Jared J. Greiner

Influence of Metabolic Syndrome on Biomarkers of Oxidative Stress and Inflammation in Obese Adults

Objective: Both obesity and the metabolic syndrome (MetS) have been independently linked with increased oxidative and inflammatory stress. This study tested the hypothesis that obesity with MetS is associated with greater oxidative and inflammatory burden compared with obesity alone.

Endothelin-1 Vasoconstrictor Tone Increases With Age in Healthy Men But Can Be Reduced by Regular Aerobic Exercise

Increased endothelin-1–mediated vasoconstrictor tone has been linked to the etiology of a number of pathologies associated with human aging, including hypertension, congestive heart failure, and coronary artery disease. However, it is currently unclear whether aging, per se, is associated with enhanced endothelin-1 system activity. We hypothesized that endothelin-1 vasoconstrictor activity is greater in healthy older compared with young men and that regular aerobic exercise is an effective intervention for reducing endothelin-1 vasoconstrictor tone in older previously sedentary men. Forearm blood flow (plethysmography) responses to intra-arterial infusion of endothelin-1 (5 pmol/min; for 20 minutes) and selective (BQ-123; 100 nmol/min; for 60 minutes) and nonselective (BQ-123+BQ-788; 100 nmol/min; for 60 minutes) endothelin-1 receptor blockade were determined in 28 healthy, sedentary men: 13 younger (age: 27±1 years) and 15 older (age: 62±2 years). The vasoconstrictor response to endothelin-1 was significantly blunted (≈65%) in the older versus younger men. In response to BQ-123, resting forearm blood flow increased (≈20%; P<0.05) in the older but not in the younger men. The addition of BQ-788 to BQ-123 did not significantly affect the blood flow responses to BQ-123 in either group. Eight of the 15 older sedentary men completed a 3-month aerobic exercise intervention. After the intervention, the vasoconstrictor response to endothelin-1 was markedly increased (225%; P<0.05), whereas BQ-123 resulted in modest vasoconstriction in the previously sedentary older men. These results demonstrate that endothelin-1–mediated vasoconstrictor tone increases with age in healthy men but can be alleviated with regular aerobic exercise.

Effects of ageing and regular aerobic exercise on endothelial fibrinolytic capacity in humans

The capacity of the vascular endothelium locally to release tissue‐type plasminogen activator (t‐PA) is critical for effective endogenous fibrinolysis. We determined the influence of ageing and regular aerobic exercise on the net release of t‐PA across the human forearm in vivo using both cross‐sectional and intervention approaches. First, we studied 62 healthy men aged 22‐35 or 50‐75 years of age who were either sedentary or endurance exercise‐trained. Net endothelial release rates of t‐PA were calculated as the product of the arteriovenous concentration gradient and forearm plasma flow to intra‐arterial bradykinin and sodium nitroprusside. Second, we studied 10 older (60 ± 2 years) healthy sedentary men before and after a 3 month aerobic exercise intervention. Net endothelial t‐PA release was significantly blunted with age in the sedentary men. At the highest dose of bradykinin the increase in t‐PA antigen release was ≈35 % less (P < 0.05) in the older (from −1.0 ± 0.4 to 37.8 ± 3.8 ng (100 ml tissue)−1 min−1) compared with young (from 0.1 ± 0.6 to 56.6 ± 9.2 ng (100 ml tissue)−1 min−1) men. In contrast, the endurance‐trained men did not demonstrate an age‐related decline in the net release of t‐PA antigen. After the exercise intervention, the capacity of the endothelium to release t‐PA increased ≈55 % (P < 0.05) to levels similar to those of the young adults and older endurance‐trained men. Regulated endothelial t‐PA release declines with age in sedentary men. Regular aerobic exercise may not only prevent, but could also reverse the age‐related loss in endothelial fibrinolytic function.

