Aerobic exercise training reduces ET-1-mediated vasoconstriction and improves endothelium-dependent vasodilation in postmenopausal women.

Endothelin-1 (ET-1) contributes to vascular dysfunction in postmenopausal women (PMW). Although aerobic exercise is beneficial in reducing ET-1-mediated vasoconstrictor tone in men, it is unknown whether this favorable vascular effect occurs in women. We tested the hypothesis that aerobic exercise training reduces ET-1-mediated vasoconstriction in PMW. We further hypothesized that reductions in ET-1 vasoconstrictor tone underly exercise-induced improvements in endothelium-dependent vasodilatation in PMW. Forearm blood flow (FBF) responses to intra-arterial infusion of selective ETA receptor blockade (BQ-123, 100 nmol/min for 60 min) and acetylcholine (4.0, 8.0, and 16.0 µg/100 mL tissue/min) in the absence and presence of ETA receptor blockade were determined before and after a 12-wk aerobic exercise training intervention in 18 healthy, sedentary PMW (58 ± 4 yr). Women exercised an average of 4.9 ± 0.7 day/wk for 51 ± 7 min/day at 71 ± 3% of maximal heart rate. Before exercise, BQ-123 significantly increased FBF (∼25%) in sedentary PMW; however, this effect was abolished following the exercise intervention. FBF responses to acetylcholine were also significantly higher after exercise training (from 4.2 ± 1.2 to 14.0 ± 3.8 mL/100 mL tissue/min) versus before (from 4.1 ± 1.0 to 11.4 ± 3.3 mL/100 mL tissue/min; ∼25% increase; P < 0.05). Before exercise training, coinfusion of BQ-123 with acetylcholine enhanced (∼25%; P < 0.05) the vasodilator response (from 4.4 ± 1.1 to 13.9 ± 4.2 mL/100 mL tissue/min) compared with acetylcholine alone; after exercise training, the presence of BQ-123 did not significantly affect the vasodilator response to acetylcholine. Aerobic exercise training reduces ET-1-mediated vasoconstriction in PMW. Furthermore, decreased ET-1-mediated vasoconstriction is an important mechanism underlying aerobic exercise-induced improvement in endothelium-dependent vasodilation in PMW.NEW & NOTEWORTHY Endothelin-1 (ET-1) contributes to declines in endothelial function in postmenopausal women. To our knowledge, we show for the first time that aerobic exercise reduces ET-1-mediated vasoconstriction in previously sedentary postmenopausal women. Moreover, aerobic exercise improved endothelial-dependent dilation due in part to the reductions in ET-1-mediated vasoconstriction.

Regular aerobic exercise counteracts endothelial vasomotor dysfunction associated with insufficient sleep.

Insufficient sleep is associated with endothelial vasomotor dysfunction and increased cardiovascular risk. Regular aerobic exercise is an effective lifestyle strategy for improving endothelial function and, in turn, reducing cardiovascular risk. We tested the hypotheses that regular aerobic exercise would 1) improve endothelial vasodilation and 2) decrease endothelin (ET)-1-mediated vasoconstrictor tone in middle-aged adults who chronically sleep <7 h/night. Thirty-six healthy, middle-aged adults were studied: 16 with normal sleep duration (age: 57 ± 2 yr; sleep duration: 7.4 ± 0.1 h/night) and 20 with short sleep duration (age: 56 ± 1 yr; sleep duration: 6.2 ± 0.1 h/night). The 20 short sleepers completed a 3-mo aerobic exercise training intervention. Forearm blood flow was determined (via plethysmography) in response to intra-arterial acetylcholine (ACh), BQ-123 (ETA receptor antagonist), ACh + BQ-123, and sodium nitroprusside. Forearm blood flow responses to ACh were lower (∼20%; P < 0.05) in the short (from 4.2 ± 0.2 to 10.5 ± 0.6 mL/100 mL tissue/min) versus normal (4.2 ± 0.2 to 12.7 ± 0.6 mL/100 mL tissue/min) sleepers. In response to BQ-123, the short-sleep group had a significantly greater increase in resting forearm blood flow than the normal-sleep group (∼25% vs. ∼8%). ACh + BQ-123 resulted in a significant (∼25%) increase in the ACh-mediated vasodilation in the short-sleep group only. After exercise training, although nightly sleep duration was unchanged (6.4 ± 0.1 h/night), ACh-mediated vasodilation was significantly higher (∼20%), ET-1-mediated vasoconstriction was significantly lower (∼80%), and the vasodilator response to ACh was not increased with ETA receptor blockade. Regular aerobic exercise, independent of changes in nightly sleep duration, can counteract insufficient sleep-related endothelial vasomotor dysfunction.NEW & NOTEWORTHY Habitual insufficient nightly sleep (<7 h/night) is associated with increased risk of cardiovascular disease and events. Endothelial dysfunction, specifically reduced endothelium-dependent vasodilation and increased endothelin (ET)-1-mediated vasoconstriction, is considered to be a major contributing mechanism underlying increased vascular risk with insufficient sleep. In contrast to insufficient sleep, regular aerobic exercise enhances endothelial vasomotor function, reducing the risk of cardiovascular disease and associated events. In the present study, we determined the effects of aerobic exercise training on endothelium-dependent vasodilation and ET-1 vasoconstriction in adults who habitually sleep <7 h/night. After exercise training, although nightly sleep duration was unchanged, endothelium-dependent vasodilation was significantly enhanced and ET-1-mediated vasoconstrictor tone was significantly reduced in adults who sleep <7 h/night. Regular aerobic exercise training can mitigate insufficient sleep-related endothelial vasomotor dysfunction and, in turn, potentially reduce the cardiovascular risk associated with habitual insufficient nightly sleep.

