Both sICAM-1 and sE-selectin were significantly elevated as a function of group (ANOVA p=0.02 and p=0.04). A post hoc Bonferroni test showed that Group 3 had significantly higher in both sICAM-1 and sE-selectin levels compared to Group 1 (234.0 vs. 151.5 ng/ml, P=0.02 and 53.4 vs. 27.6 ng/ml, P<0.01, respectively). Hemoglobin A1c was significantly elevated across the groups (ANOVA p<0.01). A post hoc Bonferroni test showed that Group 3 had significantly higher hemoglobin A1c level compared to Group 1 (7.9 vs. 5.6 % , P<0.01). There were no significant associations found between baseline markers of inflammation and baseline retinal hemodynamics across all groups. The Δ velocity was correlated with the baseline sICAM-1 (r=0.42, p=0.02) and A1c levels (r=0.37, p=0.04) in patients with NPDR. After adjustment for all other variables (A1c, hsCRP and vWF), Δ velocity, sICAM-1 and A1c were found not to be reliable predictors of baseline retinal hemodynamics. For Chapter 4: There were no significant differences in magnitude of retinal vascular reactivity in hemodynamic parameters between groups at visit 1 or visit 2. Over 6 months time, compliance was found to be significantly reduced in patients of Group 3 compared to Group 2 (-0.4 vs. 0.1, t-test p<0.01). Both sICAM-1 and sE-selectin were significantly elevated as a function of group (ANOVA p=0.02 and p<0.01). A post hoc Bonferroni test showed that Group 3 had significantly higher in both sICAM-1 and sE-selectin levels compared to Group 1 (243.4 vs. 157.3ngml, P<0.01 and 57.0 vs. 29.3 ng/ml, P<0.01, respectively). Hemoglobin A1c was significantly elevated across the groups (ANOVA p<0.01). A post hoc Bonferroni test showed that Group 3 had significantly higher hemoglobin A1c level compared to Group 1 (8.8 vs. 5.6 % , P<0.01). Baseline VR in blood velocity weakly correlates with sE-selectin (r=0.31, p=0.04) across all groups while sVCAM-1 was associated with VR in terms of blood flow (r=-0.62, p<0.01) in patients with mild-to-moderate NPDR. The ∆ blood flow after 6 months was found to be weakly associated with sE-selectin (r=0.46, p=0.03) across all groups. Finally, the ∆ blood velocity after 6 month time was found to be moderately correlated with baseline vWF Ag level (r=-0.78, p=0.02). Multiple regression analysis found that vascular inflammatory and endothelial function markers had weak predictive power for Δ hemodynamic parameters. Conclusions Chapter 3: We found weak associations between circulating markers and baseline or the disturbance in retinal hemodynamics after 6 months time. Overall, we found both an increase in rigidity of the arteriolar circulation and elevated inflammatory adhesion markers (sICAM-1 and sE-selectin) within the same population sample. Change in velocity over the follow-up period was correlated with sICAM-1 and A1c levels in patients with NPDR but the level of association was such that neither sICAM-1 nor A1c proved to reliably predict retinal hemodynamics. Finally, in Chapter 4 we demonstrated two important characteristics in early NPDR; 1) a disturbance in vascular reactivity in terms of compliance and 2) an increase in systemic markers of inflammation were found in patients with NPDR. Although systemic markers of vascular inflammation and endothelial dysfunction are not predictive of hemodynamic parameters, our study found moderate associations between baseline and disturbances in VR after 6 months time. Therefore, there is evidence that inflammation and vascular function may be related with respect to their development in NPDR.

Smoking and environmental tobacco smoke (ETS) exposure are major risk factors for cardiovascular disease (CVD), although the exact components and pathophysiological mechanisms responsible for this association remain unclear. Polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (BaP), are ubiquitous environmental contaminants that form during organic material combustion and are thus found in cigarette smoke, vehicle exhaust particles, and air pollution. We hypothesize that PAHs are key agents responsible for mediating the cigarette smoke effects in the cardiovascular system, including increased oxidative stress, inflammation, and arterial stiffness.<p> Arterial stiffness is a powerful, independent predictor of cardiovascular risk and is regulated, in part, by vasoactive mediators derived from the endothelium. The first objective of this project was to determine whether pulse wave dP/dt collected from radiotelemetry-implanted rats is a reliable indicator of changes in arterial stiffness following administration of vasoactive drugs or acute ETS exposure. Anaesthetized rats were administered a single dose of saline (vehicle control), acetylcholine, norepinephrine, and N(G)-nitro-L-arginine methyl ester (L-NAME) via the tail vein, allowing a washout period between injections. Acetylcholine decreased and norepinephrine increased dP/dt compared to saline vehicle. Injection of the nitric oxide (NO) synthase inhibitor L-NAME decreased plasma nitrate/nitrite (NOx), but transiently increased dP/dt. For the ETS experiment, rats were exposed for one hour to sham, low dose ETS, or high dose ETS. Exposure to ETS did not significantly alter dP/dt or plasma endothelin-1 (ET-1) levels, but increased plasma NOx levels at the high ETS exposure and increased plasma nitrotyrosine levels in both ETS groups. In conclusion, acute changes in NO production via acetylcholine or L-NAME alter the arterial pulse wave dP/dt consistently with the predicted changes in arterial stiffness. Although acute ETS appears to biologically inactivate NO, a concomitant increase in NO production at the high ETS exposure may explain why ETS did not acutely alter dP/dt.<p> The second objective of this project was to compare the effects of subchronic ETS and BaP exposure on circadian blood pressure patterns, arterial stiffness, and possible sources of oxidative stress in radiotelemetry-implanted rats. Pulse wave dP/dt was used as an indicator of arterial stiffness, and was compared to both structural (wall thickness) and functional (NO production and bioactivity, ET-1 levels) features of the arterial wall. In addition, histology of lung, heart, and liver were examined as well as pulmonary and hepatic detoxifying enzyme activity (cytochrome P450 specifically CYP1A1). Daily ETS exposure for 28 days altered the circadian pattern of heart rate and blood pressure in rats, with a loss in the normal dipping pattern of blood pressure during sleep. Subchronic ETS exposure also increased dP/dt in the absence of any structural modifications in the arterial wall. Although NO production and ET-1 levels were not altered by ETS, there was increased biological inactivation of NO via peroxynitrite production (as indicated by increased plasma nitrotyrosine levels). Thus, vascular stiffness and failure of blood pressure to dip precede structural changes in rats exposed to ETS for 28 days. Exposure to ETS also caused increased number of lung neutrophils as well as increased CYP1A1 activity in lung microsomes.<p> Since ETS-induced increases in arterial stiffness occurred as early as day 7, radiotelemetry-implanted rats were exposed daily to intranasal BaP for 7 days. Similar to ETS, BaP exposure altered circadian blood pressure patterns and reduced blood pressure dipping during sleep. Thus, in support of part of our hypothesis, the PAH component of cigarette smoke may be responsible for the ETS-induced increase in blood pressure and the loss of dipping pattern during sleep. Increased neutrophil recruitment was observed in the lungs of both ETS- and BaP-exposed rats, suggesting that lung inflammatory reactions may be involved in the disruption of circadian blood pressure rhythms. Unlike ETS however, BaP exposure did not significantly alter pulse wave dP/dt, endothelial function, or lung CYP1A1 activity. Thus, contrary to our hypothesis, the reduction in NO bioactivity and increased arterial stiffness caused by ETS cannot be explained by BaP at the dose and length of the exposure in the current study. Production of reactive metabolites in the lung following ETS exposure may be responsible, at least in part, for the increases in oxidative stress in the vasculature, leading to reduced NO bioactivity and increased arterial stiffness. Oxidative stress caused by BaP exposure may have been insufficient to reduce NO bioactivity in the peripheral vasculature. Therefore arterial stiffness was not increased and factors other than NO may be responsible for the increase in blood pressure observed with ETS and BaP exposure.

