1Integrative Vascular Biology Laboratory, Department of Integrative Physiology University of Colorado, Boulder; and 2Department of Medicine, University of Colorado, Health Sciences Center, Denver, Colorado
Submitted 22 February 2005 ; accepted in final form 22 June 2005
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ABSTRACT |
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endothelium; fibrinolysis; tissue-type plasminogen activator
It is now well accepted that endothelial dysfunction plays an important role in the initiation and progression of atherosclerosis (5, 45). A potential mechanism underlying the heightened atherothrombotic risk with obesity is impaired endothelial control of fibrinolysis. Endothelial cells are the principal site of synthesis and release of tissue-type plasminogen activator (t-PA), the key enzyme in initiating an endogenous fibrinolytic response, due to its ability to preferentially activate plasminogen on the surface of developing thrombi (15). Experimental and clinical data indicate that it is the capacity of the endothelium to release t-PA rapidly and acutely from intracellular storage pools, and not circulating plasma fibrinolytic concentrations, that determines the efficacy of endogenous fibrinolysis (42, 49a). Indeed, the thrombolytic potential of t-PA is greatest if it is readily available and incorporated during, rather than after, thrombus formation (4, 22). Moreover, diminished endothelial t-PA release has been linked with accelerated thrombogenesis (6, 10). Currently, it is unknown whether the capacity of the endothelium to acutely release t-PA is compromised in overweight and obese adult humans. If so, this may contribute mechanistically to their increased risk of thrombosis.
Regular aerobic exercise is associated with a reduction in cardiovascular events in overweight and obese adults, independent of weight loss (2). One of the possible mechanisms by which regular aerobic exercise confers this cardioprotection is via its availing effects on endothelial function. Indeed, improved endothelial vasomotor function has been linked to a reduction in cardiovascular events (32). Exercise-induced enhancement in the capacity of the endothelium to release t-PA might also contribute to the reduced cardiovascular risk observed in physically active overweight adults (32).
Accordingly, we tested the following hypotheses: 1) the capacity of the vascular endothelium to acutely release t-PA is impaired in overweight and obese adults; and 2) regular aerobic exercise training would improve endothelial t-PA release in previously sedentary overweight and obese adults. To address these hypotheses, we employed both cross-sectional and intervention study designs. First, we used a cross-sectional model to determine the influence of overweight and obesity on the capacity of the endothelium to release t-PA. We then performed an intervention study to determine the effects of moderate aerobic exercise training on endothelial t-PA release rates in sedentary overweight and obese adults.
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METHODS |
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Cross-sectional study.
Sixty-six sedentary adults (37 males, 29 females) were studied: 28 normal-weight (BMI <25 kg/m2); 22 overweight (BMI 25 and <30 kg/m2); and 16 obese (BMI
30 kg/m2) adults. All subjects were sedentary and had not participated in a regular aerobic exercise program for
2 yr before the start of the study. Subjects were excluded from the study if they presented a history or evidence of hepatic, renal, or hematological disease; peripheral vascular disease; stroke; diabetes [fasting plasma glucose >7.0 mmol/l (1)]; dyslipoproteinemia [total cholesterol
6.0 mmol/l, LDL-cholesterol
4.5 mmol/l, triglycerides
2.5 mmol/l (19)]; and hypertension [BP
140/90 mmHg (9)]. All subjects were screened for clinical evidence of coronary artery disease with resting and maximal exercise electrocardiograms and blood pressure. None of the subjects smoked or were taking medication, including vitamins. All of the women were
1 yr postmenopausal (mean: 7 ± 1 yr) and had never taken or had discontinued use of hormone replacement therapy
1 yr before the start of the study. Before participation, all of the subjects had the research study and its potential risks and benefits explained fully before providing written informed consent. This study was approved by the Human Research Committee of the University of Colorado at Boulder.
Intervention study. Seventeen of the 38 overweight and obese adults (11 male, 6 female) who participated in the cross-sectional study enrolled in a 3-mo aerobic exercise training program. All baseline measurements were subsequently repeated in these subjects after the exercise intervention.
Measurements
Body composition. Body mass was measured to the nearest 0.1 kg using a medical beam balance (Detecto, Webb City, MO). Percent body fat was determined by dual-energy X-ray absorptiometry (Lunar Radiation, Madison, WI). BMI was calculated as weight (kg) divided by height (m) squared. Minimal waist circumference was measured according to previously published guidelines (33).
Treadmill exercise test.
