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The Journal of Physiology logoLink to The Journal of Physiology
. 2002 Nov 15;546(Pt 1):289–298. doi: 10.1113/jphysiol.2002.027870

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

Derek T Smith *, Greta L Hoetzer *, Jared J Greiner *, Brian L Stauffer *,†, Christopher A DeSouza *,‡
PMCID: PMC2342457  PMID: 12509496

Abstract

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.

Once thought to be an inert barrier lining the arterial wall, the vascular endothelium is now recognized to play an important role in cardiovascular homeostasis. In addition to contributing to the regulation of blood flow, the vascular endothelium plays a central role in the control of fibrinolysis. Endothelial cells are the primary site of synthesis and release of tissue-type plasminogen activator (t-PA), the main plasminogen activator in fibrinolysis (Lijnen & Collen, 1997). The ability of the endothelium locally to release t-PA is critical to the fibrinolytic process. Indeed, endogenous thrombolysis is more effective if active t-PA is readily available during, rather than after, thrombogenesis due to its ability to preferentially activate plasminogen bound to fibrin, thus increasing fibrinolytic activity on the surface of a clot (Brommer, 1984; Fox et al. 1984). Moreover, the inhibitory interaction between PAI-1 and t-PA has a second-order rate constant of approximately 107m−1 s−1 (Ouimet & Loscalzo, 1994), therefore local rapid release of t-PA is vital to ensure that active unbound t-PA reaches the thrombus. It has been suggested that t-PA release represents a major feature of endothelial function with respect to preventing thrombosis (Muldowney & Vaughan, 2002).

Advancing age is associated with a progressive loss in endothelial vasodilator function (Taddei et al. 1997; DeSouza et al. 2000). Currently, it is unknown whether the capacity of the endothelium to release t-PA is similarly impaired. If so, this may contribute mechanistically to the increased risk of thrombosis with ageing.

Contrary to advancing age, habitual physical activity is associated with a reduced risk of atherothrombosis (Powell et al. 1987; Blair et al. 1989). We have previously reported that in addition to favourably modifying traditional cardiovascular risk factors, such as blood pressure (Seals et al. 2001), regular aerobic exercise also prevents and reverses the age-related reduction in acetylcholine-mediated endothelium-dependent vasodilatation (DeSouza et al. 2000). To date, studies regarding the effects of exercise on the vascular endothelium have primarily focused on vasodilatation, and there is no information regarding whether regular aerobic exercise beneficially affects t-PA release.

Accordingly, we tested the following associated hypotheses: (1) the capacity of the vascular endothelium acutely to release t-PA declines with age in healthy sedentary men; (2) compared with healthy sedentary men, endothelial t-PA release either would not be reduced or would be reduced to a lesser extent with age in men who regularly perform endurance exercise; and (3) regular aerobic exercise would at least partially reverse age-associated reductions in endothelial t-PA release in previously sedentary older men. To test these hypotheses we used two complimentary experimental approaches. First, we used a cross-sectional study design to determine the influence, and potential interaction, of age and habitual aerobic exercise on the capacity of the endothelium to release t-PA. We then performed an intervention study to determine the effects of aerobic exercise training on endothelial t-PA release in sedentary older men.

Methods

Subjects

A total of 62 healthy men aged 22-35 or 50-75 years participated in the cross-sectional study: 10 young and 20 older sedentary men and 14 young and 18 older endurance exercise-trained (distance runners and/or triathletes) men. The sedentary men had performed no regular physical exercise for at least 6 months. The endurance-trained young and older men performed vigorous aerobic-endurance exercise > 5 times per week, were active in local road races, and had been training for 13 ± 1 and 38 ± 2 years, respectively. In the intervention study, 10 of the 20 sedentary middle-aged and older men from the cross-sectional investigation were studied before and after 3 months of aerobic exercise training.