Abnormal leukocyte adhesion (i.e. leukostasis) to retinal vascular endothelial cells occurs in early diabetes. The processes of leukostasis have been clearly demonstrated in the vascular endothelium of patients with diabetes. In non-proliferative DR, clinical outcomes are manifested by excessive permeability from inflammatory progression leading to inner blood retinal barrier disruption, endothelial cell damage and widespread capillary nonperfusion. Diabetes promotes vascular leakage in DR by upregulation of adhesion molecules. Moreover, many of the pathological changes in NPDR are related to abnormalities in retinal blood flow. Studies have shown that specific circulating markers of inflammatory activity and endothelial dysfunction are associated with clinical signs of diabetic retinopathy. However, few have found an association between circulating levels of inflammatory and endothelial dysfunctional markers and abnormal retinal hemodynamics in patients with non-proliferative DR. The specific aims of this thesis are as follows: (Chapter 3)To correlate baseline levels of inflammatory and endothelial dysfunction markers and 1) baseline retinal arteriolar hemodynamics and 2) any disturbance in retinal hemodynamics over 6-month time in terms of vessel diameter, blood velocity, maximum-to-minimum velocity ratio and volumetric flow. In Chapter 4: To correlate circulating levels of inflammatory and endothelial dysfunction markers and 1) baseline vascular reactivity and 2) any disturbance in vascular reactivity after 6-month time in terms of vessel diameter, blood velocity, maximum-to-minimum velocity ratio and volumetric flow in patients with increasing non-proliferative diabetic retinopathy (NPDR) severity. Methods for Chapter 3: Diabetes subjects were stratified into either mild-to-moderate (Group 2) or moderate-to-severe (Group 3) NPDR based on their retinopathy status. Age-matched non-diabetics were recruited as controls (Group 1). Forearm blood sample was collected to determine baseline levels of inflammatory and endothelial dysfunctional markers. At visit 1, baseline retinal hemodynamics was acquired using Canon Laser Blood Flowmeter. Patients returned for a visit 2 (6 month follow-up visit) and retinal hemodynamics was reassessed. Baseline levels of inflammatory and endothelial dysfunctional markers compared between groups and correlated with both baseline and change in retinal hemodynamic parameters over 6-month time. For Chapter 4: Diabetes subjects were stratified into either mild-to-moderate NPDR or moderate-to-severe NPDR based on their retinopathy status. Age-matched non-diabetics were recruited as controls. At visit 1, forearm blood sample was collected to determine levels of inflammatory and endothelial dysfunctional markers and baseline vascular reactivity response was acquired. Retinal blood flow data was acquired while subjects breathed air. Retinal blood flow measurements were then acquired after exposure to isocapnic hyperoxic stimuli. At visit 2 (6 month follow-up), retinal vascular reactivity was reassessed. Baseline levels of inflammatory and endothelial dysfunctional markers compared between groups and correlated with both magnitude of baseline and change in vascular reactivity in terms of retinal hemodynamics. Results of Chapter 3: Maximum-to-minimum velocity ratio (max: min) was found to be significantly elevated in the group 3 compared to group 1 at baseline (0.72 vs. 0.49, after Bonferroni correction P<0.01). Both sICAM-1 and sE-selectin were significantly elevated as a function of group (ANOVA p=0.02 and p=0.04). A post hoc Bonferroni test showed that Group 3 had significantly higher in both sICAM-1 and sE-selectin levels compared to Group 1 (234.0 vs. 151.5 ng/ml, P=0.02 and 53.4 vs. 27.6 ng/ml, P<0.01, respectively). Hemoglobin A1c was significantly elevated across the groups (ANOVA p<0.01). A post hoc Bonferroni test showed that Group 3 had significantly higher hemoglobin A1c level compared to Group 1 (7.9 vs. 5.6 % , P<0.01). There were no significant associations found between baseline markers of inflammation and baseline retinal hemodynamics across all groups. The Δ velocity was correlated with the baseline sICAM-1 (r=0.42, p=0.02) and A1c levels (r=0.37, p=0.04) in patients with NPDR. After adjustment for all other variables (A1c, hsCRP and vWF), Δ velocity, sICAM-1 and A1c were found not to be reliable predictors of baseline retinal hemodynamics. For Chapter 4: There were no significant differences in magnitude of retinal vascular reactivity in hemodynamic parameters between groups at visit 1 or visit 2. Over 6 months time, compliance was found to be significantly reduced in patients of Group 3 compared to Group 2 (-0.4 vs. 0.1, t-test p<0.01). Both sICAM-1 and sE-selectin were significantly elevated as a function of group (ANOVA p=0.02 and p<0.01). A post hoc Bonferroni test showed that Group 3 had significantly higher in both sICAM-1 and sE-selectin levels compared to Group 1 (243.4 vs. 157.3ngml, P<0.01 and 57.0 vs. 29.3 ng/ml, P<0.01, respectively). Hemoglobin A1c was significantly elevated across the groups (ANOVA p<0.01). A post hoc Bonferroni test showed that Group 3 had significantly higher hemoglobin A1c level compared to Group 1 (8.8 vs. 5.6 % , P<0.01). Baseline VR in blood velocity weakly correlates with sE-selectin (r=0.31, p=0.04) across all groups while sVCAM-1 was associated with VR in terms of blood flow (r=-0.62, p<0.01) in patients with mild-to-moderate NPDR. The ∆ blood flow after 6 months was found to be weakly associated with sE-selectin (r=0.46, p=0.03) across all groups. Finally, the ∆ blood velocity after 6 month time was found to be moderately correlated with baseline vWF Ag level (r=-0.78, p=0.02). Multiple regression analysis found that vascular inflammatory and endothelial function markers had weak predictive power for Δ hemodynamic parameters. Conclusions Chapter 3: We found weak associations between circulating markers and baseline or the disturbance in retinal hemodynamics after 6 months time. Overall, we found both an increase in rigidity of the arteriolar circulation and elevated inflammatory adhesion markers (sICAM-1 and sE-selectin) within the same population sample. Change in velocity over the follow-up period was correlated with sICAM-1 and A1c levels in patients with NPDR but the level of association was such that neither sICAM-1 nor A1c proved to reliably predict retinal hemodynamics. Finally, in Chapter 4 we demonstrated two important characteristics in early NPDR; 1) a disturbance in vascular reactivity in terms of compliance and 2) an increase in systemic markers of inflammation were found in patients with NPDR. Although systemic markers of vascular inflammation and endothelial dysfunction are not predictive of hemodynamic parameters, our study found moderate associations between baseline and disturbances in VR after 6 months time. Therefore, there is evidence that inflammation and vascular function may be related with respect to their development in NPDR.

