Protein kinase C-ß 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-ß (PKCß) and nuclear factor κB, 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. PKCß expression was higher in diabetic patients and was associated with lower flow-mediated dilation (r=-0.541, P=0.02). Inhibition of PKCß with LY379196 reduced basal eNOS phosphorylation and improved insulin-mediated eNOS activation in patients with diabetes mellitus. Endothelial nuclear factor κB activation was higher in diabetes mellitus and was reduced with PKCß 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 PKCß activity in endothelial insulin resistance.

De novo lipogenesis maintains vascular homeostasis through endothelial nitric-oxide synthase (eNOS) palmitoylation.

Endothelial dysfunction leads to lethal vascular complications in diabetes and related metabolic disorders. Here, we demonstrate that de novo lipogenesis, an insulin-dependent process driven by the multifunctional enzyme fatty-acid synthase (FAS), maintains endothelial function by targeting endothelial nitric-oxide synthase (eNOS) to the plasma membrane. In mice with endothelial inactivation of FAS (FASTie mice), eNOS membrane content and activity were decreased. eNOS and FAS were physically associated; eNOS palmitoylation was decreased in FAS-deficient cells, and incorporation of labeled carbon into eNOS-associated palmitate was FAS-dependent. FASTie mice manifested a proinflammatory state reflected as increases in vascular permeability, endothelial inflammatory markers, leukocyte migration, and susceptibility to LPS-induced death that was reversed with an NO donor. FAS-deficient endothelial cells showed deficient migratory capacity, and angiogenesis was decreased in FASTie mice subjected to hindlimb ischemia. Insulin induced FAS in endothelial cells freshly isolated from humans, and eNOS palmitoylation was decreased in mice with insulin-deficient or insulin-resistant diabetes. Thus, disrupting eNOS bioavailability through impaired lipogenesis identifies a novel mechanism coordinating nutritional status and tissue repair that may contribute to diabetic vascular disease.

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.

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.

The brachial artery remodels to maintain local shear stress despite the presence of cardiovascular risk factors.

OBJECTIVE: Under physiological conditions, arteries remodel in response to changes in blood flow to maintain local shear stress. Risk factors and developing atherosclerosis may be associated with maladaptive remodeling that produces relatively large arteries with low levels of shear stress. Recent studies have shown that the brachial artery and other peripheral arteries are enlarged in patients with risk factors and cardiovascular disease, and we tested the hypothesis that this finding represents maladaptive remodeling. METHODS AND RESULTS: We measured brachial artery diameter and flow by ultrasound and calculated shear stress in a diverse cohort of 1583 subjects (age 53+/-17 years, 62% male, and 51% with coronary artery disease and/or peripheral arterial disease). In a stepwise linear regression model, age (P<0.001), gender (P<0.001), body mass index (P<0.001), hypertension (P=0.005), and hypercholesterolemia (P=0.02) were associated with larger brachial diameter. Older age was associated with lower shear stress (P<0.01), consistent with maladaptive remodeling. However, body mass index, hypertension, hypercholesterolemia, and prevalent atherosclerosis were associated with proportionate changes in blood flow and no difference in shear stress compared to reference groups, suggesting adaptive remodeling. CONCLUSIONS: These findings suggest that enlargement of the brachial artery in the setting of obesity, hypertension, hypercholesterolemia, and atherosclerosis reflects adaptive remodeling. The results provide further support for the concept that arterial remodeling is an important homeostatic response that is maintained despite the presence of risk factors and developing atherosclerosis.

Flow-induced arterial remodeling relates to endothelial function in the human forearm.

BACKGROUND: Chronic changes in blood flow stimulate arterial remodeling, which contributes to the maintenance of vascular homeostasis. Experimental studies suggest that remodeling represents a response to local changes in endothelial shear stress and is nitric oxide-dependent. METHODS AND RESULTS: To investigate determinants of outward arterial remodeling in humans, we measured ulnar artery flow, diameter, and flow-mediated dilation before and after removal of the adjacent radial artery in 53 patients who were undergoing coronary bypass surgery (age 60+/-11 years; 13% female). Removal of the radial artery increased ulnar artery blood flow by 35% (P=0.009) and increased ulnar artery diameter by 9% (P<0.001) 4 to 8 weeks after surgery. At 1 week, ulnar artery shear stress was increased by 58% (P<0.001), but it was no longer different from baseline at longer-term follow-up. The contralateral ulnar artery was unaffected, which suggests that these findings were not attributable to the systemic effects of medications or the postoperative state. Extent of outward remodeling correlated with the increase in blood flow (r=0.50, P=0.001) and with flow-mediated dilation at baseline (r=0.50, P=0.001). Remodeling correlated inversely with baseline endothelial expression of P-selectin in the radial artery (r=-0.76, P=0.004, n=14). CONCLUSIONS: A sustained increase in blood flow in the ulnar artery induced outward arterial remodeling despite the presence of risk factors and coronary artery disease. The remodeling response was related to endothelial phenotype, as reflected by flow-mediated dilation and expression of P-selectin. These findings provide evidence that the endothelium plays an important role in the regulation of vascular structure in humans.