All subjects were normotensive (arterial blood pressure < 140/90 mmHg) and free of overt disease as assessed by medical history, physical examination and fasting blood chemistries (e.g. plasma glucose concentrations < 6.4 mmol l−1 and total cholesterol < 6.2 mmol l−1). Sedentary and endurance-trained men over the age of 50 years were further evaluated for clinical evidence of coronary artery disease with electrocardiograms and blood pressure at rest and during incremental exercise performed to exhaustion. All subjects were non-smokers and not taking medication. The experimental protocol was approved by the Human Research Committee at the University of Colorado at Boulder; voluntary written informed consent was obtained from each subject after the nature, purpose and risks of the study had been explained to them. All experiments conformed to the Declaration of Helsinki.

Measurements

Endurance-trained subjects and subjects who completed the 3 month exercise intervention were studied 20-24 h after their last exercise training session to avoid the immediate (acute) effects of exercise, while still representing their normal physiological state (i.e. habitually exercising).

Body composition

Percentage body fat was determined by dual energy X-ray absorptiometry (DXA, Model DPX-IQ Lunar Radiation Corporation). Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. Minimal waist circumference was measured according to published guidelines (Lohman et al. 1988).

Treadmill exercise test

To assess aerobic fitness subjects performed incremental treadmill exercise using a modified Balke protocol as previously described (Evans et al. 1995). Maximal oxygen consumption (V̇O2,max) was measured using on-line computer-assisted open circuit spirometry. In addition, heart rate and rating of perceived exertion (RPE) were measured throughout exercise (Borg, 1970) and total exercise time to exhaustion was recorded.

Metabolic measurements

Fasting plasma lipid and lipoprotein, glucose and insulin concentrations were determined by the clinical laboratory affiliated with the General Clinical Research Center, as previously described (DeSouza et al. 1998).

Intra-arterial fibrinolytic protocol

All measurements were performed between 7 and 10 a.m. after a 12 h overnight fast in a temperature-controlled room. An intravenous catheter was placed in a deep antecubital vein of the non-dominant arm. Thereafter, a 5-cm, 20 gauge catheter was introduced into the brachial artery of the same arm under local anaesthesia (1 % lidocaine). After catheterization the subjects were allowed to rest 20 min before baseline measurements were made. Forearm blood flow (FBF) was measured using strain-gauge venous occlusion plethysmography (D.E. Hokanson, Bellevue, WA), as previously described by our laboratory (DeSouza et al. 2000). Drug infusion rates were normalized per 100 ml of forearm tissue and infused at 5 ml min−1 by a syringe pump. Following the measurement of resting blood flow for 5 min, bradykinin was infused intra-arterially at 12.5, 25 and 50 ng (100 ml tissue)−1 min−1 and sodium nitroprusside at 1.0, 2.0 and 4.0 μg (100 ml tissue)−1 min−1 for 5 min at each dose. To avoid an order effect, the sequence of drug administration was randomized. Bradykinin was chosen to stimulate t-PA release because it has been repeatedly shown to be a specific, reliable and potent stimulant of endothelial t-PA release, in both the forearm and coronary vasculature, in adult humans (Brown et al. 1997, 1999, 2000; Minai et al. 2001). In contrast, acetylcholine, for example, has been shown to be neither a reliable nor effective stimulus for t-PA release (Brown et al. 1999; Dell'Omo et al. 1999).

Net endothelial release of t-PA antigen and PAI-1 antigen in response to bradykinin and sodium nitroprusside was calculated according to Jern et al. (1997). Briefly, arteriovenous concentration gradients were determined by subtracting the measured values in simultaneously collected venous and arterial blood. For both t-PA and PAI-1, a positive difference indicated a net release and a negative difference, net uptake. Net release or uptake rates were calculated as follows:

graphic file with name tjp0546-0289-mu1.jpg

where CV and CA represent the concentration in the vein and artery, respectively. Haematocrit was measured in triplicate using the standard microhaematocrit technique and corrected for trapped plasma volume within the trapped erythrocytes (Chaplin & Mollison, 1952). The total amount of t-PA antigen released across the forearm in response to bradykinin was calculated as the total area under each curve above baseline using a trapezoidal model.