In 2008 the global prevalence of hypertension [high blood pressure (BP), systolic ≥140 mmHg and/or diastolic ≥90 mmHg] was around 40% in adults > 25 yrs of age, according to the 2013 WHO statistics. Hypertension is a major risk factor for myocardial infarction, heart failure and stroke. Currently, around 20% of the Canadian population is affected by hypertension. Hypertension is more closely associated with diabetes. More than two thirds of people with diabetes have hypertension, alongwith increased activity of the renin angiotensin aldosterone (RAAS) system. The RAAS plays a major role in maintaining fluid balance, vascular tone and BP. The components of the RAAS include the hormone renin, which cleaves angiotensinogen, a circulating inactive peptide into angiotensin I. Angiotensin converting enzyme (ACE) converts angiotensin I into the active peptide angiotensin II (Ang II). Ang II causes vasoconstriction, sodium reabsorption from the kidney tubules and also release of the hormone, aldosterone, from the adrenal cortex. The epidemic of hypertension, diabetes and obesity is widely attributed to a high carbohydrate diet, containing mainly high fructose corn syrup and sucrose. However, the underlying molecular mechanisms are far from clear. A high fructose diet increases BP in Sprague-Dawley (SD) rats; along with elevated plasma and aortic levels of methylglyoxal (MG). MG is a reactive dicarbonyl compound mainly formed as an intermediate during glycolysis. Small amounts of MG are also formed during amino acid (threonine) and fatty acid metabolism. MG reacts with certain proteins to form irreversible advanced glycation end products (AGEs). MG has high affinity for arginine, lysine and cysteine. Plasma MG levels are increased in hypertensive rats and diabetic patients. However, it is not yet clear whether MG is the cause or effect of hypertension. Moreover, safe and specific MG scavengers are not available. The aim of the project was to determine the effect of MG and a high fructose diet on the RAAS and the BP in male SD rats. The hypothesis that L-arginine, and its inactive isomer D-arginine, can efficiently scavenge MG in vitro, was also tested. Male SD rats were treated with a continuous infusion of MG with a subcutaneous minipump for 4 weeks, or with a high fructose diet (60% of total calories) for 16 weeks. We also used isolated aortic rings from 12 week old normal male SD rats to study endothelial function. Organs / tissues, cultured human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs) were used for molecular studies. HPLC, Western blotting and Q-PCR were used to measure MG, reduced glutathione (GSH), proteins and mRNA, respectively. siRNA for angiotensinogen and the receptor for advanced glycation endproducts (RAGE) were used to study mechanisms. MG treated rats developed a significant increase in BP and plasma levels of aldosterone, renin, angiotensin and catecholamines. MG level, and protein and mRNA for angiotensin, AT1 receptor, adrenergic α1D receptor and renin were significantly increased in the aorta and/or kidney of MG treated rats, a novel finding. Alagebrium, a MG scavenger and AGEs breaker, attenuated the above effects of MG. Treatment of cultured VSMCs with MG or high glucose (25mM) significantly increased cellular MG, and protein and mRNA for nuclear factor kappa B (NF-κB), angiotensin, AT1 and α1D receptors, which were prevented by inhibition of NF-κB, and by alagebrium. Silencing of mRNA for RAGE prevented the increase in NF-kB induced by MG. Silencing of mRNA for angiotensinogen prevented the increase in NF-κB, angiotensin, AT1 and α1D receptors’ protein. Fructose treated rats developed a significant increase in BP. MG level and protein and mRNA for angiotensin II, AT1 receptor, adrenergic α1D receptor and renin were significantly increased, whereas GSH levels were decreased, in the aorta and/or kidney of fructose fed rats. The protein expression of the receptor for AGEs (RAGE) and NF-κB were also significantly increased in the aorta of fructose fed rats. MG treated VSMCs showed increased protein for angiotensin II, AT1 receptor, and α1D receptor. The effects of fructose and MG were attenuated by metformin, a MG scavenger and AGEs inhibitor. In experiments to test the MG scavenging action of arginine, both D-arginine and L-arginine prevented the attenuation of acetylcholine-induced endothelium-dependent vasorelaxation by MG and high glucose. However, the inhibitory effect of the NOS inhibitor, Nω-nitro-L-arginine methyl ester, on vasorelaxation was prevented only by L-arginine, but not by D-arginine. MG and high glucose increased protein expression of arginase, a novel finding, and also of NADPH oxidase 4 and NF-κB, and production of reactive oxygen species in HUVECs and VSMCs, which were attenuated by D- and L-arginine. However, D- and L-arginine did not attenuate MG and high glucose-induced increased arginase activity in VSMCs and the aorta. D- and L-arginine also attenuated the increased formation of the MG-specific AGE, Nε-carboxyethyl lysine, caused by MG and high glucose in VSMCs. In conclusion, MG activates NF-κB through RAGE and thereby increases renin angiotensin levels, a novel finding, and a probable mechanism of increase in BP. There is a strong association between elevated levels of MG, RAGE, NF-κB, mediators of the RAAS and BP in high fructose diet fed rats. Arginine attenuates the increased arginase expression, oxidative stress, endothelial dysfunction and AGEs formation induced by MG and high glucose, by an endothelial NOS independent mechanism.