Physical inactivity rapidly induces insulin resistance and microvascular dysfunction in healthy volunteers.

OBJECTIVE: Sedentary lifestyle increases the risk of cardiovascular disease and diabetes. Vascular dysfunction contributes to atherogenesis and has been linked to insulin resistance. METHODS AND RESULTS: We measured insulin sensitivity by glucose tolerance test and vascular function by ultrasound and venous occlusion plethysmography in 20 healthy subjects (14 men, 6 women) at baseline and during 5 days of bed rest. Bed rest led to a 67% increase in the insulin response to glucose loading (P<0.001) suggesting increased insulin resistance and produced increases in total cholesterol and triglycerides. Bed rest led to decreased reactive hyperemia in the forearm (1317+/-404 to 1112+/-260 mL/min, P=0.01) and the calf (28.5+/-7.0 to 22.2+/-8.7 mL/min/dL, P=0.003) indicating impaired microvascular function. Bed rest decreased brachial artery diameter and increased systolic blood pressure suggesting increased basal arterial tone. There were no changes in circulating inflammatory markers arguing against systemic inflammation as a mechanism for vascular dysfunction in this setting. CONCLUSIONS: Physical inactivity was associated with the development of insulin resistance, dyslipidemia, increased blood pressure, and impaired microvascular function in healthy volunteers. Our findings may provide insight into the pathogenesis of vascular disease in sedentary individuals and emphasize that even short-term physical inactivity may have adverse metabolic and vascular consequences.

Long-term inhibition of the central alpha(2B)-adrenergic receptor gene via recombinant AAV-delivered antisense in hypertensive rats.

BACKGROUND: Salt-induced hypertension is mediated via the alpha(2B)-adrenergic receptor (AR) subtype. In alpha(2B)-AR gene knockout mice, blood pressure (BP) does not rise with salt loading, and in rats with salt-induced hypertension, BP decreases transiently with antisense (AS) treatment targeting the alpha(2B)-AR gene. The present experiments were designed to explore the possibility of gene transfection in the brain by intracerebroventricular (ICV) delivery of AS-DNA via adeno-associated virus (AAV) to prolong alpha(2B)-AR inhibition and hence reversal of salt-dependent hypertension. METHODS: A recombinant AAV (rAAV) vector preparation encoding the alpha(2B)-AS fragment (previously tested in vitro for inhibition of alpha(2B)-AR protein production in cells) and containing green fluorescence protein (GFP) for visualization was injected ICV into subtotally nephrectomized, salt-fed rats. Control rats received rAAV-GFP (n = 8 per group). RESULTS: We observed that BP rose from a baseline of 120 +/- 10 to 184 +/- 12 mm Hg. Injection of rAAV-alpha(2B)-AS produced a 35 +/- 12 mm Hg fall in BP, lasting without evidence of diminishing for at least 16 days, whereas rAAV-GFP-injected rats showed a continued rise in BP. Rats treated with rAAV-alpha(2B)-AS treated had a 45% to 65% decrease in alpha(2B)-AR protein levels in key regulatory regions of the brain. Neither group had signs of immunologic response to the virus injection. CONCLUSIONS: These results indicate that our construct, when given by ICV means, could reach multiple sites of the central nervous system relevant to BP regulation and could safely inhibit the central alpha(2B)-adrenergic receptor, thereby achieving prolonged reversal of salt-induced hypertension.

Effects of cranberry juice consumption on vascular function in patients with coronary artery disease.