A questionnaire designed to detect and document recent infection/inflammation (< 2 weeks) was administered prior to the phlebotomies. Subjects with a history of recent infection/inflammation did not undergo the fibrinolytic protocol in order to avoid confounding effects from potential infection/inflammation-associated fibrinolytic changes (Macko et al. 1996).

Blood sampling and fibrinolytic assays

All phlebotomies were performed with minimal venostasis. Arterial and venous blood samples were collected simultaneously at baseline and the end of each drug dose to determine t-PA and PAI-1 antigen concentrations. The first 3 ml of blood from both the artery and vein were discarded before each sample was collected. All samples were collected in tubes containing 0.45 m sodium citrate buffer, pH 4.3 (final dilution volume 1:10; Stabilyte, Biopool AB, Sweden). Within 15 min of collection, samples were centrifuged for 20 min at 6000 g at 4 °C. Platelet-poor plasma was aliquoted and stored at −70 °C until assayed at the end of the study. All assays were performed in duplicate with a maximum of one freeze-thaw cycle. Plasma concentrations of t-PA antigen and PAI-1 antigen were determined by enzyme immunoassay (Biopool International, Venutra CA, USA; Diagnostic Stago, France).

Exercise intervention

The 3 month aerobic exercise-training programme employed in the present study has been described in detail previously by our laboratory (DeSouza et al. 2000). Briefly, after completion of baseline measurements, subjects underwent a supervised orientation; thereafter they exercised on their own. Subjects were asked to exercise 5-7 days per week, 40-50 min per day, at 60-75 % of their individual maximum heart rate, as determined during maximal exercise testing. Most subjects walked, but some integrated jogging into their exercise session, as their fitness improved, in order to maintain their heart rate within the prescribed range. Adherence to the exercise programme was documented every 2 weeks based on data downloaded directly from heart rate monitors (Polar Electro Inc., Woodbury, NY, USA) and from exercise logs.

Statistical analysis

Data for the cross-sectional study were analysed by multi-factor analysis of variance (ANOVA; age - training status). When indicated by a significant F-value, a post-hoc test using the Newman-Keuls method was performed to identify significant group differences. Group differences in FBF and endothelial t-PA and PAI-1 release to bradykinin and sodium nitroprusside were determined by repeated measures ANOVA. Relations 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. All data are expressed as mean ± s.e.m. Statistical significance was set a priori at P < 0.05.

Results

Cross-sectional study

Selected subject characteristics are presented in Table 1. The mean age difference between the young and older men was 33 years. Body mass was not related to age, but was lower (P < 0.05) in endurance-trained than sedentary men. Body fat percentage, waist circumference and total cholesterol increased (P < 0.01) with age in both the sedentary and endurance-trained groups. Maximal oxygen consumption was higher in the endurance-trained men than in the sedentary men at both ages (P < 0.05). The older sedentary men demonstrated the highest insulin concentrations of all groups (P < 0.05). There were no significant differences among the groups in the forearm vasodilator responses to bradykinin and sodium nitroprusside (Fig. 1).

Table 1.