Enhanced endothelin-1 system activity with overweight and obesity

Endothelin (ET)-1-mediated vasoconstrictor tone contributes to the development and progression of several adiposity-related conditions, including hypertension and atherosclerotic vascular disease. The aims of the present study were to determine 1) whether endogenous ET-1 vasoconstrictor activity is elevated in overweight and obese adults, and, if so, 2) whether increased ET-1-mediated vasoconstriction contributes to the adiposity-related impairment in endothelium-dependent vasodilation. Seventy-nine adults were studied: 34 normal weight [body mass index (BMI) < 25 kg/m(2)], 22 overweight (BMI ≥ 25 and < 30 kg/m(2)), and 23 obese (BMI ≥ 30 kg/m(2)). Forearm blood flow (FBF) responses to intra-arterial infusion of ET-1 (5 pmol/min for 20 min) and selective ET-1 receptor blockade (BQ-123, 100 nmol/min for 60 min) were determined. In a subset of the study population, FBF responses to ACh (4.0, 8.0, and 16.0 μg·100 ml tissue(-1)·min(-1)) were measured in the absence and presence of selective ET-1 receptor blockade. The vasoconstrictor response to ET-1 was significantly blunted in overweight and obese adults (∼ 70%) compared with normal weight adults. Selective ET-1 receptor blockade elicited a significant vasodilator response (∼ 20%) in overweight and obese adults but did not alter FBF in normal weight adults. Coinfusion of BQ-123 did not affect FBF responses to ACh in normal weight adults but resulted in an ∼ 20% increase (P < 0.05) in ACh-induced vasodilation in overweight and obese adults. These results demonstrate that overweight and obesity are associated with enhanced ET-1-mediated vasoconstriction that contributes to endothelial vasodilator dysfunction and may play a role in the increased prevalence of hypertension with increased adiposity.

Impaired endothelium-dependent vasodilation in overweight and obese adult humans is not limited to muscarinic receptor agonists

Muscarinic receptor agonists have primarily been used to characterize endothelium-dependent vasodilator dysfunction with overweight/obesity. Reliance on a single class of agonist, however, yields limited, and potentially misleading, information regarding endothelial vasodilator capacity. The aims of this study were to determine 1) whether the overweight/obesity-related reduction in endothelium-dependent vasodilation extends beyond muscarinic receptor agonists and 2) whether the contribution of nitric oxide (NO) to endothelium-dependent vasodilation is reduced in overweight/obese adults. Eighty-six middle-aged and older adults were studied: 42 normal-weight (54 +/- 1 yr, 21 men and 21 women, body mass index = 23.4 +/- 0.3 kg/m(2)) and 44 overweight/obese (54 +/- 1 yr, 28 men and 16 women, body mass index = 30.3 +/- 0.6 kg/m(2)) subjects. Forearm blood flow (FBF) responses to intra-arterial infusions of acetylcholine in the absence and presence of the endothelial NO synthase inhibitor N(G)-monomethyl-l-arginine, methacholine, bradykinin, substance P, isoproterenol, and sodium nitroprusside were measured by strain-gauge plethysmography. FBF responses to each endothelial agonist were significantly blunted in the overweight/obese adults. Total FBF (area under the curve) to acetylcholine (50 +/- 5 vs. 79 +/- 4 ml/100 ml tissue), methacholine (55 +/- 4 vs. 86 +/- 5 ml/100 ml tissue), bradykinin (62 +/- 5 vs. 85 +/- 4 ml/100 ml tissue), substance P (37 +/- 4 vs. 57 +/- 5 ml/100 ml tissue), and isoproterenol (62 +/- 4 vs. 82 +/- 6 ml/100 ml tissue) were 30%-40% lower in the overweight/obese than normal-weight adults. N(G)-monomethyl-l-arginine significantly reduced the FBF response to acetylcholine to the same extent in both groups. There were no differences between the groups in the FBF responses to sodium nitroprusside. These results indicate that agonist-stimulated endothelium-dependent vasodilation is universally impaired with overweight/obesity. Moreover, this impairment appears to be independent of NO.

Endothelial progenitor cell number and colony-forming capacity in overweight and obese adults