To assess aerobic fitness, subjects performed incremental treadmill exercise using a modified Balke protocol as previously described (11). Maximal oxygen consumption (O2 max) was measured using online computer-assisted open-circuit spirometry. In addition, heart rate and rating of perceived exertion were measured throughout exercise, and total exercise time to exhaustion was recorded.
Metabolic measurements. Fasting plasma lipid and lipoprotein, glucose, and insulin concentrations were determined using standard techniques by the clinical laboratory affiliated with the General Clinical Research Center. Insulin resistance was estimated using the homeostasis model assessment (HOMA-IR) derived from fasting glucose and insulin concentrations (35).
Markers of inflammation.
Fasting plasma concentrations of C-reactive protein (CRP), interleukin (IL)-6, IL-18, and tumor necrosis factor (TNF)-, were determined by ELISA (ALPCO Diagnostics, R&D Systems). Inter- and intra-assay variability for all assays were <7 and <8%, respectively.
Intra-Arterial Fibrinolytic Protocol.
All measurements were performed in a temperature-controlled room between 7 and 10 AM after a 12-h overnight fast, as previously described by our laboratory (24, 48). Briefly, an intravenous catheter was placed in a deep antecubital vein of the nondominant arm. Thereafter, a 5-cm, 20-gauge catheter was introduced into the brachial artery of the same arm under local anesthesia (1% lidocaine). Forearm blood flow (FBF) was measured using strain-gauge venous occlusion plethysmography (D. E. Hokanson, Bellevue, WA) and presented as milliliters per 100 milliliters forearm volume per minute. After the measurement of resting blood flow for 5 min, bradykinin was infused intra-arterially at rates of 12.5, 25, and 50 ng·100 ml tissue1·min1 and sodium nitroprusside at 1.0, 2.0, and 4.0 µg·100 ml tissue1·min1 for 5 min at each dose, as previously described (24). To avoid an order effect, the sequence of drug administration was randomized. Forearm volume was determined by the water displacement method.
Net endothelial release of t-PA antigen and plasminogen activator inhibitor-1 (PAI-1) antigen in response to bradykinin and sodium nitroprusside was calculated according to Jern et al. (27) using the following equation:
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Exercise Intervention.
The 3-mo home-based aerobic exercise training program employed in the present study has been described previously by our laboratory (11). Briefly, after completion of baseline measurements, subjects underwent a supervised orientation, after which they exercised on their own. Subjects were asked to exercise 57 days/wk, 4050 min/day, at 6075% of their individual maximum heart rate, determined during maximal exercise testing. Most subjects walked, but some integrated jogging into their exercise session as their fitness improved, to maintain their heart rate within the prescribed range. Compliance with the exercise program was documented every 2 wk using data downloaded directly from heart rate monitors (Polar Electro, Woodbury, NY) and from exercise logs.
Statistical Analysis.
Differences in subject baseline characteristics and area under the curve data were determined by between-groups analysis of variance (ANOVA). Group differences in FBF and endothelial t-PA and PAI-1 antigen release in response to bradykinin and sodium nitroprusside were determined by repeated-measures ANOVA. When indicated by a significant F value, a post hoc test using the Newman-Keuls method was performed to identify differences among the groups. Relationships between variables of interest were assessed by means of Pearson's correlation coefficient and linear regression analysis. Changes in the dependent variables resulting from the exercise intervention were assessed by repeated-measures ANOVA. Importantly, there were no significant gender interactions in any of the key outcome variables; therefore, the data were pooled and presented together. Because of the skewed distribution of plasma CRP concentrations, the data were log-transformed to satisfy basic assumptions for parametric testing. However, per joint American College of Cardiology and Centers for Disease Control recommendations (43), the absolute values for CRP are presented to facilitate clinical interpretation. All data are expressed as means ± SE. Statistical significance was set a priori at P < 0.05.
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RESULTS |
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Table 1 presents selected subject characteristics. By design, body mass, BMI, and waist circumference were higher (P < 0.01) in the overweight and obese subjects compared with normal-weight controls. There were no differences in age, resting blood pressure, O2 max, and plasma lipid and lipoprotein concentrations between the groups. Although within clinically normal levels, plasma glucose and insulin concentrations, as well as HOMA-IR levels, were highest in the obese group. Plasma concentrations of inflammatory markers are shown in Table 2. Circulating CRP and IL-18 levels were higher (P < 0.05) in the overweight and obese compared with normal-weight subjects.
