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J Appl Physiol 95: 2390-2397, 2003. First published August 15, 2003; doi:10.1152/japplphysiol.00403.2003
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Role of TGF-{beta}1 in relation to exercise-induced type I collagen synthesis in human tendinous tissue

Katja Heinemeier, Henning Langberg, Jens L. Olesen, and Michael Kjaer

Sports Medicine Research Unit, Department of Rheumatology H, Bispebjerg Hospital, DK-2400 Copenhagen, Denmark

Submitted 23 April 2003 ; accepted in final form 8 August 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
Mechanical loading of tissue is known to influence local collagen synthesis, and microdialysis studies indicate that mechanical loading of human tendon during exercise elevates tendinous type I collagen production. Transforming growth factor-{beta}1 (TGF-{beta}1), a potent stimulator of type I collagen synthesis, is released from cultured tendon fibroblasts in response to mechanical loading. Thus TGF-{beta}1 could link mechanical loading and collagen synthesis in tendon tissue in vivo. Tissue levels of TGF-{beta}1 and type I collagen metabolism markers [procollagen I COOH-terminal propeptide (PICP) and COOH-terminal telopeptide of type I collagen (ICTP)] were measured by microdialysis in peritendinous tissue of the Achilles' tendon in six male volunteers before and after treadmill running (1 h, 12 km/h, 3% uphill). In addition, blood levels of TGF-{beta}1, PICP, and ICTP were obtained. PICP levels increased 68 h after exercise (P < 0.05). Dialysate levels of TGF-{beta}1 changed from 303 ± 46 pg/ml (at rest) to 423 ± 86 pg/ml 3 h postexercise. This change was nonsignificant, but the decay of tissue TGF-{beta}1 after catheter insertion was markedly delayed by exercise compared with the decay seen in resting subjects. Plasma concentrations of TGF-{beta}1 rose 30% in response to exercise (P < 0.05 vs. pre). Our observations indicate an increased local production of type I collagen in human peritendinous tissue in response to uphill running. Although not conclusive, changes in circulating and local TGF-{beta}1, in response to exercise, suggest a role for TGF-{beta}1 in mechanical regulation of local collagen type I synthesis in tendon-related connective tissue in vivo.

extracellular matrix; tendon; mechanical loading; transforming growth factor-{beta}1

FIBROUS TYPE I COLLAGEN IS an important tensile strengthcarrying part of the extracellular matrix (5), allowing for transmission of force generated by, e.g., skeletal muscle into movement (22). One mechanism known to regulate collagen homeostasis is the mechanical loading of tissue (7), and collagen expression and/or production has been shown to rise in several different cell types and tissues, including renal (53), cardiac (6, 49), vascular (38), pulmonary (3, 47), bone (19), ligament (24), and tendinous (36) cells and/or tissue, in response to strain. These findings are almost entirely based on in vitro studies, and only recently it has been indicated, by microdialysis studies, that the type I collagen accumulation increases in the peritendinous tissue of human Achilles tendon in the days after acute exercise involving mechanical loading of the tendon (29). The regulation of this increased collagen production is only partly understood. Regulatory hormones and growth factors are, together with integrins, cytoskeletal proteins, and certain ion channels, thought to be responsible for mechanically induced cell signaling (7). Studies have indicated that mechanical loading increases the expression of several growth factors and cytokines, such as IGF-I (39), IL-6 (8, 26), and transforming growth factor-{beta}1 (TGF-{beta}1) (18). With regards to TGF-{beta}1, in vitro experiments with human and animal cell cultures as well as in vivo animal studies have shown an increased expression of TGF-{beta}1 in response to mechanical stimuli in a number of cell and tissue types (8, 9, 18, 38, 40, 42, 45, 49). Furthermore, it has been found that mechanically induced type I collagen expression in ligament fibroblasts, cardiac fibroblasts, and human intestinal smooth muscle cells is directly dependent on TGF-{beta}1 activity (18, 24, 33). Thus it is possible that TGF-{beta}1 is involved in the regulation of type I collagen synthesis in human tendon tissue in a similar manner and that the increase in local type I collagen production found in human tendinous tissue in response to exercise (27, 29) is based, at least in part, on a local increase in the concentration of TGF-{beta}1 caused by mechanical stimuli of tendon fibroblasts. To investigate this hypothesis in humans, it was studied whether exercise that induced mechanical loading of the Achilles tendon resulted in any increase in systemic and local tissue TGF-{beta}1 concentrations along with an increase in local type I collagen production, as determined by microdialysis, in the peritendinous area of the human Achilles tendon.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
Microdialysis for Determination of Tissue TGF-{beta}1 and Markers of Type I Collagen Turnover