O Diabetes Mellitus tipo 1 (DM1) está associado a uma incidência aumentada de doença micro e macrovascular. Estudos sugerem que a doença vascular no DM1 tenha como evento precursor a disfunção endotelial (DE). A hiperglicemia parece causar DE no DM1 através da geração do estresse oxidativo. O momento exato do surgimento da DE na história natural do DM1, assim como a influência do controle glicêmico de curto e longo prazo ainda não estão estabelecidos. Objetivo: O objetivo principal do presente estudo foi avaliar a função endotelial através da Dilatação Arterial Mediada por Fluxo (DMF) em indivíduos com Diabetes Mellitus tipo1. Os objetivos secundários foram analisar os fatores que possam estar envolvidos com a disfunção endotelial no DM1, como o tempo de diabetes, o controle glicêmico e a presença de complicações microvasculares, como a microalbuminúria. Métodos: Estudo prospectivo transversal com 57 pacientes com DM1 e 10 indivíduos não diabéticos, consecutivamente alocados e comparados quanto à presença de DE, através da DMF, aferida pela dilatação da artéria braquial após hiperemia reativa (dilatação endotélio-dependente) e após dilatação mediada por uso de nitrato sublingual (dilatação endotélio-independente). Considerou-se como DE quando valores de DMF foram menores ou iguais a 8% em relação ao valor basal. Os pacientes foram orientados a fazer monitorização glicêmica capilar intensiva nos 30 dias que antecederam a avaliação vascular. No 30º dia, houve coleta de exames laboratoriais e a avaliação vascular foi realizada. Dados prospectivos e históricos de hemoglobina glicosilada (HbA1c), através da técnica de imunoturbidimetria (Cobas Integra 400; Roche), foram obtidos aos 3, 6, 9,12,15,18 e 24 meses anteriores ao teste para DMF. Os critérios de exclusão foram: tabagismo, hipertensão, obesidade, hipotireoidismo, uso de estatina, gestação, história de neoplasia ou doença vascular. Resultados: Em 57 pacientes com DM1 estudados, 28 (49%) apresentaram DE. A média da dilatação endotélio-dependente foi significativamente menor nos pacientes com DM1, comparados aos indivíduos não-diabéticos (9,48±6,48% vs.14,56±5,60%, p=0,02). A dilatação endotélio-independente foi significativamente menor nos pacientes com DM1 em relação aos controles (22,26±9,2% vs. 29,31±4,2%, p=0,02, VR: acima de 8%), mas não houve diferença entre os DM1 com ou sem DE (p= 0,72). O tempo de DM1 (meses) foi maior nos pacientes com DE do que nos sem DE (105,4±74,7 vs. 66,3±48,0, p=0.02) e houve correlação linear negativa entre duração do DM e presença de DE (r-0,28, p=0,02). A média da HbA1c (%) coletada no momento da avaliação vascular foi semelhante entre pacientes com DM1 com DE e sem DE (8,97%±1.85 vs 8,23%±1.45, p=0.10) e não houve correlação significativa com a DMF (r=-0,128 p=0,34). Todavia, quando as HbA1c históricas foram avaliadas, houve correlação significativa com a HbA1c aos 15 meses (r=-0,303, p=0,02) e no período de 12-24 meses anteriores ao exame vascular (r=-0,289, p=0,03), mas não com a HbA1c média de 0-12m (r=-0,181 p=0,18). A DMF foi menor nos pacientes com microalbuminúria em relação aos normoalbuminúrcos (4,83±3,81% vs 10,35±6,50%, p=0,015). A microalbuminúria também foi mais prevalente nos DM1 com DE do que sem DE (22,2% vs 3,5%, p=0,04). Considerando apenas os pacientes com DM1 com tempo de DM menor que 5 anos, 10/28 (35,7%) apresentaram DE. Com relação a dilatação não-dependente de endotélio (%), não houve diferença em relação aos controles (p=0,16) e nem entre os DM1 com e sem DE (p=0,27). A média da HbA1c na época do exame vascular também não foi diferente nos pacientes com e sem DE (8,20±0,94% vs. 7,99±1,37%, p=0,66). As correlações de Pearson entre a DMF e as HbA1c históricas foram negativas aos 12 meses (r=-0,419, p=0,03), aos 15 meses (r=-0,437, p=0,03) e com a HbA1c média de12-24 meses (r=-0,426, p=0,027). Conclusões: Pacientes com DM1 apresentam prejuízo na função endotelial, quando comparados a controles não diabéticos. A DE é um evento precoce na história natural do DM1, e está presente nos pacientes antes dos 5 anos de doença, estando associada, ao tempo de DM1, à presença de microalbuminúria e ao controle metabólico de longo-prazo. A ausência de disfunção de músculo liso endotelial no grupo com menos de 5 anos de DM, com valores de dilatação não-endotéliodependente semelhantes aos controles, sugere ser a DE um fenômeno ainda reversível nos primeiros anos de doença. / Patients with Type 1 diabetes (T1DM) are at high-risk for developing micro and macrovascular complications. Endothelial dysfunction (ED) has been suggested to be a precursor of both complications in Type 1 diabetes. Hyperglycemia may be associated to ED through generation of oxidative stress. The exactly moment when ED occurs in T1DM is until not well established. Also we do not known if long-term rather than short term metabolic control have a greater impact in ED. Objective: The aim of this study was to assess endothelial function by Flow Mediated Dilation (FMD) in (T1DM) patients and compare with non- diabetic controls. Secondary objectives were to analyze factors that could be associated to ED: duration of T1DM, glycemic control and microvascular complications like microalbuminuria. Research design and methods: In a cross-sectional study 57 adolescents with T1DM and 10 non-diabetic controls, were recruited and compared for the presence of ED by FMD with evaluation of reactive hyperemia (endothelium-dependent dilatation) and after using sublingual nitrate spray for assessed non-endothelialdependent dilatation. ED was considered when FMD ≤ 8% in relation to basal value. Patients performed intensive self monitoring blood glucose for 30 days before vascular studies. At day 30, blood was drawn for biochemical determinations and endothelial function was carried out. Historical data from Glycated hemoglobin (HbA1c), determined by immunoturbidimetry (Cobas Integra 400; Roche) were collected at 3, 6, 9,12,15,18 and 24 months before the test for FMD. Excluding criteria were any time tobacco use, clinical hypertension, obesity, hypothyroidism, statin use, current pregnancy and any history of previous neoplasia or vascular disease. Results: Of 57 T1DM patients studied, 28 (49%) presented ED. FMD was significantly decreased in T1DM compared to controls (9.48±6.48% vs. 14.56±5.60%, p=0.02). Nitrate-mediated dilation (%) was decreased in T1DM compared to controls (22.26±9.2% vs. 29.31±4.2%, p=0.02, RV= >8%), but it was not different between T1DM with or without ED (p=0.72). The duration of T1DM was