Selected subject characteristics of the cross-sectional study

Sedentary Endurance-trained
Variable Young (n = 10) Older (n = 20) Young (n = 14) Older (n = 18)
Age (years) 28 ± 1 62 ± 1* 29 ± 1 62 ± 1*
Body mass (kg) 82.9 ± 5.5 88.3 ± 1.9 713 ± 1.5† 78.3 ± 2.1†
Body fat (%) 16.2 ± 1.7 29.2 ± 1.1* 9.2 ± 1.2† 19.2 ± 1.1*†
Waist circumference (cm) 89.7 ± 4.3 104.0 ± 2.1* 78.8 ± 1.0† 90.0 ± 2.1†
Body mass index (kg m−2) 24.9 ± 1.6 28.6 ± 0.7 22.1 ± 0.5† 24.2 ± 0.5†
Systolic BP (mmHg) 115 ± 3 124 ± 2 106 ± 5 123 ± 3*
Diastolic BP (mmHg) 64 ± 3 81. ± 2* 70 ± 4 75 ± 2†
V̇O2,max (ml kg−1 min−1) 49.6 ± 1.7 29.7 ± 1.4* 63.8 ± 1.2† 40.0 ± 1.1*†
Total cholesterol (mmol l−1) 3.7 ± 0.3 4.5 ± 0.2* 4.0 ± 0.2 4.7 ± 0.3*
HDL-C (mmol l−1) 1.0 ± 0.1 1.1 ± 0.1 1.4 ± 0.1 1.4 ± 0.1
LDL-C (mmol l−1) 2.2 ± 0.2 2.7 ± 0.1 2.3 ± 0.2 2.7 ± 0.4
Triglycerides (mmol l−1) 1.1 ± 0.2 1.3 ± 0.1* 0.8 ± 0.1 0.9 ± 0.1
Glucose (mmol l−1) 5.2 ± 0.2 5.3 ± 0.1 5.1 ± 0.2 5.3 ± 0.2
Insulin (pmol l−1) 37.8.1 ± 7.2 40.8 ± 4.2* 26.4 ± 5.4† 24.6 ± 1.8†
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V̇O2,max, maximal oxygen consumption; BP, blood pressure; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. Values are means ± s.e.m.

*

P < 0.05 vs, young of same training status.

†

P < 0.05 vs. age-matched sedentary.

Figure 1.

Figure 1

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Forearm blood flow responses to bradykinin and sodium nitroprusside in sedentary and endurance-trained men (both P = 0.20). Values are mean ± s.e.m.

Baseline venous t-PA antigen and PAI-1 antigen concentrations are shown in Table 2. Circulating plasma t-PA antigen and PAI-1 antigen levels were significantly higher with age in both the sedentary and endurance-trained men. However, the endurance-trained men demonstrated lower (P < 0.01) t-PA and PAI-1 antigen levels compared with their sedentary counterparts of similar age. Figure 2 shows the net release rates of t-PA antigen in the forearm to bradykinin in the sedentary and endurance-trained subjects. Net endothelial t-PA release in response to bradykinin was significantly blunted with age in the sedentary men. At the highest dose of bradykinin (50 ng (100 ml tissue)−1 min−1) the increase in net endothelial release of t-PA antigen was ≈35 % (P < 0.05) less 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 addition, the area under the curve for net t-PA antigen release was ≈50 % lower in the older than young men (190 ± 17 vs. 342 ± 60 ng (100 ml tissue)−1: P < 0.05). In stark contrast, the endurance-trained men did not demonstrate an age-related decline in the net release of t-PA antigen. In fact, the magnitude of increase in the net release of t-PA antigen in response to bradykinin was slightly (but not significantly) higher in the older (from 0.1 ± 0.3 to 72.4 ± 11.1 ng (100 ml tissue)−1 min−1) versus young (from 0.9 ± 0.4 to 62.3 ± 11.7 ng (100 ml tissue)−1 min−1) endurance-trained subjects. The area under the curve for net t-PA antigen release also was not different (P = 0.51) in the older (364 ± 56 ng (100 ml tissue)−1) versus young (323 ± 46 ng (100 ml tissue)−1) endurance-trained men. As a result, both the net release of t-PA antigen and the area under the curve were significantly greater in the older endurance-trained men compared with their age-matched sedentary counterparts. Net release rates of t-PA antigen in response to bradykinin were similar between the young sedentary and endurance-trained men. There were no significant differences in the forearm t-PA responses to sodium nitroprusside among the groups (data not shown). In addition, the infusion of bradykinin and sodium nitroprusside produced no consistent or significant changes in the net release of PAI-1 antigen among the groups. For example, at the highest dose of bradykinin the young (9.6 ± 7.4 IU (100 ml tissue)−1 min−1) and older (6.6 ± 5.2 IU (100 ml tissue)−1 min−1) sedentary and the older endurance-trained (11.8 ± 3.6 IU (100 ml tissue)−1 min−1) groups demonstrated some net release whereas the young endurance-trained (-1.2 ± 2 IU (100 ml tissue)−1 min−1) men demonstrated net uptake of PAI-1 antigen.

