Alissa A. Frame

Altered Mitochondrial Dynamics Contributes to Endothelial Dysfunction in Diabetes Mellitus

Background— Endothelial dysfunction contributes to the development of atherosclerosis in patients with diabetes mellitus, but the mechanisms of endothelial dysfunction in this setting are incompletely understood. Recent studies have shown altered mitochondrial dynamics in diabetes mellitus with increased mitochondrial fission and production of reactive oxygen species. We investigated the contribution of altered dynamics to endothelial dysfunction in diabetes mellitus. Methods and Results— We observed mitochondrial fragmentation (P=0.002) and increased expression of fission-1 protein (Fis1; P<0.0001) in venous endothelial cells freshly isolated from patients with diabetes mellitus (n=10) compared with healthy control subjects (n=9). In cultured human aortic endothelial cells exposed to 30 mmol/L glucose, we observed a similar loss of mitochondrial networks and increased expression of Fis1 and dynamin-related protein-1 (Drp1), proteins required for mitochondrial fission. Altered mitochondrial dynamics was associated with increased mitochondrial reactive oxygen species production and a marked impairment of agonist-stimulated activation of endothelial nitric oxide synthase and cGMP production. Silencing Fis1 or Drp1 expression with siRNA blunted high glucose–induced alterations in mitochondrial networks, reactive oxygen species production, endothelial nitric oxide synthase activation, and cGMP production. An intracellular reactive oxygen species scavenger provided no additional benefit, suggesting that increased mitochondrial fission may impair endothelial function via increased reactive oxygen species. Conclusion— These findings implicate increased mitochondrial fission as a contributing mechanism for endothelial dysfunction in diabetic states.

Protein Kinase C-β Contributes to Impaired Endothelial Insulin Signaling in Humans With Diabetes Mellitus

Background— Abnormal endothelial function promotes atherosclerotic vascular disease in diabetes. Experimental studies indicate that disruption of endothelial insulin signaling, through the activity of protein kinase C-&bgr; (PKC&bgr;) and nuclear factor &kgr;B, reduces nitric oxide availability. We sought to establish whether similar mechanisms operate in the endothelium in human diabetes mellitus. Methods and Results— We measured protein expression and insulin response in freshly isolated endothelial cells from patients with type 2 diabetes mellitus (n=40) and nondiabetic controls (n=36). Unexpectedly, we observed 1.7-fold higher basal endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 in patients with diabetes mellitus (P=0.007) without a difference in total eNOS expression. Insulin stimulation increased eNOS phosphorylation in nondiabetic subjects but not in diabetic patients (P=0.003), consistent with endothelial insulin resistance. Nitrotyrosine levels were higher in diabetic patients, indicating endothelial oxidative stress. PKC&bgr; expression was higher in diabetic patients and was associated with lower flow-mediated dilation (r=−0.541, P=0.02). Inhibition of PKC&bgr; with LY379196 reduced basal eNOS phosphorylation and improved insulin-mediated eNOS activation in patients with diabetes mellitus. Endothelial nuclear factor &kgr;B activation was higher in diabetes mellitus and was reduced with PKC&bgr; inhibition. Conclusions— We provide evidence for the presence of altered eNOS activation, reduced insulin action, and inflammatory activation in the endothelium of patients with diabetes mellitus. Our findings implicate PKC&bgr; activity in endothelial insulin resistance.

Interaction of tau with the RNA-Binding Protein TIA1 Regulates tau Pathophysiology and Toxicity

Dendritic mislocalization of microtubule associated protein tau is a hallmark of tauopathies, but the role of dendritic tau is unknown. We now report that tau interacts with the RNA-binding protein (RBP) TIA1 in brain tissue, and we present the brain-protein interactome network for TIA1. Analysis of the TIA1 interactome in brain tissue from wild-type (WT) and tau knockout mice demonstrates that tau is required for normal interactions of TIA1 with proteins linked to RNA metabolism, including ribosomal proteins and RBPs. Expression studies show that tau regulates the distribution of TIA1, and tau accelerates stress granule (SG) formation. Conversely, TIA1 knockdown or knockout inhibits tau misfolding and associated toxicity in cultured hippocampal neurons, while overexpressing TIA1 induces tau misfolding and stimulates neurodegeneration. Pharmacological interventions that prevent SG formation also inhibit tau pathophysiology. These studies suggest that the pathophysiology of tauopathy requires an intimate interaction with RNA-binding proteins.