longer in ED vs. Non- ED patients: 105.4±74.7 vs. 66.3±48.0 months, p= 0.02 and presented negative linear correlation between duration of T1DM and FMD (r=-0.284, p=0.03). HbA1c at the moment of the vascular analysis did not differ between ED and Non-ED patients (8.97±1.85% vs. 8.23±1.44%, p= 0.10) and it was not associated with FMD (r=-0.128, p=0.34). However, we found significant negative correlation between HbA1c and FMD at 15 months (r=-0.303, p=0.02) and at 12-24 months before vascular study, but not with median HbA1c of 0-12m (r=-0.181 p=0.8). Microalbuminuria was more prevalent in T1DM patients with ED than Non-ED (22.2% vs. 3.5%, p=0.04). FMD was decreased in microalbuminuric compared to normoalbuminuric patients (4.83±3.81% vs 10.35±6.50%, p=0.015). In T1DM patients with less than 5 years of disease, 10 of 28 (35.7%) presented ED. Nitrate-mediated dilation, in this group, was not decreased compared to controls (p=0.16) and it was not different in T1DM patients with or without ED (p=0.27). HbA1c at the moment of vascular analysis did not significantly differ in ED compared to Non-ED patients (8.20±0.94% vs.7.99±1.37%, p=0.66). Pearson’s correlation between FMD and historical HbA1c was negative with HbA1c at 12 (r=-0.419, p=0.03), at 15 (r=-0.437, p=0.03) and 12-24 months before vascular analysis (r=- 0.426, p=0.02). Conclusions: Endothelial function is impaired in T1DM patients compared to nondiabetic controls. ED is a phenomenon that can occur quite early in the natural history of T1DM, presented before 5 years of disease and is related to duration of disease, long- term metabolic control and microalbuminúria. Vascular smooth muscle was not impaired in T1DM patients with less than 5 years of disease, with values of non-endothelial-dependent dilation similar to controls, suggesting that ED can be a reversible event in this first years of disease.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A apneia obstrutiva do sono (AOS) é considerada um fator de risco independente para as doenças cardiovasculares. Existem evidências de que indivíduos com apneia obstrutiva do sono podem apresentar elevação nos mediadores inflamatórios, alterações no perfil metabólico, aumento na atividade do sistema nervoso simpático, com consequente elevação da pressão arterial e disfunção endotelial. Nos últimos anos, inúmeros estudos tem apontado a AOS como um dos fatores responsáveis pela hipertensão resistente. O objetivo do estudo foi avaliar a presença da apneia obstrutiva do sono e o comportamento da função endotelial em pacientes com hipertensão resistente, comparando com hipertensos apresentando pressão arterial controlada com até 3 classes diferentes de fármacos anti-hipertensivos. Trata-se de um estudo transversal com 40 pacientes hipertensos: 20 com hipertensão arterial resistente (HAR) e 20 com pressão arterial controlada por medicação (hipertensão arterial controlada; HAC), sem distinção de raça ou gênero, com idade entre 18 e 75 anos. A pressão arterial casual e a monitorização ambulatorial da pressão arterial foram aferidas por método oscilométrico em aparelhos automáticos. A função endotelial e a presença da apneia obstrutiva do sono foram avaliadas através da tonometria arterial periférica pelos equipamentos Endo-PAT2000 e o aparelho portátil Watch-PAT200, respectivamente. A avaliação antropométrica foi realizada através das aferições das circunferências da cintura e do pescoço, índice de massa corporal (IMC), e relação cintura-estatura. A composição corporal foi avaliada por bioimpedância elétrica BIODYNAMICS 450. As análises estatísticas foram realizadas pelo software GraphPad PRISM, versão 6.01. A prevalência de AOS no grupo com HAR foi de 85% (Índice de apneia-hipopneia [AHI]= 12,391,89) e de 80% no grupo com HAC (AHI =20,744,69), sendo mais frequente em homens (p=0,04; OR=3,86; 95% IC 0,99 a 14,52). Os dois grupos apresentaram valores semelhantes das variáveis antropométricas avaliadas. A função endotelial avaliada pelo índice de hiperemia reativa foi similar nos dois grupos (grupo HAR: 1,880,09 vs. grupo HAC: 2,030,09; p=0,28). Apesar do número de dessaturações de oxigênio >4% ter apresentado diferença significativa entre os grupos (grupo HAR: 28,755,08 vs. grupo HAC: 64,1516,97; p=0,04), o tempo total de sono (grupo HAR: 309,515,27 vs. grupo HAC: 323,318,74 min) e a saturação mínima da oxi-hemoglobina (grupo HAR: 87,80,85 vs. grupo HAC: 83,32,37%) não mostraram essa diferença. Considerando todos os pacientes hipertensos, o AHI apresentou correlação significativa com o peso corporal (r=0,51; p=0,0007), o IMC (r=0,41; p=0,007), a circunferência da cintura (r=0,44; p=0,005), a circunferência do pescoço (r=0,38; p=0,01) e a relação cintura-estatura (r=0,39; p=0,01). Os pacientes sem AOS em comparação com os pacientes com AOS, apresentaram risco significativamente menor de apresentar comprometimento da função endotelial (OR=0,17; 95% IC 0,04-0,72; p=0,03). Os achados do presente estudo sugerem que a prevalência de AOS em pacientes com hipertensão resistente é elevada, porém semelhante a de indivíduos com hipertensão controlada. Pacientes com hipertensão resistente e controlada não apresentaram diferenças significativas em relação à função endotelial. A gravidade de AOS no grupo total de hipertensos se associou com maior risco de comprometimento da função endotelial. / Obstructive sleep apnea (OSA) is considered an independent risk factor for cardiovascular disease. There is evidence that individuals with OSA may have increased inflammatory mediators, changes in the metabolic profile, increased sympathetic activity with consequent elevation of blood pressure (BP) and endothelial dysfunction. Resistant hypertension (RH) is defined as uncontrolled blood pressure (BP &#8805; 140/90mmHg) despite the current use of three hypotensive drugs at full doses, including a diuretic, or the need for >3 medications to control BP. OSA has been reported as the most common secondary cause of high blood pressure maintenance. The objective was to determine the prevalence of OSA and verify its association with endothelial function and anthropometric parameters in patients with resistant hypertension (RHGroup) and BP controlled by medication (CHGroup). It was a cross-sectional study involving 40 hypertensive patients (20 in RHG and 20 in CHG), aged between 18 and 75 years. Endothelial function and OSA were assessed by peripheral arterial tonometry. BP was measured by oscillometric method on automatic device. Endothelial function was assessed by peripheral arterial tonometry (PAT) by EndoPAT2000 and the OSA diagnosis also through PAT, using the portable device WatchPAT200. Anthropometric evaluation was performed through measurements of waist (WaC), hip and neck circumference (NC), BMI, waist to height ratio (WHtR), and body composition assessed by bioelectrical impedance. Patients were generally late middle-aged (54.95 2.39 in the RH group and 56.15 2.42 in the controlled hypertension [CH] group. The prevalence of OSA in RHG was 85% (17 of 20) [apnea-hypopnea index=12.391.89], and 80% (16 of 20) in CHG (AHI=20.744.69) and it was more frequent in men (93.7% [15 of 16] vs 75% [16 of 24]; p=0.04, OR=3.86; 95% IC 0.99 to 14.92). Both groups presented similar anthropometric parameters values. Endothelial function evaluated by reactive hyperemia index was similar in both groups (RHG: 1.880.09 vs CHG: 2.030.09; p=0.28). Although we found differences in oxygen desaturation> 4% (RHG: 28.75 5.08 vs CHG: 64.15 16.97, p = 0.04), total sleep time (RHG: 309.5 15.27 vs CHG: 323.3 18.74 min) and minimum saturation (RHG: 87.80.85 vs CHG: 83.32.37%) was not different. In general, OSA was correlated with weight (r = 0.51, p = 0.0007), BMI (r = 0.41, p = 0.0078), WaC (r = 0, 44, p = 0.005), NC (r = 0.38, p = 0.01) and WHtR (r = 0.39, p = 0.01) and independently associated with impairment of endothelial function (p = 0.0297, OR = 0.17, 95% CI 0.04 to 0.72). In conclusion, the findings of this study show high prevalence of OSA in patients with resistant hypertension, similar to that of controlled hypertension group. There were no significant differences in endothelial function between resistant and controlled hypertension patients. The presence of OSA in the total group of hypertensive patients was associated with increased risk of impaired endothelial function.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A hipertensão é uma das mais importantes causas de morte prematura no mundo. Estudos sobre a Euterpe oleracea Mart. (açaí), uma planta típica do Brasil e rica em polifenóis, têm mostrado grande potencial terapêutico contra a hipertensão, uma vez que seus benefícios podem ser associados às ações antioxidante, vasodilatadora e anti-hipertensiva. O rato espontaneamente hipertenso (SHR) é um modelo experimental utilizado para o estudo da hipertensão essencial. Neste estudo, investigamos o efeito do tratamento crônico do extrato hidroalcoólico do caroço de açaí (ASE) sobre a hipertensão de SHR. Animais SHR e Wistar receberam tratamento com ASE (200 mg/Kg/dia) na água de beber, ou veículo, desde 21 dias até 4 meses de idade e tiveram a pressão arterial sistólica (PAS) aferida por pletismografia de cauda. Os efeitos vasodilatadores da acetilcolina (ACh) e nitroglicerina (NG) foram estudados em leito arterial mesentérico (LAM) perfundido e pré-contraído com norepinefrina. A atividade das enzimas superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx), os níveis de malondialdeído (MDA), a carbonilação de proteínas e os níveis de nitrito foram avaliados em plasma, LAM, coração e rim por espectrofotometria. A expressão das proteínas SOD e eNOS foram avaliadas por western blot em LAM e as alterações vasculares pela espessura da túnica média em aorta. A PAS foi maior (p<0.05) nos animais SHR, e reduzida pelo tratamento com ASE. O efeito vasodilatador reduzido da ACh em SHR foi recuperado pelo ASE e o da NG não foi diferente entre os grupos. Não houve diferença nos níveis de glicose e insulina em SHR comparados aos controles. Entretanto, a insulina se apresentou reduzida no grupo SHR+ASE. O nível de renina foi maior nos SHR e normalizado pelo ASE (p<0.05). Os níveis de MDA não foram diferentes entre SHR e controles, entretanto o tratamento com ASE reduziu esses níveis em rim de SHR (p<0.05). Os níveis de carbonilação de proteínas foram maiores em amostras de rim e coração de SHR e o ASE reduziu o dano sobre proteínas (p<0.05), não tendo diferença em plasma e LAM. A atividade da SOD foi menor em amostras de rim nos animais SHR e aumentada pelo tratamento com ASE (p<0.05). Entretanto, a atividade aumentada da SOD em coração e LAM dos SHR, foi reduzida pelo tratamento com ASE, não havendo diferença em amostras de plasma. Não houve diferença na atividade da GPx em amostras de LAM e coração dos diferentes grupos, porém sua atividade foi aumentada em rim dos SHR, e o tratamento com ASE normalizou essa atividade. Em plasma, a atividade da GPx foi reduzida em SHR e aumentada pelo tratamento (p<0.05). A atividade da enzima CAT foi reduzida em plasma e rim de SHR e o ASE aumentou sua atividade. Não houve diferença em amostras de LAM, entretanto em amostras de coração o tratamento aumentou a atividade da CAT em SHR (p<0.05). Em amostras de plasma, coração e rim, não houve diferença nos níveis de nitrito entre os diferentes grupos, porém em amostras de LAM foram menores em SHR e SHR+ASE (p<0.05). A expressão das proteínas eNOS e SOD apresentaram-se aumentadas em SHR (p<0.05) sem alteração com o tratamento. Os SHR apresentaram um aumento na espessura da camada média da aorta que foi reduzido (p<0.05) pelo ASE. Este estudo demonstrou que o tratamento crônico com ASE em SHR reduziu a hipertensão, preveniu a disfunção endotelial e o remodelamento vascular. O aumento da defesa antioxidante e redução do dano oxidativo devem contribuir para os efeitos benéficos de ASE. Portanto, sugerimos que o ASE pode ser uma ferramenta importante para o tratamento das alterações cardiovasculares associadas à hipertensão essencial. / Hypertension is a major cause of premature death worldwide. Studies on the Euterpe oleracea Mart. (açaí), a typical plant of Brazil, rich in polyphenols, have shown great therapeutic potential against hypertension, since its benefits can be associated with antioxidant action, vasodilator and antihypertensive. The spontaneously hypertensive rat (SHR) is an experimental model used for the study of essential hypertension. Therefore, this study investigated the effect of chronic treatment of hydroalcoholic extract of stone of the açaí (ASE) on hypertension in SHR. For this purpose, SHR and Wistar rats were treated with ASE (200 mg.kg-1.day-) in drinking water, or vehicle, from 21 days to 4 months of age and had their systolic blood pressure (SBP) measured by tail plethysmography. The vasodilatory effects of acetylcholine (ACh) and nitroglycerin (NG) were studied in perfused mesenteric arterial bed (LAM) pre-contracted with norepinephrine. The activity of the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), levels of malondialdehyde (MDA), protein