Nebivolol, But Not Metoprolol, Treatment Improves Endothelial Fibrinolytic Capacity in Adults With Elevated Blood Pressure.

BACKGROUND: Vascular endothelial fibrinolytic function is impaired in adults with prehypertension and hypertension and plays a mechanistic role in the development of atherothrombotic events. The influence of ß-blockers on endothelial fibrinolysis is unknown. This study compared the effects of chronic nebivolol and metoprolol treatment on endothelial tissue-type plasminogen activator (t-PA) release in adults with elevated blood pressure (BP). METHODS AND RESULTS: Forty-four middle-aged adults (36% women) with elevated BP completed a 3-month, double-blind, randomized, placebo-controlled trial comparing nebivolol (5 mg/d), metoprolol succinate (100 mg/d), and placebo. Net endothelial t-PA release was determined in vivo in response to intrabrachial infusions of bradykinin and sodium nitroprusside before and after each intervention. In a subset, the dose-response curves to bradykinin and sodium nitroprusside were repeated with a coinfusion of the antioxidant vitamin C. At baseline, resting BP and endothelial t-PA release were comparable between the 3 groups. BP decreased to a similar extent (≈10 mm Hg) in the nebivolol- and metoprolol-treated groups. There was a substantial increase (≈30%; P<0.05) in the capacity of the endothelium to release t-PA following chronic treatment with nebivolol but not metoprolol or placebo. Mitigating oxidant stress with vitamin C coinfusion potentiated t-PA release (90%; P<0.05) at baseline in all groups. However, after the intervention, t-PA release was unchanged by vitamin C coinfusion in the nebivolol group only. CONCLUSIONS: Nebivolol but not metoprolol improves endothelial t-PA release in adults with elevated BP. This may be an important vascular benefit of nebivolol. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01595516.

Regular aerobic exercise reduces endothelin-1-mediated vasoconstrictor tone in overweight and obese adults.