Table 2.

Baseline t-PA antigen and PAI-1 antigen concentrations for subjects in the cross-sectional study

Sedentary Endurance-trained
Variable Young (n = 10) Older (n-20) Young (n = 14) Older (n = 18)
t-PA antigen (ng ml−1) 5.9 ± 1.2 9.8 ± 0.8* 3.8 ± 0.3† 6.6 ± 0.6*†
PAI-1 antigen (ng ml−1) 9.9 ± 2.7 20.3 ± 4.0* 2.2 ± 0.2† 7.1 ± 1.7*†
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Values are mean ± s.e.m.

*

P < 0.05vs. young of same training status.

†

P < 0.05vs. age-matched sedentary.

Figure 2.

Figure 2

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Net release rate of t-PA antigen across the forearm in response to bradykinin in sedentary (P < 0.05) and endurance-trained (P = 0.42) men and total amount of t-PA antigen released (area under the curve; *P < 0.05vs. young sedentary). Values are mean ± s.e.m.

In the overall study population net t-PA antigen release at the highest dose of bradykinin was related to HDL-cholesterol (r = 0.29; P < 0.05) only, and no other significant relations were observed with net t-PA antigen release or area under the t-PA antigen curve.

Intervention study

All 10 older men (age 60 ± 2 years) completed the exercise intervention study. On average, the men walked 5.3 ± 0.3 days week−1, 45 ± 2 min day−1, at an intensity of 66 ± 1 % of individually determined maximal heart rate. There were no significant changes in body mass, adiposity, blood pressure, heart rate at rest, maximal oxygen consumption or plasma cholesterol, glucose or insulin concentrations (Table 3). However, heart rate at a standardized submaximal workload of ≈70 % of the initial (baseline) maximal oxygen consumption decreased (P < 0.05) and maximal treadmill walking time increased (P < 0.05) in response to exercise training (Table 3). There were no significant changes in the FBF responses to bradykinin and sodium nitroprusside after exercise training (Fig. 3).

Table 3.

Selected subject characterstics of the exercise intervention study

Variable Before training After training
Body mass (kg) 87.9 ± 2.3 88.3 ± 2.3
Body fat (%) 27.9 ± 1.8 29.6 ± 2.0
Waist circumference (cm) 103.6 ± 2.9 101.0 ± 3.0
Body mass index (kg m−2) 28.9 ± 0.8 28.9 ± 0.9
Systolic BP (mm Hg) 117 ± 2 121 ± 1
Diastolic BP (mm Hg) 66 ± 3 66 ± 2
Heart rate (beats min−1) 79 ± 3 76 ± 2
V̇o2,max (ml kg−1 min−1) 4.7 ± 0.4 31.0 ± 1.6
Total cholesterol (mmol l−1) 4.7 ± 0.4 5.4 ± 0.2
HDL-C (mmol l−1) 1.1 ± 0.1 1.3 ± 0.2
LDL-C (mmol l−1) 2.8 ± 0.3 3.2 ± 0.2
Triglycerides (mmol l−1) 1.7 ± 0.4 1.9 ± 0.4
Glucose (mmol l−1) 5.4 ± 0.0 5.3 ± 0.1
Insulin (pmol l−1) 44.4 ± 6.6 48.0 ± 7.2
Treadmill exercise time (min) 9.9 ± 0.5 11.4 ± 0.5*
Submaximal heart rate (beats min−1) 159 ± 4 147 ± 4*
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V̇O2,max, maximal oxygen consumption; MAP, mean arterial blood; HDL-C, high- density lipoprotein cholesterol; LDL-C low-density lipoprotein cholesterol. Values are means ± s.e.m.

*

P < 0.05vs. before training

Figure 3.

Figure 3

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Forearm blood flow responses to bradykinin (P = 0.51) and sodium nitroprusside (P = 0.79) before and after 3 months of aerobic exercise training. Values are mean ± s.e.m.