Microdialysis catheters were constructed of an 80-cm inlet of polyethylene tubing (0.76 mm ID), connected to 30 mm of permeable membrane [hollow plasma separator tubes (Asahi, Japan); diameter 4 mm; molecular mass cutoff 3,000 kDa], by 7.5 mm of nylon tubing (0.50 mm ID, 0.63 mm OD). Identical nylon tubing was used for the outlet (8 cm). A stainless steel wire was placed inside the catheter to increase mechanical stability. Before use the catheters were sterilized by use of ethylene oxide (ETO-sterilization; ABATOX, Herlev, DK & Mærsk Medical a/s, Lynge, Denmark). During each microdialysis run, two microdialysis catheters were placed diagonally, with an angle of 45° to the tendon and just ventral to both left and right Achilles tendon under ultrasound guidance, leaving the outlet of catheter at the medial side of the tendon. The catheters were perfused at a rate of 5 µl/min, by use of a high-precision syringe pump (CMA/100, Carnegie, Solna, Sweden), with Ringer-acetate containing 3H-labeled human type IV collagen (130 kDa; NEN, Boston, MA) with a perfusate activity of 5.6 kBq/ml. The in vivo relative recovery for TGF-{beta}1 was not measured, because no radioactive TGF-{beta} for human use was commercially available. Therefore the microdialysis results on TGF-{beta} are presented as dialysate concentrations. As for the measurement of type I collagen turnover, levels of procollagen I COOH-terminal propeptide (PICP) and COOH-terminal telopeptide of type I collagen (ICTP) were measured in the dialysate. The level of PICP is correlated to type I collagen production, as one PICP is cleaved from every newly secreted type I collagen triple helix, whereas ICTP is a degradation product of type I collagen breakdown. The relative loss of 3H-labeled human type IV collagen was used to estimate the in vivo relative recovery of PICP and ICTP according to the internal reference method (44), and, on the basis of the relative recoveries, the tissue level of PICP and ICTP was estimated as described previously in detail by Langberg et al. (29).

Control Experiment

To investigate possible fluctuations in basal tissue levels of TGF-{beta}1 during the microdialysis experimental procedure (described above), separate control experiments were carried out. Dialysate fluid was sampled from probes placed along the Achilles tendon for a period of 3.5 h at rest in six healthy, moderately trained male volunteers, with a mean age of 25 yr (range 23–27 yr) and a mean body mass of 81 kg (range 72–86 kg). All subjects gave informed consent, and the study was approved by the Ethical Committee of Copenhagen (KF 11-088/01). Subjects fasted from 10 PM on the day preceding the experiment and were told not to engage in any type of exercise or training for 48 h before the study. To rule out any influence of dietary collagen on circulating and local levels of type I collagen metabolism markers, the participants were given a list of gelatin-rich foods and informed not to consume any of these for 4 days leading up to the experiment and during the experiment. The experiment lasted for a total time of 3.5 h after insertion of the microdialysis fibers, and dialysate was sampled at 30-min intervals for the first 1.5 h and at 1-h intervals for the last 2 h. Samples were weighed to eliminate the possibility of ultrafiltration, and afterward they were immediately frozen at -80°C until analysis.