Effects of Concord grape juice on ambulatory blood pressure in prehypertension and stage 1 hypertension

BACKGROUND Consumption of flavonoid-containing foods may be useful for the management of hypertension. OBJECTIVE We investigated whether 100% Concord grape juice lowers blood pressure in patients with prehypertension and stage 1 hypertension. DESIGN We conducted a double-blind crossover study to compare the effects of grape juice (7 mL · kg⁻¹ · d⁻¹) and matched placebo beverage on 24-h ambulatory blood pressure, stress-induced changes in blood pressure, and biochemical profile. Participants consumed each beverage for 8 wk with a 4-wk rest period between beverages. They ceased consumption of grapes and other flavonoid-containing beverages throughout the study. RESULTS We enrolled 64 otherwise healthy patients taking no antihypertensive medications (31% women, 42% black, age 43 ± 12 y). Baseline mean (± SD) cuff blood pressure was 138 ± 7 (systolic)/82 ± 7 (diastolic) mm Hg. No effects on the primary endpoint of 24-h mean systolic blood pressure, diastolic blood pressure, or stress-induced changes in blood pressure were observed. A secondary endpoint was nocturnal dip in systolic pressure. At baseline, nocturnal pressure was 8.3 ± 7.1% lower at night than during daytime. The mean nocturnal dip increased 1.4 percentage points after grape juice and decreased 2.3 percentage points after placebo (P = 0.005). Fasting blood glucose was 91 ± 10 mg/dL at baseline for the entire cohort. Glucose decreased 2 mg/dL after consumption of grape juice and increased 1 mg/dL after consuming the placebo (P = 0.03). CONCLUSIONS We observed no effect of grape juice on ambulatory blood pressure in this cohort of relatively healthy individuals with modestly elevated blood pressure. Secondary analyses suggested favorable effects on nocturnal dip and glucose homeostasis that may merit further investigation. This trial was registered at clinicaltrials.gov as NCT00302809.

Relation of mitochondrial oxygen consumption in peripheral blood mononuclear cells to vascular function in type 2 diabetes mellitus

Recent studies have shown mitochondrial dysfunction and increased production of reactive oxygen species in peripheral blood mononuclear cells (PBMCs) and endothelial cells from patients with diabetes mellitus. Mitochondria oxygen consumption is coupled to adenosine triphosphate (ATP) production and also occurs in an uncoupled fashion during formation of reactive oxygen species by components of the electron transport chain and other enzymatic sites. We therefore hypothesized that diabetes would be associated with higher total and uncoupled oxygen consumption in PBMCs that would correlate with endothelial dysfunction. We developed a method to measure oxygen consumption in freshly isolated PBMCs and applied it to 26 patients with type 2 diabetes mellitus and 28 non-diabetic controls. Basal (192±47 vs 161±44 pmoles/min, p=0.01), uncoupled (64±16 vs 53±13 pmoles/min, p=0.007), and maximal (795±87 vs 715±128 pmoles/min, p=0.01) oxygen consumption rates were higher in diabetic patients compared to controls. There were no significant correlations between oxygen consumption rates and endothelium-dependent flow-mediated dilation measured by vascular ultrasound. Non-endothelium-dependent nitroglycerin-mediated dilation was lower in diabetics (10.1±6.6 vs 15.8±4.8%, p=0.03) and correlated with maximal oxygen consumption (r = −0.64, p=0.001). In summary, we found that diabetes mellitus is associated with a pattern of mitochondrial oxygen consumption consistent with higher production of reactive oxygen species. The correlation between oxygen consumption and nitroglycerin-mediated dilation may suggest a link between mitochondrial dysfunction and vascular smooth muscle cell dysfunction that merits further study. Finally, the described method may have utility for the assessment of mitochondrial function in larger scale observational and interventional studies in humans.

Intravenous Lipid Infusion Induces Endoplasmic Reticulum Stress in Endothelial Cells and Blood Mononuclear Cells of Healthy Adults

Background Endoplasmic reticulum (ER) stress and the subsequent unfolded protein response may initially be protective, but when prolonged, have been implicated in atherogenesis in diabetic conditions. Triglycerides and free fatty acids (FFAs) are elevated in patients with diabetes and may contribute to ER stress. We sought to evaluate the effect of acute FFA elevation on ER stress in endothelial and circulating white cells. Methods and Results Twenty‐one healthy subjects were treated with intralipid (20%; 45 mL/h) plus heparin (12 U/kg/h) infusion for 5 hours. Along with increased triglyceride and FFA levels, intralipid/heparin infusion reduced the calf reactive hyperemic response without a change in conduit artery flow‐mediated dilation consistent with microvascular dysfunction. To investigate the short‐term effects of elevated triglycerides and FFA, we measured markers of ER stress in peripheral blood mononuclear cells (PBMCs) and vascular endothelial cells (VECs). In VECs, activating transcription factor 6 (ATF6) and phospho‐inositol requiring kinase 1 (pIRE1) proteins were elevated after infusion (both P<0.05). In PBMCs, ATF6 and spliced X‐box‐binding protein 1 (XBP‐1) gene expression increased by 2.0‐ and 2.5‐fold, respectively (both P<0.05), whereas CHOP and GADD34 decreased by ≈67% and 74%, respectively (both P<0.01). ATF6 and pIRE1 protein levels also increased (both P<0.05), and confocal microscopy revealed the nuclear localization of ATF6 after infusion, suggesting activation. Conclusions Along with microvascular dysfunction, intralipid infusion induced an early protective ER stress response evidenced by activation of ATF6 and IRE1 in both leukocytes and endothelial cells. Our results suggest a potential link between metabolic disturbances and ER stress that may be relevant to vascular disease.