NEW FINDINGS: What is the central question of this study? Does aerobic exercise training reduce endothelin-1 (ET-1)-mediated vasoconstrictor tone in overweight/obese adults? And, if so, does lower ET-1 vasoconstriction underlie the exercise-related enhancement in endothelium-dependent vasodilatation in overweight/obese adults? What is the main finding and its importance? Regular aerobic exercise reduces ET-1-mediated vasoconstrictor tone in previously sedentary overweight/obese adults, independent of weight loss. Decreased ET-1 vasoconstriction is an important mechanism underlying the aerobic exercise-induced improvement in endothelium-dependent vasodilator function in overweight/obese adults. Endothelin-1 (ET-1)-mediated vasoconstrictor tone is elevated in overweight and obese adults, contributing to vasomotor dysfunction and increased cardiovascular disease risk. Although the effects of habitual aerobic exercise on endothelium-dependent vasodilatation in overweight/obese adults have been studied, little is known regarding ET-1-mediated vasoconstriction. Accordingly, the aims of the present study were to determine the following: (i) whether regular aerobic exercise training reduces ET-1-mediated vasoconstrictor tone in overweight and obese adults; and, if so, (ii) whether the reduction in ET-1-mediated vasoconstriction contributes to exercise-induced improvement in endothelium-dependent vasodilatation in this population. Forearm blood flow (FBF) in response to intra-arterial infusion of selective ETA receptor blockade (BQ-123, 100 nmol min-1 for 60 min), acetylcholine [4.0, 8.0 and 16.0 µg (100 ml tissue)-1  min-1 ] in the absence and presence of ETA receptor blockade and sodium nitroprusside [1.0, 2.0 and 4.0 µg (100 ml tissue)-1  min-1 ] were determined before and after a 3 month aerobic exercise training intervention in 25 (16 men and nine women) overweight/obese (body mass index 30.1 ± 0.5 kg m-2 ) adults. The vasodilator response to BQ-123 was significantly lower (∼25%) and the FBF responses to acetylcholine were ∼35% higher after exercise training. Before the exercise intervention, the co-infusion of acetylcholine plus BQ-123 resulted in a greater vasodilator response than acetylcholine alone; however, after the exercise intervention the FBF response to acetylcholine was not significantly increased by ETA receptor blockade. These results demonstrate that regular aerobic exercise reduces ET-1-mediated vasoconstrictor tone in previously sedentary overweight and obese adults. Moreover, decreased ET-1-mediated vasoconstriction is an important mechanism underlying the aerobic exercise-induced improvement in endothelium-dependent vasodilator function in overweight/obese adults.

Endothelial vasodilator function in normal-weight adults with metabolic syndrome.

Metabolic syndrome (MetS) typically presents with obesity; however, obesity is not a requisite characteristic for MetS classification and related vascular risk. We tested the hypothesis that MetS, independent of excess adiposity, is associated with impaired endothelial vasodilator dysfunction. Thirty-two sedentary, middle-aged adults were studied: 11 normal weight (9 male and 2 female; body mass index (BMI), 24.0 ± 0.3 kg/m2); 11 normal weight with MetS (9 male and 2 female; BMI, 24.7 ± 0.3 kg/m2); and 10 obese without MetS (8 male and 2 female; BMI, 31.4 ± 0.5 kg/m2). MetS was established according to National Cholesterol Education Program Adult Treatment Panel III criteria. Forearm blood flow (FBF) responses to intra-arterial infusions of acetylcholine and sodium nitroprusside were measured via strain-gauge plethysmography. FBF responses to acetylcholine were ∼20% lower (P < 0.05) in the normal-weight subjects with MetS (from 4.0 ± 0.3 to 13.0 ± 1.0 mL/(100 mL tissue·min)) and obese subjects (from 4.8 ± 0.2 to 12.2 ± 1.1 mL/(100 mL tissue·min)) compared with the normal-weight subjects (from 4.6 ± 0.4 to 15.8 ± 0.7 mL/(100 mL tissue·min)). Of note, FBF responses to acetylcholine were similar between the normal-weight adults with MetS and the obese adults. There were no differences among groups in FBF response to sodium nitroprusside. These data indicate that the presence of MetS, independent of obesity, is associated with diminished endothelium-dependent vasodilation. Endothelial vasodilator dysfunction may underlie the increased cardiovascular risk in normal-weight adults with MetS.

Chronic Nebivolol Treatment Suppresses Endothelin-1-Mediated Vasoconstrictor Tone in Adults With Elevated Blood Pressure.

UNLABELLED: Endothelin-1 (ET-1) plays a major role in the pathophysiology of hypertension and its associated cardiovascular risk. We tested the hypothesis that chronic nebivolol treatment reduces ET-1-mediated vasoconstrictor tone in adult humans with elevated blood pressure (BP). Furthermore, reducing ET-1 vasoconstrictor activity contributes to the improvement in endothelial vasodilator function associated with nebivolol treatment. Forty-two middle-aged adults with elevated BP (systolic BP ≥130 mm Hg or diastolic BP ≥85 mm Hg) completed a 3-month, double-blind, randomized, placebo controlled trial: 14 received nebivolol (8 men/6 women; 5 mg per day); 14 received metoprolol succinate (9 men/5 women; 100 mg per day); and 14 received placebo (9 men/5 women). Forearm blood flow (plethysmography) responses to selective (BQ-123: 100 nmol/min; 60 minutes) and nonselective (BQ-123+BQ-788 [50 nmol/min]; 60 minutes) ET-1 receptor blockade, as well as acetylcholine (4.0, 8.0, and 16.0 µg per 100 mL of tissue per minute) in the absence and presence of nonselective ET-1 receptor blockade were determined before and after each treatment intervention. Forearm blood flow responses to BQ-123 and BQ-123+BQ-788 were similarly and significantly elevated (≈30% and 60%, respectively) from baseline in all 3 groups. Nebivolol, but not metoprolol or placebo, therapy resulted in a marked (≈25% and 45%; P<0.05) reduction in forearm blood flow response to BQ-123 and BQ-123+BQ-788. Moreover, after nebivolol therapy only, vasodilator response to acetylcholine was not significantly increased by ET-1 receptor blockade. These results demonstrate that nebivolol, but not metoprolol, treatment reduces ET-1-mediated vasoconstrictor tone in adult humans with elevated BP. In addition, nebivolol-induced reduction in ET-1-mediated vasoconstrictor tone underlies the favorable effects of this ß-blocker on endothelial vasodilation. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01395329.