Exercise Experiment

Six healthy, moderately trained male volunteers with a mean age of 26 yr (range 25–28 yr) and a mean body mass of 77 kg (range 70–80 kg) were included in the study. All subjects gave informed consent, and the study was approved by the Ethical Committee of Copenhagen [KF 11-088/01].

Experiments were started at 8 AM on all days. Subjects fasted from 10 PM on days preceding experiment days and were told not to engage in any type of exercise or training for 48 h before the first day of the study and for the entire length of the experiment. Dietary intake of gelatin was restricted as in the control experiment. The first day of the experiment consisted of a 3-h preexercise rest period, a 1-h exercise period, and a postexercise recovery period of 6 h. Additionally, microdialysis was sampled for 4-h periods at 20 and 68 h after exercise (Fig. 1). The exercise intervention consisted of a 10-min warm-up treadmill run at 10 km/h, followed by 1 h of 3% uphill treadmill running at 12 km/h. No direct determination of the individuals maximal oxygen uptake was made, but considering that all individuals were moderately trained and young and that they were all completely exhausted after 1 h indicates a relative work intensity of 70–80% of maximal oxygen uptake (1). Microdialysis was performed throughout the rest periods, during which subjects were placed in a supine position at room temperature. Microdialysis catheters were always removed before running, and afterward new catheters were inserted. During microdialysis, dialysate samples were collected at 30-min intervals for the first hour and at 60-min intervals for the remaining periods. Samples were weighed to eliminate the possibility of ultrafiltration, and afterward they were immediately frozen at -80°C until analysis. To minimize the influence of the insertion trauma on measured concentrations of TGF-{beta}, only samples collected after the first 2 h of microdialysis were used for analysis in the exercise experiment.



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  		Fig. 1. Experimental design. In the exercise experiment, microdialysate was sampled from the peritendinous tissue of the Achilles tendon during rest periods before and after 1 h of uphill treadmill running (hatched bars). Additionally, blood was sampled before (Rest) and at different times after (post-exe) exercise (arrows).

 

Blood Samples

On the basis of preliminary experiments (results not shown) and on recommendations given by Wakefield et al. (50), a reliable method for sampling and handling of blood for TGF-{beta} determination was established. Blood samples were obtained before exercise, immediately after, and 6, 20, and 68 h after exercise (Fig. 1) by use of a wide-gauge butterfly needle (21-G) and removing the tourniquet immediately after needle insertion. Samples were collected in tubes containing 70 µl K3EDTA, immediately cooled on ice for 30 min, and then centrifuged at 3,000 g for 15 min, whereafter the supernatant was collected and frozen at -80°C until analysis. Blood used for determination of type I collagen metabolism markers (PICP and ICTP) was sampled as described above and collected in 10-ml vacutainers, allowed to coagulate for 30 min, and then centrifuged at 3,000 g for 15 min. The resulting supernatant (serum) was stored at -80°C until analysis. Hematocrit values were determined by the microhematocrit method at all times of blood sampling, and the measured blood concentrations of TGF-{beta}1, PICP, and ICTP were corrected for changes in plasma volume according to the Van Beaumont formula (48).

Sample Analysis

TGF-{beta}1. Samples were analyzed for TGF-{beta}1 by using a commercially available ELISA kit (DuoSet, R & D Systems, Minneapolis, MN). Representative dialysate samples were tested for the presence of active TGF-{beta}1, but the concentration was under the detection limit of the assay. Accordingly, all samples were activated by acidification to separate TGF-{beta}1 from its binding proteins, allowing for measurement of total TGF-{beta}1. For activation of plasma samples, 2.5 M acetic acid-10 M urea were used, and 1 M HCl was used for microdialysis samples (as recommended by the supplier). Activated samples were neutralized by use of 2.7 M NaOH-1 M HEPES for plasma samples and 1.2 M NaOH-0.5 M HEPES for microdialysis (as recommended by the supplier). Samples were loaded on ELISA plates immediately after neutralization. All samples were measured in duplicate, and samples obtained from each subject were measured in the same assay. The average coefficient of variation on duplicate measurements was found to be 2.0% (range 0.02–10.6%). The intra-assay precision was found to be 5.9% at 1,619 pg/ml (plasma), whereas the interassay precision was 7.6% at 1,724 pg/ml (plasma).