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Exercise Intervention Study
All 17 overweight and obese adults (age 57 ± 1 yr) completed the 3-mo exercise intervention study. Subjects exercised an average of 5.0 ± 0.3 days/wk for 44 ± 2 min/day at an intensity of 68 ± 1% of maximal heart rate. There were no significant changes in body mass, adiposity, heart rate at rest, arterial blood pressure, O2 max, plasma cholesterol, glucose or insulin concentrations, and HOMA-IR levels (Table 4). However, aerobic exercise training increased exercise time to exhaustion by
20% (P < 0.01) and significantly decreased heart rate and ratings of perceived exertion at the same absolute submaximal level of exercise (
70% of baseline
O2 max). Plasma concentrations of CRP, IL-6, IL-18, and TNF-
were not affected by exercise training (Table 2). In addition, FBF responses to bradykinin were higher but not significantly different after exercise training (Table 3).
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DISCUSSION |
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Overweight and obesity have been associated with increased cardiovascular morbidity and mortality (39, 40) resulting from atherothrombotic events. Indeed, overweight persons demonstrate accelerated atherosclerosis (36) and increased rates of myocardial infarction and stroke (40). In addition, excess adiposity has been linked to an increased risk of recurrent coronary events after an acute myocardial infarction (46, 50). The mechanisms underlying this thrombotic tendency are not completely understood. Much attention has been focused on the effects of adiposity on the fibrinolytic system, specifically differences in plasma concentrations of t-PA antigen and PAI-1 antigen in obese and normal-weight adults (17, 31, 47). However, systemic fibrinolytic concentrations provide an indirect, nonspecific, and in some cases misleading assessment of vascular fibrinolytic potential (26, 27). Plasma concentrations of t-PA and PAI-1 are highly dependent on the rate of t-PA/PAI-1 complex formation and, in turn, hepatic clearance (41). Moreover, we (23, 48) and others (27) have demonstrated that circulating t-PA and PAI-1 concentrations do not reflect the capacity of the endothelium to acutely release t-PA, the primary determinant of endogenous fibrinolytic activity.
The results of the present study demonstrate, for the first time, that the ability of the endothelium to release t-PA is markedly reduced in both overweight and obese adults. Both the rate and total amount of t-PA antigen released from the endothelium in response to bradykinin were 50% lower in the overweight and obese adults compared with normal-weight controls of similar age. This loss in endothelial fibrinolytic function may have important clinical consequences. Indeed, the ability of endothelial cells to release t-PA is a vital defense mechanism against thrombosis; disruption of this process is considered to be an important antecedent to atherothrombotic disease (38, 41). For example, in animal models, t-PA-deficient mice demonstrate accelerated atherosclerotic fibrin deposition and extensive myocardial tissue necrosis (6, 10). In humans, reduced endothelial t-PA release rates have been linked to increased atheromatous plaque in cigarette smokers and myocardial infarctions in patients with acute coronary syndromes (25). Thus impaired endothelial release of t-PA may be an important factor underlying the prothrombotic state associated with overweight and obesity.
The mechanisms responsible for impaired endothelial t-PA release in overweight and obese adults are not clear. Although the FBF responses to bradykinin were significantly lower in the overweight and obese subjects compared with normal-weight controls, it is unlikely that the overweight/obesity-related differences in endothelial t-PA release were a blood flow-related phenomenon. Consistent with previous studies (24, 27, 48) we observed no significant effect of sodium nitroprusside on t-PA antigen release across the forearm despite a marked increase in limb blood flow in each group. In addition, we observed no correlation between bradykinin-induced changes in blood flow and t-PA release in either the overweight, obese, or normal-weight groups. The lack of differences in PAI-1 antigen release among the groups in response to bradykinin rules out the potential confounding effects of t-PA-PAI-1 complex formation. It has been suggested that increased inflammatory burden may contribute to the heightened risk of atherothrombosis with obesity (16, 52). Indeed, inflammatory mediators such as CRP, IL-6, and IL-18 have been linked to arterial thrombogenesis (3, 51). Although the overweight and obese subjects in the present study demonstrated significantly higher plasma concentrations of CRP, IL-18, and TNF- compared with the normal-weight controls, only a weak, albeit significant, relation (r = 0.29) was observed with IL-18 and endothelial t-PA release. CRP, IL-6, and TNF-
were not associated with t-PA release. On the basis of these correlative findings, it would be unreasonable to dismiss the possibility that local inflammatory processes at the vascular wall might adversely affect endothelial function. However, the results of the present study indicate that impaired endothelial t-PA release in overweight and obese adults is largely unrelated to circulating inflammatory proteins. It is possible that overweight/obesity-related increases in oxidative stress may contribute to diminished endothelial t-PA release. Ex vivo studies have shown that oxidative stress not only inhibits t-PA release from cultured endothelial cells but also damages the fibrin-binding affinity of t-PA reducing its thrombolytic capacity (20, 30). In vivo, oxidative stress has been linked to impaired endothelial vasodilator function in overweight adults (44) and might also negatively impact endothelial t-PA release. We are currently investigating the effects of oxidative stress on endothelial t-PA release in obese adults.