Elevations in C-reactive protein and endothelin-1 system activity in humans.

AIMS: C-reactive protein (CRP) is an inflammatory cytokine that has been shown to be an independent predictor of future atherothrombotic events. Hyperactivity of endothelin-1 (ET-1), a potent vasoconstrictor peptide produced by the endothelium, is linked with cardiovascular disease development and progression. ET-1 is sensitive to inflammatory stimuli, though the influence of CRP on ET-1 system activity is unknown. We tested the hypothesis that ET-1-mediated vasoconstrictor tone is enhanced in adults with elevated plasma CRP concentrations. MATERIALS AND METHODS: Sixty non-obese adults (43-70years) were studied: 20 with hsCRP<1.0mg/L (low CRP; 0.5±0.1mg/L); 20 with hsCRP 1.0-3.0mg/L (moderate CRP; 2.0±0.1mg/L); and 20 with hsCRP>3.0mg/L (high CRP; 6.3±0.5mg/L). Forearm blood flow (FBF; plethysmography) was determined in response to intra-arterial infusions of ET-1 (5pmol/min for 20min) and selective ETA receptor blockade (BQ-123, 100nmol/min for 60min). KEY FINDINGS: In response to ET-1, FBF decreased ~10% in the low (-10.0±2.3%), moderate (-10.7±4.0%), and high (-6.6±5.2%) CRP groups, with no significant differences between groups. Additionally, all groups demonstrated a marginal, though significant (~10%), vasodilator response to BQ-123; however, there were no differences in the FBF response to BQ-123 across CRP groups. There were no significant correlations between plasma CRP concentrations and peak FBF response to either ET-1 or BQ-123. SIGNIFICANCE: These results indicate that ET-1 system activity is not influenced by elevations in CRP. Enhanced ET-1 system activity may not be involved in the increased cardiovascular disease risk associated with elevations in plasma CRP concentrations.

Influence of habitual high dietary fat intake on endothelium-dependent vasodilation.

High-fat diets are associated with an increased risk of cardiovascular disease. A potential underlying mechanism for the increased cardiovascular risk is endothelial dysfunction. Nitric oxide (NO)-mediated endothelium-dependent vasodilation is critical in the regulation of vascular tone and overall vascular health. The aim of this study was to determine the influence of dietary fat intake on endothelium-dependent vasodilation. Forty-four middle-aged and older sedentary, healthy adults were studied: 24 consumed a lower fat diet (LFD; 29% ± 1% calories from fat) and 20 consumed a high-fat diet (HFD; 41% ± 1% calories from fat). Four-day diet records were used to assess fat intake, and classifications were based on American Heart Association guidelines (<35% of total calories from fat). Forearm blood flow (FBF) responses to acetylcholine, in the absence and presence of the endothelial NO synthase inhibitor N(G)-monomethyl-l-arginine (L-NMMA), as well as responses to sodium nitroprusside were determined by plethysmography. The FBF response to acetylcholine was lower (∼15%; P < 0.05) in the HFD group (4.5 ± 0.2 to 12.1 ± 0.8 mL/100 mL tissue/min) than in the LFD group (4.6 ± 0.2 to 14.4 ± 0.6 mL/100 mL tissue/min). L-NMMA significantly reduced the FBF response to acetylcholine in the LFD group (∼25%) but not in the HFD group. There were no differences between groups in the vasodilator response to sodium nitroprusside. These data indicate that a high-fat diet is associated with endothelium-dependent vasodilator dysfunction due, in part, to diminished NO bioavailability. Impaired NO-mediated endothelium-dependent vasodilation may contribute to the increased cardiovascular risk with high dietary fat intake.