Objective:To investigate whether adiposity influences endothelial progenitor cell (EPC) number and colony-forming capacity.Design:Cross-sectional study of normal weight, overweight and obese adult humans.Participants:Sixty-seven sedentary adults (aged 45–65 years): 25 normal weight (body mass index (BMI) ⩽25 kg/m2; 12 males/13 females); 18 overweight (BMI=25–29.9 kg/m2; 12 males/6 females); and 24 obese (BMI ⩾30 kg/m2; 18 males/6 females). All participants were non-smokers and free of overt cardiometabolic disease.Measurements:Peripheral blood samples were collected and circulating EPC number was assessed by flow cytometry. Putative EPCs were defined as CD45−/CD34+/VEGFR-2+/CD133+ or CD45−/CD34+ cells. EPC colony-forming capacity was measured in vitro using a colony-forming unit (CFU) assay.Results:Number of circulating putative EPCs (either CD45−/CD34+/VEGFR-2+/CD133+ or CD45−/CD34+ cells) was lower (P<0.05) in obese (0.0007±0.0001%; 0.050±0.006%) compared with overweight (0.0016±0.0004%; 0.089±0.019%) and normal weight (0.0015±0.0003%; 0.082±0.008%) adults. There were no differences in EPC number between the overweight and normal weight groups. EPC colony formation was significantly less in the obese (6±1) and overweight (4±1) compared with normal weight (9±2) adults.Conclusion:These results indicate that: (1) the number of circulating EPCs is lower in obese compared with overweight and normal weight adults; and (2) EPC colony-forming capacity is blunted in overweight and obese adults compared with normal weight adults. Impairments in EPC number and function may contribute to adiposity-related cardiovascular risk.

Sex differences in endothelin-1-mediated vasoconstrictor tone in middle-aged and older adults

The prevalence of cardiovascular disease is lower in middle-aged and older women than men. Increased endothelin-1-mediated vasoconstriction has been linked to the etiology of a number of cardiovascular diseases, including atherosclerosis, heart failure, and hypertension. It is unknown whether a sex difference in endothelin-1-mediated vasoconstrictor tone exists in middle-aged and older adults. Therefore, we tested the hypothesis that middle-aged and older men would demonstrate greater ET-1-mediated vasoconstrictor tone than age-matched women. Forearm blood flow in response to intra-arterial infusions of endothelin (ET)-1, BQ-123 (a selective ET(A) receptor antagonist), and BQ-788 (a selective ET(B) receptor antagonist) was assessed by venous occlusion plethysmography in 21 women (age: 58 + or - 1 yr; body mass index: 26.0 + or - 1.0 kg/m(2)) and 25 men (age: 57 + or - 2 yr; body mass index: 26.8 + or - 0.7 kg/m(2)). In response to BQ-123, the increase in forearm blood flow from baseline was significantly higher in the men than the women (24 + or - 5% vs. 9 + or - 5%; P < 0.05). In contrast, the increase in forearm blood flow in response to BQ-123 coinfused with BQ-788 was greater in the women than the men, such that the maximum vasodilation to dual endothelin receptor blockade was similar between men and women (approximately 25%). There was no difference in the vasoconstrictor response to ET-1 between the sexes. These results indicate that middle-aged and older men are under greater ET(A) receptor-mediated vasoconstrictor tone than age-matched women. Since the ET(A) receptor is the predominant receptor subtype in the coronary vasculature, this sex difference in vasoconstrictor tone may be a mechanism contributing to the sex difference in the prevalence of coronary heart disease in middle-aged and older adults.

Aging Is Associated with a Proapoptotic Endothelial Progenitor Cell Phenotype

The aim of this study was to determine if aging is associated with enhanced endothelial progenitor cell (EPC) sensitivity to apoptosis. Cells with phenotypic EPC characteristics were isolated from healthy, nonobese young (age 25 ± 1 years) and older (61 ± 1 years) men. Intracellular active caspase-3 concentrations in response to staurosporine stimulation were approximately 35% higher (p < 0.05) in EPCs from older (3.15 ± 0.29 pg/ml) compared with young (2.33 ± 0.24 pg/ml) men. Protein expression of Akt, p70 S6-kinase and Bcl-2 was markedly lower (approx. 35, 75 and 60%, respectively, all p < 0.05) in EPCs from older compared with young men, whereas there were no age-related differences in either 14-3-3Ε or Bax expression. Additionally, EPC telomerase activity was 57% lower (p < 0.05) in older (0.18 ± 0.11 AU) versus young (0.43 ± 0.11 AU) men. These results indicate that aging is associated with a proapoptotic EPC phenotype characterized by decreased expression of key antiapoptotic proteins associated with the PI-3-kinase signaling pathway and reduced telomerase activity. These age-related changes likely contribute, in part, to the diminished ability of EPCs to resist an apoptotic stimulus in older men. Increased susceptibility to apoptosis may contribute to the numerical and functional impairments observed in EPCs with aging.