BACKGROUND: Cranberry juice contains polyphenolic compounds that could improve endothelial function and reduce cardiovascular disease risk. OBJECTIVE: The objective was to examine the effects of cranberry juice on vascular function in subjects with coronary artery disease. DESIGN: We completed an acute pilot study with no placebo (n = 15) and a chronic placebo-controlled crossover study (n = 44) that examined the effects of cranberry juice on vascular function in subjects with coronary artery disease. RESULTS: In the chronic crossover study, subjects with coronary heart disease consumed a research preparation of double-strength cranberry juice (54% juice, 835 mg total polyphenols, and 94 mg anthocyanins) or a matched placebo beverage (480 mL/d) for 4 wk each with a 2-wk rest period between beverages. Beverage order was randomly assigned, and participants refrained from consuming other flavonoid-containing beverages during the study. Vascular function was measured before and after each beverage, with follow-up testing ≥12 h after consumption of the last beverage. Mean (±SD) carotid-femoral pulse wave velocity, a measure of central aortic stiffness, decreased after cranberry juice (8.3 ± 2.3 to 7.8 ± 2.2 m/s) in contrast with an increase after placebo (8.0 ± 2.0 to 8.4 ± 2.8 m/s) (P = 0.003). Brachial artery flow-mediated dilation, digital pulse amplitude tonometry, blood pressure, and carotid-radial pulse wave velocity did not change. In the uncontrolled pilot study, we observed improved brachial artery flow-mediated dilation (7.7 ± 2.9% to 8.7 ± 3.1%, P = 0.01) and digital pulse amplitude tonometry ratio (0.10 ± 0.12 to 0.23 ± 0.16, P = 0.001) 4 h after consumption of a single 480-mL portion of cranberry juice. CONCLUSIONS: Chronic cranberry juice consumption reduced carotid femoral pulse wave velocity-a clinically relevant measure of arterial stiffness. The uncontrolled pilot study suggested an acute benefit; however, no chronic effect on measures of endothelial vasodilator function was found. This trial was registered at clinicaltrials.gov as NCT00553904.

Altered mitochondrial membrane potential, mass, and morphology in the mononuclear cells of humans with type 2 diabetes.

Mitochondrial membrane hyperpolarization and morphologic changes are important in inflammatory cell activation. Despite the pathophysiologic relevance, no valid and reproducible method for measuring mitochondrial homeostasis in human inflammatory cells is available currently. The purpose of this study was to define and validate reproducible methods for measuring relevant mitochondrial perturbations and to determine whether these methods could discern mitochondrial perturbations in type 2 diabetes mellitus (T2DM), which is a condition associated with altered mitochondrial homeostasis. We employed 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzamidazol-carboncyanine (JC-1) to estimate mitochondrial membrane potential (Psi(m)) and acridine orange 10-nonyl bromide (NAO) to assess mitochondrial mass in human mononuclear cells isolated from blood. Both assays were reproducible. We validated our findings by electron microscopy and pharmacologic manipulation of Psi(m). We measured JC-1 and NAO fluorescence in the mononuclear cells of 27 T2DM patients and 32 controls. Mitochondria were more polarized (P = 0.02) and mitochondrial mass was lower in T2DM (P = 0.008). Electron microscopy demonstrated diabetic mitochondria were smaller, were more spherical, and occupied less cellular area in T2DM. Mitochondrial superoxide production was higher in T2DM (P = 0.01). Valid and reproducible measurements of mitochondrial homeostasis can be made in human mononuclear cells using these fluorophores. Furthermore, potentially clinically relevant perturbations in mitochondrial homeostasis in T2DM human mononuclear cells can be detected.

Effects of flavonoid-containing beverages and EGCG on endothelial function.

Abnormalities of the vascular endothelium contribute to all stages of atherosclerosis from lesion development to clinical cardiovascular disease events. Recognized risk factors, including diabetes mellitus, hypertension, dyslipidemia, cigarette smoking, and sedentary lifestyle are associated with endothelial dysfunction. A variety of pharmacological and behavioral interventions have been shown to reverse endothelial dysfunction in patients with cardiovascular disease. A large number of epidemiological studies suggest that dietary factors, including increased intake of flavonoid-containing foods and beverages, reduce cardiovascular risk, and recent studies have shown that such beverages have favorable effects on endothelial function. These studies have engendered interest in the development of dietary supplements or drugs that would allow for more convenient and higher dose administration of flavonoids and might prove useful for prevention or treatment of cardiovascular disease. In this paper, we will review the contribution of endothelial dysfunction to the pathogenesis and clinical expression of atherosclerosis and recent data linking flavonoid and EGCG consumption to improved endothelial function and reduced cardiovascular risk.