Circulating plasma concentrations of t-PA antigen (13.3 ± 1.6 vs. 11.5 ± 1.5 ng ml−1) and PAI-1 antigen (20.5 ± 5.4 vs. 29.6 ± 6.6 ng ml−1) were not significantly changed after compared with before the aerobic exercise-training programme. However, regular exercise significantly increased the capacity of the vascular endothelium to release t-PA in response to bradykinin. Net endothelial release of t-PA antigen across the forearm to bradykinin was ≈60 % higher (P < 0.05) after (from 2.0 ± 1.7 to 68.2 ± 8.5 ng (100 ml tissue)−1 min−1) versus before (-1.1 ± 0.9 to 40.3 ± 6.9 ng (100 ml tissue)−1 min−1) exercise training (Fig. 4). Moreover, the total amount of t-PA antigen released (area under the curve) in response to bradykinin increased ≈45 % (from 227 ± 29 to 327 ± 37 ng (100 ml tissue)−1; P < 0.05) with the exercise intervention. Of note, the rate of net release and total amount of t-PA antigen released across the forearm after exercise training were not significantly different from those observed in the young adults and older endurance-trained men who participated in the cross-sectional study. There was a modest, albeit significant, change in net t-PA antigen release in response to sodium nitroprusside after (from −0.5 ± 0.5 to 5.2 ± 2.7 ng (100 ml tissue)−1 min−1) compared with before (-0.8 ± 0.5 to −0.7 ± 1.5 ng (100 ml tissue)−1 min−1) exercise training. The net release of PAI-1 antigen was unaffected by exercise. For example, at the highest dose of bradykinin, net release of PAI-1 antigen was not significantly different after exercise training (5.4 ± 8.5 vs. 8.7 ± 5.3 IU (100 ml tissue) min−1). There were no significant correlates of the improvement in endothelial t-PA release in response to exercise training.

Figure 4.

Figure 4

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Net release rate of t-PA antigen (P < 0.05) across the forearm in response to bradykinin and total amount of t-PA antigen released before and after 3 months of aerobic exercise training. Values are mean ± s.e.m. *P < 0.05vs. before training.

Discussion

The primary new findings of the present study are as follows. First, the capacity of the endothelium to release t-PA antigen declines significantly with age in healthy sedentary men. Second, in contrast to sedentary men, endothelial t-PA antigen release is well preserved with age in men who perform regular endurance exercise. Third, a relatively brief (13 week) period of aerobic exercise training can reverse the age-associated loss in the capacity of the endothelium to release t-PA in previously sedentary older men. Taken together these findings indicate that although endothelial t-PA release is reduced in older sedentary men, this impairment does not appear to be an inevitable or irreversible consequence of biological ageing.