Influence of dietary saturated fat intake on endothelial fibrinolytic capacity in adults.

Approximately 50% of middle-aged and older adults in the United States regularly consume a diet high in saturated fat. High dietary saturated fat intake has been linked to promote atherothrombotic vascular disease. We tested the hypothesis that endothelial fibrinolytic function is diminished in middle-aged and older adults who habitually consume a diet high in saturated fat. Twenty-four healthy, sedentary middle-aged, and older adults (54 to 71 years) were studied: 10 (8 men and 2 women) with a dietary saturated fat intake <10% (lower saturated fat) of total calories and 14 (9 men and 5 women) with a dietary saturated fat intake ≥10% of total calories (high saturated fat). Net endothelial release of tissue-type plasminogen activator (t-PA), the primary activator of fibrinolysis, was determined, in vivo, in response to intrabrachial infusions of bradykinin (12.5 to 50.0 ng/100 ml tissue/min) and sodium nitroprusside (1.0 to 4.0 µg/100 ml tissue/min). Capacity of the endothelium to release t-PA in response to bradykinin was ∼30% less (p <0.05) in the high (from -0.7 ± 0.6 to 36.9 ± 3.3 ng/100 ml tissue/min) compared with the lower (from -0.3 ± 0.3 to 53.4 ± 7.8 ng/100 ml tissue/min) dietary saturated fat group. Moreover, total amount of t-PA released was significantly less (∼30%) (201 ± 22 vs 274 ± 29 ng/100 ml tissue) in the adults who reported consuming a diet high in saturated fat. These results indicate that the capacity of the endothelium to release t-PA is lower in middle-aged and older adults who habitually consume a diet high in saturated fat. In conclusion, endothelial fibrinolytic dysfunction may underlie the increased atherothrombotic disease risk with a diet high in saturated fat.

Predictors of improvement in cardiometabolic risk factors with weight loss in women.

BACKGROUND: Weight loss is associated with improvements in cardiometabolic risk factors, including serum glucose, insulin, C-reactive protein, and blood lipids. Few studies have evaluated the long-term (>18 months) effect of weight loss on these risk factors or sought to identify factors associated with sustained improvements in these measures. METHODS AND RESULTS: In 417 overweight/obese women (mean [SD] age, 44 [10] years) participating in a weight loss trial, we sought to identify predictors of weight loss-associated cardiometabolic risk factors after 12 and 24 months of intervention. Total cholesterol (TC), low-density lipoprotein (LDL)-cholesterol (LDL-C), high-density lipoprotein (HDL)-cholesterol, non-HDL-cholesterol, triglycerides (TG), insulin, glucose, C-reactive protein (CRP), and cardiopulmonary fitness were measured at baseline and at 12 and 24 months. After 24 months, significant reductions in body weight, waist circumference, CRP, TC, HDL-cholesterol, and non-HDL-cholesterol were observed (P<0.01). After 24 months, mean TC and non-HDL-cholesterol were reduced regardless of the amount of weight lost, whereas reductions in LDL-cholesterol, CRP, insulin, and TG were observed only in those who lost ≥10% body weight. Step-test performance improved only in those who lost ≥10% body weight after 24 months. Change in weight demonstrated a positive predictive value for change in cholesterol, insulin, glucose, and triglycerides. Baseline level of the biomarker showed the greatest predictive value for follow-up measures for insulin, cholesterol, glucose, and triglycerides. CONCLUSIONS: Our data extend the results from short-term weight loss trials and suggest that the magnitude of weight loss and baseline values for risk factors are associated with improvements in cardiometabolic risk factors even after 24 months. CLINICAL TRIALS REGISTRATION INFORMATION URL: http://www.clinicaltrials.gov/. Unique identifier: NCT00640900.

Daily consumption of grapefruit for 6 weeks reduces urine F2-isoprostanes in overweight adults with high baseline values but has no effect on plasma high-sensitivity C-reactive protein or soluble vascular cellular adhesion molecule 1.