carbonylation and nitrite were evaluated in plasma, LAM, heart and kidney by spectrophotometry. The expression of eNOS and SOD protein were evaluated by western blot and vascular changes by the thickness of the tunica media in aorta. SBP was higher (p <0.05) in SHR, and reduced by treatment with ASE. The reduced vasodilator effect of ACh in SHR was recovered by ASE and of the NG was not different between groups. There was no difference in the levels of glucose and insulin in SHR compared to controls. However, insulin was reduced in the SHR+ASE group. The level of renin was higher in SHR and normalized by the ASE (p <0.05). MDA levels were not different between SHR and controls, however treatment with ASE reduced these levels in kidney samples of SHR (p <0.05). The levels of protein carbonylation were higher in samples of kidney and heart of SHR and the protein damage was reduced by treatment with ASE (p <0.05), with no difference in plasma samples and LAM. The SOD activity was lower in samples of kidney in SHR and increased by treatment with ASE (p <0.05). However, the increased activity of SOD in samples of heart and LAM of SHR was reduced by treatment with ASE, with no difference in plasma. There was no difference in GPx activity in samples of LAM and hearts of different groups, but its activity was increased in kidney of SHR, and ASE treatment normalized this activity. In plasma GPx activity was reduced in SHR and increased by treatment (p <0.05). The CAT enzyme activity was reduced in samples of plasma and kidney of SHR and ASE increased its activity. There was no difference in samples of LAM, but in samples of heart treatment increased the activity of CAT in SHR (p <0.05). In plasma samples, heart and kidney, there was no difference in nitrite levels between different groups, but it was reduced in samples of LAM of SHR and SHR+ASE (p <0.05). The expression of eNOS and SOD protein was increased in SHR (p <0.05) and unchanged by treatment. The SHR exhibited an increase in medial thickness of the aorta which was reduced (p <0.05) by ASE. This study showed that chronic treatment with ASE reduced hypertension in the SHR and prevented the endothelial dysfunction and vascular remodeling. The increase in antioxidant defense and reduction of oxidative damage may contribute to the beneficial effects of ASE. Therefore, we suggest that the ASE can be an important tool for the treatment of cardiovascular disorders associated with essential hypertension.

O Diabetes Mellitus tipo 1 (DM1) está associado a uma incidência aumentada de doença micro e macrovascular. Estudos sugerem que a doença vascular no DM1 tenha como evento precursor a disfunção endotelial (DE). A hiperglicemia parece causar DE no DM1 através da geração do estresse oxidativo. O momento exato do surgimento da DE na história natural do DM1, assim como a influência do controle glicêmico de curto e longo prazo ainda não estão estabelecidos. Objetivo: O objetivo principal do presente estudo foi avaliar a função endotelial através da Dilatação Arterial Mediada por Fluxo (DMF) em indivíduos com Diabetes Mellitus tipo1. Os objetivos secundários foram analisar os fatores que possam estar envolvidos com a disfunção endotelial no DM1, como o tempo de diabetes, o controle glicêmico e a presença de complicações microvasculares, como a microalbuminúria. Métodos: Estudo prospectivo transversal com 57 pacientes com DM1 e 10 indivíduos não diabéticos, consecutivamente alocados e comparados quanto à presença de DE, através da DMF, aferida pela dilatação da artéria braquial após hiperemia reativa (dilatação endotélio-dependente) e após dilatação mediada por uso de nitrato sublingual (dilatação endotélio-independente). Considerou-se como DE quando valores de DMF foram menores ou iguais a 8% em relação ao valor basal. Os pacientes foram orientados a fazer monitorização glicêmica capilar intensiva nos 30 dias que antecederam a avaliação vascular. No 30º dia, houve coleta de exames laboratoriais e a avaliação vascular foi realizada. Dados prospectivos e históricos de hemoglobina glicosilada (HbA1c), através da técnica de imunoturbidimetria (Cobas Integra 400; Roche), foram obtidos aos 3, 6, 9,12,15,18 e 24 meses anteriores ao teste para DMF. Os critérios de exclusão foram: tabagismo, hipertensão, obesidade, hipotireoidismo, uso de estatina, gestação, história de neoplasia ou doença vascular. Resultados: Em 57 pacientes com DM1 estudados, 28 (49%) apresentaram DE. A média da dilatação endotélio-dependente foi significativamente menor nos pacientes com DM1, comparados aos indivíduos não-diabéticos (9,48±6,48% vs.14,56±5,60%, p=0,02). A dilatação endotélio-independente foi significativamente menor nos pacientes com DM1 em relação aos controles (22,26±9,2% vs. 29,31±4,2%, p=0,02, VR: acima de 8%), mas não houve diferença entre os DM1 com ou sem DE (p= 0,72). O tempo de DM1 (meses) foi maior nos pacientes com DE do que nos sem DE (105,4±74,7 vs. 66,3±48,0, p=0.02) e houve correlação linear negativa entre duração do DM e presença de DE (r-0,28, p=0,02). A média da HbA1c (%) coletada no momento da avaliação vascular foi semelhante entre pacientes com DM1 com DE e sem DE (8,97%±1.85 vs 8,23%±1.45, p=0.10) e não houve correlação significativa com a DMF (r=-0,128 p=0,34). Todavia, quando as HbA1c históricas foram avaliadas, houve correlação significativa com a HbA1c aos 15 meses (r=-0,303, p=0,02) e no período de 12-24 meses anteriores ao exame vascular (r=-0,289, p=0,03), mas não com a HbA1c média de 0-12m (r=-0,181 p=0,18). A DMF foi menor nos pacientes com microalbuminúria em relação aos normoalbuminúrcos (4,83±3,81% vs 10,35±6,50%, p=0,015). A microalbuminúria também foi mais prevalente nos DM1 com DE do que sem DE (22,2% vs 3,5%, p=0,04). Considerando apenas os pacientes com DM1 com tempo de DM menor que 5 anos, 10/28 (35,7%) apresentaram DE. Com relação a dilatação não-dependente de endotélio (%), não houve diferença em relação aos controles (p=0,16) e nem entre os DM1 com e sem DE (p=0,27). A média da HbA1c na época do exame vascular também não foi diferente nos pacientes com e sem DE (8,20±0,94% vs. 7,99±1,37%, p=0,66). As correlações de Pearson entre a DMF e as HbA1c históricas foram negativas aos 12 meses (r=-0,419, p=0,03), aos 15 meses (r=-0,437, p=0,03) e com a HbA1c média de12-24 meses (r=-0,426, p=0,027). Conclusões: Pacientes com DM1 apresentam prejuízo na função endotelial, quando comparados a controles não diabéticos. A DE é um evento precoce na história natural do DM1, e está presente nos pacientes antes