We (DeSouza et al. 1998) and others (Hasimoto et al. 1987; Hamsten, 1993) have previously shown that sedentary ageing is associated with reduced plasma fibrinolytic activity characterized by increased systemic concentrations of t-PA antigen, PAI-1 antigen and PAI-1 activity. Indeed, in the present study basal plasma concentrations of t-PA antigen and PAI-1 antigen were significantly higher in the older compared with young sedentary men. However, although the influence of age on plasma levels of t-PA antigen is well documented, it is the local endothelial release rate of t-PA and not systemic plasma concentration that determines endogenous thrombolysis potential (Kooistra et al. 1994; Jern et al. 1999). To our knowledge, this is the first study to demonstrate that the capacity of the endothelium acutely to release t-PA antigen is significantly blunted with advancing age. Specifically, we observed a 35 % age-related reduction in forearm endothelial t-PA antigen release in healthy sedentary men. This degree of impairment in acute endothelial t-PA antigen release in response to bradykinin with ageing is similar to that reported in other at-risk populations, including hypertensive patients and cigarette smokers (Jern et al. 1997; Pretorius et al. 2002). The fact that we observed a diminished rate of t-PA antigen release in our older sedentary men despite higher circulating plasma concentrations provides further evidence that systemic t-PA antigen levels do not reflect the ability of the endothelium rapidly and locally to release t-PA (Newby et al. 1997; Jern et al. 1999). Reduced capacity of the endothelium to release t-PA with age may have important physiological and clinical implications. t-PA is considered to be critical for dissolving embolized clots (Bugge et al. 1996) and managing fibrin deposition in the vasculature (Christie et al. 1999). Studies in chimpanzees and baboons have shown that rapid local release of t-PA in response to a thrombogenic stimulus results in pronounced fibrin degradation (Giles et al. 1990; Kruithof et al. 1997). In stark contrast, t-PA-deficient mice demonstrate accelerated atherogenesis characterized by extensive fibrin deposition and severe myocardial tissue necrosis (Carmeliet et al. 1994; Christie et al. 1999). In adult humans, Newby et al. (2001) recently reported that reduced coronary endothelial t-PA release was associated with increased atheromatous plaque burden in cigarette smokers. It should be noted that, although we assessed acute endothelial t-PA antigen release in the forearm, a vascular bed usually devoid of diffuse atherosclerosis, there is evidence that this site provides an excellent surrogate measure of t-PA release in the coronary circulation (Newby et al. 1999, 2001). Thus, impaired regulated release of t-PA from the vascular endothelium may represent an important mechanism underlying the increased incidence of thrombotic cardiovascular events in middle-aged and older sedentary men.

Another novel finding of our cross-sectional study was that contrary to the sedentary men, endothelial t-PA antigen release was not impaired with age in men who regularly performed aerobic endurance exercise. There were no significant differences in either net endothelial t-PA release or area under the t-PA curve in response to bradykinin between the older and young endurance-trained men. As a result, the older endurance-trained men demonstrated significantly higher t-PA antigen release compared with their sedentary peers of similar age. Since there were no differences in t-PA antigen release rates between the young adult groups, our cross-sectional findings suggest that the age-related decline in the capacity of the endothelium to release t-PA may be prevented in men who engage in habitual endurance exercise. From a primary prevention perspective, preserved endothelial fibrinolytic capacity may play a major role in the lower incidence of atherothrombotic events observed in middle-aged and older men who exercise regularly (Blair et al. 1989).

To confirm the favourable effects of regular aerobic exercise on endothelial t-PA release observed in our cross-sectional study, we performed a follow-up exercise-training intervention in previously sedentary older men. This allowed us to examine the direct effect of exercise on the capacity of the endothelium to release t-PA by determining whether improvements in t-PA antigen release, if observed, were dependent upon concomitant changes in other factors known to affect endothelial function such as arterial blood pressure (Panza et al. 1993) and plasma lipid and lipoproteins (Casino et al. 1995).

The results of our intervention study corroborate and extend our cross-sectional observations. After only 3 months of regular aerobic exercise (primarily walking) we observed an ≈50 % increase in net endothelial t-PA antigen release across the forearm in response to bradykinin in previously sedentary older men. Of note, this improvement was not associated with changes in body mass, adiposity, arterial blood pressure, plasma cholesterol or maximal aerobic capacity, indicating a primary modulatory effect of aerobic exercise on endothelial regulation of t-PA release. We have previously shown that the intensity (moderate) and mode (primarily walking) of exercise training employed in the present study can restore acetylcholine-mediated forearm endothelium-dependent vasodilatation and central arterial compliance to normal levels in healthy middle-aged and older men (DeSouza et al. 2000; Tanaka et al. 2000). The results of this study complement these findings. Indeed, after exercise training, both the rate and total amount of t-PA antigen released to bradykinin were not different from those observed in the young adults and older endurance-trained athletes in the cross-sectional study. From a clinical perspective, it is noteworthy that the improvements observed in endothelial t-PA antigen release were accomplished with a moderate, home-based aerobic exercise training programme that can be safely performed by most, if not all, sedentary healthy middle-aged and older adults (DeSouza et al. 2000; Seals et al. 2001).