Individuals with obesity and metabolic syndrome (MetS) are at increased risk of cardiovascular disease, in part due to heightened inflammatory/oxidative processes. Results from epidemiologic and experimental studies suggest that citrus, and grapefruit in particular, may have a role in promoting vascular health, although clinical trial data are lacking. Here, we evaluated the anti-inflammatory/antioxidant effects of habitual grapefruit consumption in 69 overweight/obese men and women and in a subsample of participants with MetS (n = 29). Participants were randomly assigned to either a grapefruit group in which they consumed a low bioactive diet plus 1.5 grapefruit/d for 6 wk (n = 37, n = 14 with MetS) or to a control condition in which a low bioactive diet devoid of citrus was consumed (n = 32, n = 15 with MetS). Plasma soluble vascular adhesion molecule-1 (sVCAM-1), plasma high-sensitivity C-reactive protein (hsCRP), and urinary F2-isoprostanes were evaluated before and after the intervention phase. F2-isoprostane concentrations were not different in the grapefruit versus control arm after the intervention (12.4 ± 6.4 vs. 15.9 ± 9.0 ng/mg creatinine, P = 0.16), whereas plasma hsCRP concentrations tended to be lower in the grapefruit versus control arm postintervention (2.1 ± 1.5 vs. 2.8 ± 2.0 mg/L, P = 0.09). In adults with MetS, grapefruit consumption tended to result in lower postintervention F2-isoprostane concentrations compared with the control condition (12.0 ± 4.5 vs. 18.3 ± 10.9 ng/mg creatinine, P = 0.06). Furthermore, those with high baseline F2-isoprostane concentrations experienced significant reductions in this biomarker in response to grapefruit consumption (P = 0.021). Change in sVCAM-1 concentrations did not vary by treatment arm nor were there differences between arms postintervention. These results suggest that intake of grapefruit twice daily for 6 wk does not significantly reduce inflammation and oxidative stress, although there is a suggestion of favorable modulation of oxidative stress in overweight and obese adults with MetS or those with high baseline urine F2-isoprostane concentrations.

The effects of daily consumption of grapefruit on body weight, lipids, and blood pressure in healthy, overweight adults.

Folklore has suggested that consuming grapefruit may promote weight control. Sparse data exist to support this hypothesis, although there is some evidence of health promotion effects with regard to blood pressure control and modulation of circulating lipids. The aim of this randomized controlled trial was to prospectively evaluate the role of grapefruit in reducing body weight and blood pressure and in promoting improvements in the lipid profile in overweight adults (N = 74). Following a 3-week washout diet low in bioactive-rich fruits and vegetables, participants were randomized to either the control diet (n = 32) or daily grapefruit (n = 42) in the amount of one half of a fresh Rio-Red grapefruit with each meal (3× daily) for 6 weeks. No differences between group in weight, blood pressure, or lipids were demonstrated. Grapefruit consumption was associated with modest weight loss (-0.61 ± 2.23 kg, P = .097), a significant reduction in waist circumference (-2.45 ± 0.60 cm, P = .0002), and a significant reduction in systolic blood pressure (-3.21 ± 10.13 mm Hg, P = .03) compared with baseline values. Improvements were observed in circulating lipids of those consuming grapefruit, with total cholesterol and low-density lipoprotein significantly decreasing by -11.7 mg/dL (P = .002) and -18.7 mg/dL (P < .001), respectively, compared with baseline values. This study suggests that consumption of grapefruit daily for 6 weeks does not significantly decrease body weight, lipids, or blood pressure as compared with the control condition. However, the improvements in blood pressure and lipids demonstrated in the intervention group suggest that grapefruit should be further evaluated in the context of obesity and cardiovascular disease prevention.

Conformational detection of p53's oligomeric state by FlAsH Fluorescence.

The p53 tumor suppressor protein is a critical checkpoint in prevention of tumor formation, and the function of p53 is dependent on proper formation of the active tetramer. In vitro studies have shown that p53 binds DNA most efficiently as a tetramer, though inactive p53 is predicted to be monomeric in vivo. We demonstrate that FlAsH binding can be used to distinguish between oligomeric states of p53, providing a potential tool to explore p53 oligomerization in vivo. The FlAsH tetra-cysteine binding motif has been incorporated along the dimer and tetramer interfaces in the p53 tetramerization domain to create reporters for the dimeric and tetrameric states of p53, though the geometry of the four cysteines is critical for efficient FlAsH binding. Furthermore, we demonstrate that FlAsH binding can be used to monitor tetramer formation in real-time. These results demonstrate the potential for using FlAsH fluorescence to monitor protein-protein interactions in vivo.