Effect of Sulfasalazine on Inflammation and Endothelial Function in Patients with Established Coronary Artery Disease

Inflammation is critical for atherosclerosis development and may be a target for risk-reduction therapy. In experimental studies, activation of the inflammatory regulator, nuclear factor kappa B (NFlB), contributes to endothelial activation and reduced nitric oxide production. We treated patients with coronary artery disease with sulfasalazine, an inhibitor of NFκB, and placebo in a randomized, double-blind, crossover study design. Brachial artery flow-mediated dilation (FMD) and digital vascular function were measured at baseline and after each 6-week treatment period. Of the 53 patients enrolled in the crossover study, 32 (age 60 ± 10, 22% female) completed all the visits, with a high rate of study withdrawal due to gastrointestinal side effects. In a subset of 10 participants, we compared the effects of 4 days of sulfasalazine treatment (n = 5) to no treatment (n = 5) on NFκB-regulated gene expression in peripheral blood mononuclear cells. Tumor necrosis factor α-stimulated expression of CD69 and NFlB subunit p50 was significantly blunted after 4 days of sulfasalazine treatment but not after no treatment. However, FMD and digital vasodilator response did not significantly change from baseline with long-term sulfasalazine treatment. Short-term sulfasalazine inhibited NFlB activity; however, long-term treatment was poorly tolerated and did not improve endothelial function. Our findings suggest that sulfasalazine therapy is not the optimal anti-inflammatory treatment for reversing endothelial dysfunction in cardiovascular disease. Further studies are warranted to investigate the potential for NFlB inhibition to reduce cardiovascular risk.

Renal sodium handling and sodium sensitivity

The pathophysiology of hypertension, which affects over 1 billion individuals worldwide, involves the integration of the actions of multiple organ systems, including the kidney. The kidney, which governs sodium excretion via several mechanisms including pressure natriuresis and the actions of renal sodium transporters, is central to long term blood pressure regulation and the salt sensitivity of blood pressure. The impact of renal sodium handling and the salt sensitivity of blood pressure in health and hypertension is a critical public health issue owing to the excess of dietary salt consumed globally and the significant percentage of the global population exhibiting salt sensitivity. This review highlights recent advances that have provided new insight into the renal handling of sodium and the salt sensitivity of blood pressure, with a focus on genetic, inflammatory, dietary, sympathetic nervous system and oxidative stress mechanisms that influence renal sodium excretion. Increased understanding of the multiple integrated mechanisms that regulate the renal handling of sodium and the salt sensitivity of blood pressure has the potential to identify novel therapeutic targets and refine dietary guidelines designed to treat and prevent hypertension.

GNAI2 polymorphic variance associates with salt sensitivity of blood pressure in the Genetic Epidemiology Network of Salt Sensitivity study

Salt sensitivity of blood pressure (BP) increases hypertension risk and associated adverse cardiovascular outcomes. At present, there are no validated rapid tests or diagnostic markers to identify salt sensitivity of BP in clinical practice. Based on our prior animal studies that report a role for brain Gαi2 proteins in the salt sensitivity of BP and evidence that GNAI2 single nucleotide polymorphisms (SNPs) associate with hypertension risk, we investigated the hypothesis that GNAI2 SNPs associate with salt sensitivity of BP in humans. Our data provide the first evidence that a GNAI2 SNP (rs10510755) positively associates with salt sensitivity of BP in the Genetic Epidemiology of Salt Sensitivity data set (continuous phenotype P = 0.049, case-control phenotype P = 0.039; n = 968), independently of subject sex or age. These observations suggest that genotyping at GNAI2 may be a useful biomarker in identifying individuals at risk for developing salt-sensitive BP and related complications or in identifying salt sensitivity within the hypertensive population.

Mechanisms of altered renal sodium handling in age-related hypertension

The prevalence of hypertension rises with age to approximately two out of three adults over the age of 60 in the United States. Although the mechanisms underlying age-related hypertension are incompletely understood, sodium homeostasis is critical to the long-term regulation of blood pressure and there is strong evidence that aging is associated with alterations in renal sodium handling. This minireview focuses on recent advancements in our understanding of the vascular, neurohumoral, and renal mechanisms that influence sodium homeostasis and promote age-related hypertension.