It is important to note that the overweight and obese subjects in the present study were free of the cardiovascular and metabolic abnormalitites that often accompany excess body fatness, such as clinically overt coronary artery disease, hypertension, type 2 diabetes, dyslipidemia, and hyperinsulinemia (7). Thus it is tempting to speculate that diminished endothelial t-PA release might be a primary consequence of overweight or obesity. However, in addition to being overweight or obese, the subjects in the present study were sedentary. We (48) have previously demonstrated that a sedentary lifestyle is associated with impaired endothelial release of t-PA. Moreover, epidemiological studies suggest that physical inactivity might contribute significantly to the increased cardiovascular risk with overweight and obesity. Indeed, the prevalence of cardiovascular and metabolic abnormalities has been shown to be lower in overweight and obese adults who engage in regular physical activity (18, 28). The results of our exercise intervention study support this notion and demonstrate that impaired endothelial t-PA release is not an unavoidable consequence of overweight and obesity.
Indeed, 3 mo of moderate aerobic exercise training (primarily walking) resulted in a marked improvement in the capacity of the endothelium to release t-PA in previously sedentary overweight and obese adults. The rate and total amount of t-PA antigen released across the forearm in response to bradykinin increased by >50% after exercise training and, notably, were no longer significantly different from the levels observed in the normal weight controls. It is important to emphasize that the improvement in endothelial t-PA release occurred without concomitant changes in body mass and body composition and was accomplished with a moderate intensity home-based aerobic exercise training program that can be safely performed by most, if not all, overweight and obese adults. The fact that the capacity of the endothelium to release t-PA increased independent of weight loss or changes in body composition and lipid profile suggest a primary modulatory effect of aerobic exercise on endothelial fibrinolytic regulation. Several factors may underlie the exercise-induced increase in endothelial t-PA release capacity, such as shear stress-induced increases in endothelial t-PA mRNA signal expression and protein synthesis (12, 13) and elevated intracellular calcium concentrations facilitating greater t-PA release upon stimulation (29). In addition, we observed no change in CRP, IL-6, IL-18, and TNF- with exercise training, further supporting the benign influence of these circulating inflammatory factors on endothelial t-PA release. Importantly, from a public health perspective, our findings emphasize that overweight and obese persons can receive important cardioprotective effects from moderate regular aerobic exercise regardless of changes in adiposity. Improved endothelial fibrinolytic function may be a central mechanism underlying the reduced risk of cardiovascular disease morbidity and mortality observed in overweight and obese adults who engage in regular physical activity (2).
There are two important experimental limitations of the present study that should be mentioned. First, as with all cross-sectional study designs, we cannot rule out the possibility that genetic and/or lifestyle factors might have influenced the results of our group comparisons. In an effort to minimize the influence of lifestyle behaviors, we studied overweight and normal-weight adults of similar age who were nonsmokers, not currently taking medication that could influence endothelial function (i.e., statins), and who did not differ in habitual physical activity. Second, with respect to the exercise intervention, our study design is somewhat weakened by the lack of a nonexercise control group. However, the exercise training program used in the present study has consistently been shown to confer important vascular benefits, such as improvements in central arterial compliance and endothelial vasomotor regulation in healthy middle-aged and older adults (11, 49). Thus it is likely that the observed exercise-induced increase in endothelial t-PA was indeed a primary effect of the intervention.
In conclusion, the results of the present study indicate that the capacity of the endothelium to release t-PA is impaired in middle-aged overweight adults. Endothelial fibrinolytic dysfunction may contribute to the increased risk of atherothrombotic events associated with overweight and obesity. Importantly, impaired endothelial t-PA release is not an irreversible consequence of adiposity. Regular aerobic exercise, independent of weight loss, can significantly enhance endothelial fibrinolytic function in overweight and obese adults. Taken together, our findings support the postulate that regular exercise can alleviate some of the health risks associated with overweight and obesity.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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