We should emphasize that our results pertain only to the forearm. It is quite likely that if we measured t-PA release in the trained limbs (i.e. legs) the beneficial effect of exercise training would have been greater. Nevertheless, the fact that we observed significantly depressed forearm endothelial t-PA release with sedentary ageing and normalization with exercise training involving the lower limbs provides further evidence that endothelial dysfunction is systemic in nature and that regular aerobic exercise produces favourable global endothelial adaptations.

In the present study, consistent with previous reports (Newby et al. 1997; Stein et al. 1998), we observed no major effect of sodium nitroprusside on endothelial t-PA antigen release in either the cross-sectional or intervention studies. This finding, coupled with the fact that the forearm blood flow (FBF) responses to bradykinin did not differ among the groups, confirms that the age- and exercise-related differences observed in endothelial t-PA release were independent of changes in limb blood flow. In addition, there were no differences in the net release of PAI-1 antigen (to either bradykinin or sodium nitroprusside) among the young and older groups or after exercise training, thus ruling out the potential confounding effects of t-PA/PAI-1 complex formation. We can only speculate, however, on the primary mechanisms responsible for the unfavourable influence of age and beneficial effects of regular exercise on regulated t-PA release from the endothelium. With respect to ageing, it is possible that a defect in the signalling pathway triggering t-PA release or reduced t-PA synthesis and/or intracellular storage may contribute to endothelial fibrinolytic dysfunction. Several lines of evidence from animal models suggest that t-PA expression may decline with age, potentially reducing the availability of intracellular t-PA and limiting the capacity of the endothelium to release high amounts of t-PA upon stimulation (Ahn et al. 1999; Popa-Wagner et al. 2000). It is also plausible that age-related increases in oxidative stress may impair endothelial t-PA release. In vitro studies have shown that oxidative stress significantly inhibits t-PA release from cultured endothelial cells (Shatos et al. 1990; Kugiyama et al. 1993). With regard to the beneficial effects of exercise on regulated endothelial t-PA release, it is possible that mechanical alteration/deformation of the endothelium during exercise as a result of increased arterial pressure and pulsatile flow may upregulate t-PA mRNA expression and secretion. Mechanical shear stress has been shown to be a potent stimulator of t-PA transcription and protein production (Diamond et al. 1989, 1990). In addition, since an increase in cytoplasmic calcium can induce acute t-PA release (Kooistra et al. 1994), higher endothelial intracellular calcium concentrations in response to regular exercise may also contribute to greater capacity to release t-PA (Laughlin et al. 1998). Reduced oxidative stress with exercise is also an attractive hypothesis. Clearly, future studies are needed to determine the mechanisms responsible for age- and exercise-related alterations in acute endothelial t-PA release.

In conclusion, the results of this study demonstrate, for the first time, that acute t-PA release from the vascular endothelium declines with age in sedentary but not endurance exercise-trained adult men. In addition, regular aerobic exercise fully restores the capacity of the endothelium to release t-PA in previously sedentary older men. Impaired regulated release of t-PA by endothelial cells may represent a major abnormality in a primary defence mechanism against thrombosis (Kooistra et al. 1994). Importantly, the decline in the capacity of the endothelium to release t-PA does not appear to be a consequence of primary ageing, but rather a by-product of a sedentary lifestyle. Regular aerobic exercise may not only prevent, but could also reverse the deleterious effects of sedentary ageing on endothelial fibrinolytic function.

Acknowledgments

We would like to thank all of the subjects who participated in the study and Yoli Casas, Marilyn Ng and Heather Irmiger for their technical assistance. This study was supported by National Institutes of Health award HL03840, an American Diabetes Association Clinical Research Award and American Heart Association award 0060430Z (Dr DeSouza) and by award number 2 M01-RR00051 from the General Clinical Research Center Program of the National Center for Research Resources, National Institutes of Health. Dr Stauffer was supported by American Heart Association award 0120679Z. Derek Smith was supported by American Heart Association award 0110221Z.

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