Endothelin-1 vasoconstriction and the age-related decline in endothelium-dependent vasodilatation in men.

ET (endothelin)-1, a potent vasoconstrictor peptide released by the endothelium, plays an important role in vasomotor regulation and has been linked to diminished endothelial vasodilator capacity in several pathologies associated with human aging, including hypertension, Type 2 diabetes and coronary artery disease. However, it is currently unknown whether the decline in endothelial vasodilatation with advancing age is due to elevated ET-1 vasoconstrictor activity. Accordingly, we tested the hypothesis that the age-related impairment in ACh (acetylcholine)-mediated endothelium-dependent vasodilatation is due, at least in part, to increased ET-1-mediated vasoconstrictor tone. FBF (forearm blood flow) responses to ACh, SNP (sodium nitroprusside) and BQ-123 (ET(A) receptor blocker) were determined in 14 young (age, 25 ± 1 years) and 14 older (age, 61 ± 2 years) healthy non-obese men. Additionally, FBF responses to ACh were determined in the presence of ETA blockade. Vasodilatation to ACh was lower (approx. 25%; P<0.05) in the older men (from 4.9 ± 0.2 to 13.9 ± 0.9 ml·100 ml(-1) of tissue·min(-1)) compared with the young men (4.6 ± 0.3 to 17.2 ± 1.0 ml·100 ml(-1) of tissue·min(-1)). There were no differences in FBF responses to SNP between the young (4.8 ± 0.3 to 18.5 ± 0.3 ml·100 ml(-1) of tissue·min(-1)) and older (5.1 ± 0.3 to 17.3 ± 0.8 ml·100 ml(-1) of tissue·min(-1)) men. In the young men, resting FBF was not significantly altered by BQ-123, whereas, in the older men, FBF increased approx. 25% in response to BQ-123 infusion (P<0.05). Co-infusion of ACh with BQ-123 resulted in an approx. 20% increase in the ACh-induced vasodilatation in older men compared with saline. In contrast, FBF responses to ACh were not significantly altered by ET(A) blockade in the young men. In conclusion, these results demonstrate that ET-1 vasoconstrictor activity contributes, at least in part, to diminished endothelium-dependent vasodilatation in older men.

Endothelial Progenitor Cell Function, Apoptosis, and Telomere Length in Overweight/Obese Humans

Excess adiposity is associated with increased cardiovascular morbidity and mortality. Endothelial progenitor cells (EPCs) play an important role in vascular repair. We tested the hypothesis that increased adiposity is associated with EPC dysfunction, characterized by diminished capacity to release angiogenic cytokines, increased apoptotic susceptibility, reduced cell migration, and shorter telomere length. A total of 67 middle‐aged and older adults (42–67 years) were studied: 25 normal weight (normal weight; BMI: 18.5–24.9 kg/m2) and 42 overweight/obese (overweight/obese; BMI: 25.0–34.9 kg/m2). Cells with phenotypic EPC characteristics were isolated from peripheral blood. EPC release of vascular endothelial growth factor (VEGF) and granulocyte colony–stimulating factor (G‐CSF) was determined in the absence and presence of phytohemagglutinin (10 µg/ml). Intracellular active caspase‐3 and cytochrome c concentrations were determined by immunoassay. Migratory activity of EPCs in response to VEGF (2 ng/ml) and stromal cell–derived factor‐1α (SDF‐1α; 10 ng/ml) was determined by Boyden chamber. Telomere length was assessed by Southern hybridization. Phytohemagglutinin‐stimulated release of VEGF (90.6 ± 7.6 vs. 127.2 ± 11.6 pg/ml) and G‐CSF (896.1 ± 77.4 vs. 1,176.3 ± 126.3 pg/ml) was ∼25% lower (P < 0.05) in EPCs from overweight/obese vs. normal weight subjects. Staurosporine induced a ∼30% greater (P < 0.05) increase in active caspase‐3 in EPCs from overweight/obese (2.8 ± 0.2 ng/ml) compared with normal weight (2.2 ± 0.2) subjects. There were no significant differences in EPC migration to either VEGF or SDF‐1α. Telomere length did not differ between groups. These results indicate that increased adiposity adversely affects the ability of EPCs to release proangiogenic cytokines and resist apoptosis, potentially compromising their reparative potential.