dos 5 anos de doença, estando associada, ao tempo de DM1, à presença de microalbuminúria e ao controle metabólico de longo-prazo. A ausência de disfunção de músculo liso endotelial no grupo com menos de 5 anos de DM, com valores de dilatação não-endotéliodependente semelhantes aos controles, sugere ser a DE um fenômeno ainda reversível nos primeiros anos de doença. / Patients with Type 1 diabetes (T1DM) are at high-risk for developing micro and macrovascular complications. Endothelial dysfunction (ED) has been suggested to be a precursor of both complications in Type 1 diabetes. Hyperglycemia may be associated to ED through generation of oxidative stress. The exactly moment when ED occurs in T1DM is until not well established. Also we do not known if long-term rather than short term metabolic control have a greater impact in ED. Objective: The aim of this study was to assess endothelial function by Flow Mediated Dilation (FMD) in (T1DM) patients and compare with non- diabetic controls. Secondary objectives were to analyze factors that could be associated to ED: duration of T1DM, glycemic control and microvascular complications like microalbuminuria. Research design and methods: In a cross-sectional study 57 adolescents with T1DM and 10 non-diabetic controls, were recruited and compared for the presence of ED by FMD with evaluation of reactive hyperemia (endothelium-dependent dilatation) and after using sublingual nitrate spray for assessed non-endothelialdependent dilatation. ED was considered when FMD ≤ 8% in relation to basal value. Patients performed intensive self monitoring blood glucose for 30 days before vascular studies. At day 30, blood was drawn for biochemical determinations and endothelial function was carried out. Historical data from Glycated hemoglobin (HbA1c), determined by immunoturbidimetry (Cobas Integra 400; Roche) were collected at 3, 6, 9,12,15,18 and 24 months before the test for FMD. Excluding criteria were any time tobacco use, clinical hypertension, obesity, hypothyroidism, statin use, current pregnancy and any history of previous neoplasia or vascular disease. Results: Of 57 T1DM patients studied, 28 (49%) presented ED. FMD was significantly decreased in T1DM compared to controls (9.48±6.48% vs. 14.56±5.60%, p=0.02). Nitrate-mediated dilation (%) was decreased in T1DM compared to controls (22.26±9.2% vs. 29.31±4.2%, p=0.02, RV= >8%), but it was not different between T1DM with or without ED (p=0.72). The duration of T1DM was longer in ED vs. Non- ED patients: 105.4±74.7 vs. 66.3±48.0 months, p= 0.02 and presented negative linear correlation between duration of T1DM and FMD (r=-0.284, p=0.03). HbA1c at the moment of the vascular analysis did not differ between ED and Non-ED patients (8.97±1.85% vs. 8.23±1.44%, p= 0.10) and it was not associated with FMD (r=-0.128, p=0.34). However, we found significant negative correlation between HbA1c and FMD at 15 months (r=-0.303, p=0.02) and at 12-24 months before vascular study, but not with median HbA1c of 0-12m (r=-0.181 p=0.8). Microalbuminuria was more prevalent in T1DM patients with ED than Non-ED (22.2% vs. 3.5%, p=0.04). FMD was decreased in microalbuminuric compared to normoalbuminuric patients (4.83±3.81% vs 10.35±6.50%, p=0.015). In T1DM patients with less than 5 years of disease, 10 of 28 (35.7%) presented ED. Nitrate-mediated dilation, in this group, was not decreased compared to controls (p=0.16) and it was not different in T1DM patients with or without ED (p=0.27). HbA1c at the moment of vascular analysis did not significantly differ in ED compared to Non-ED patients (8.20±0.94% vs.7.99±1.37%, p=0.66). Pearson’s correlation between FMD and historical HbA1c was negative with HbA1c at 12 (r=-0.419, p=0.03), at 15 (r=-0.437, p=0.03) and 12-24 months before vascular analysis (r=- 0.426, p=0.02). Conclusions: Endothelial function is impaired in T1DM patients compared to nondiabetic controls. ED is a phenomenon that can occur quite early in the natural history of T1DM, presented before 5 years of disease and is related to duration of disease, long- term metabolic control and microalbuminúria. Vascular smooth muscle was not impaired in T1DM patients with less than 5 years of disease, with values of non-endothelial-dependent dilation similar to controls, suggesting that ED can be a reversible event in this first years of disease.

abstract: Cardiovascular disease has reached epidemic proportions resulting in its ranking as the number one cause of mortality in the Western world. A key player in the pathophysiology of vascular disease is oxidative stress due to free radical accumulation. This intervention study was conducted to evaluate any potential mediation of oxidative stress using a soil-derived organometallic compound (OMC) with suspected antioxidant properties. A 10-week study was conducted in male Sprague-Dawley rats (n = 42) fed either a high-fat diet (HFD) consisting of 60% kcal from fat or a standard Chow diet containing only 6% kcals from fat. Rats from each diet group were then subdivided into 3 subgroups (n = 6-10 each) that received 0.0 mg/mL, 0.6 mg/mL or 3.0 mg/mL OMC. Neither the diet nor OMC significantly changed protein expression of inducible nitric oxide synthase (iNOS) in isolated aortas. Plasma levels of the inflammatory marker, tumor necrosis factor alpha (TNFα) were below detection after the 10-week trial. Superoxide dismutase (SOD), a scavenger of the free radical, superoxide, was not significantly different following HFD although levels of SOD were significantly higher in Chow rats treated with 0.6 mg/mL OMC compared to HFD rats treated with the same dose (p < 0.05). Lipopolysaccharides (LPS) were significantly increased following 10 weeks of high fat intake (p < 0.05). This increase in endotoxicity was prevented by the high dose of OMC. HFD significantly increased fasting serum glucose levels at both 6 weeks (p < 0.001) and 10 weeks (p < 0.025) compared to Chow controls. The high dose of OMC significantly prevented the hyperglycemic effects of the HFD in rats at 10 weeks (p = 0.021). HFD-fed rats developed hyperinsulinemia after 10 weeks of feeding (p = 0.009), which was not prevented by OMC. The results of this study indicate that OMC may be an effective strategy to help manage diet-induced hyperglycemia and endotoxemia. However, further research is needed to determine the mechanism by which OMC helps prevent hyperglycemia as measures of inflammation (TNFα) and vascular damage (iNOS) were inconclusive. / Dissertation/Thesis / Masters Thesis Nutrition 2018