To evaluate the degree of degradation of TGF-{beta}1 in dialysate, we performed repeated measurements of samples, which were frozen and thawed between measurements (and left for up to 2 h at room temperature before refreezing); no change was observed over time in the TGF-{beta}1 level of these dialysate samples. This indicates that the degradation of TGF-{beta}1 in dialysate is negligible, at least at room temperature, and we would not expect a considerable degradation even at the slightly higher temperature of the dialysate when it is sampled from the subjects.

Type I collagen metabolism markers. Microdialysis and serum samples were analyzed for PICP and ICTP with a commercially available RIA kit (Orion Diagnostica, Espoo, Finland). Samples from each subject were measured in the same assay. According to the manufacturer, the detection level of the kit is 1.2 µg/l for PICP and 0.5 µg/l for ICTP. Intra-assay precision is given as 3.1% for PICP at 54 µg/l and 6.2% for ICTP at 3.8 µg/l, whereas the interassay precision is given as 5.8% for PICP at 52 µg/l and 7.9% for ICTP at 3.3 µg/l.

Statistics

All data are presented as means ± SE. When a significant difference was found over time (determined by the Friedman test), the Wilcoxon signed-rank test was used to compare paired observations. P < 0.05 was considered significant. Microdialysis results for TGF-{beta}1, PICP, and ICTP are given as the average of values observed in paired samples from the right and left leg of all subjects. No systematic significant difference was seen between legs with regards to the obtained values of TGF-{beta}1, PICP, and ICTP.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
Exercise-Induced Changes in TGF-{beta}1

To evaluate the influence of exercise on systemic and local TGF-{beta}1 levels, blood and dialysate samples (from peritendon tissue) were obtained from the participating subjects before, and at several times after, 1 h of uphill treadmill running (Fig. 1). A significant increase in plasma TGF-{beta}1, from 992 ± 49 pg/ml (at rest) to 1,301 ± 39 pg/ml, was found immediately after the exercise bout (P < 0.05), and levels tended to remain elevated at 6 h postexercise (1,114 ± 29 pg/ml), although this was not significant compared with the basal level (P = 0.08) (Fig. 2). In dialysate, no significant changes were seen between resting values of TGF-{beta}1 and the concentrations measured postexercise, although a value of 423 ± 86 pg/ml was observed 3 h postexercise compared with the resting value of 303 ± 46 pg/ml (P > 0.05) (Fig. 3). Analysis of successive microdialysis samples obtained during a 6-h recovery period after exercise revealed that the dialysate level of TGF-{beta}1 was unchanged until 4 h postexercise, whereafter a significant decrease was seen relative to the initial concentration at 0.5 h (from 509 ± 91 to 244 ± 91 pg/ml) (P < 0.05) (Fig. 4). In comparison, an ongoing significant decrease was found in a control experiment in which dialysate was obtained over 3.5 h in subjects who had not performed exercise (Fig. 5). Taking this into account, the constant TGF-{beta}1 level found in dialysate up to 4 h after exercise could indicate an exercise-induced increase in tissue TGF-{beta}1. This is further illustrated by the relative changes over time in dialysate levels of TGF-{beta}1 found in the postexercise recovery period compared with the relative changes seen at in the control experiment (Fig. 6).



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  		Fig. 2. Hematocrit-corrected concentrations of transforming growth factor-{beta}1 (TGF-{beta}1) in plasma before and after (post) exercise (n = 6). *P < 0.05. Error bars represent SE.

 


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  		Fig. 3. Concentrations of TGF-{beta}1 in dialysate obtained from the peritendinous tissue of the Achilles tendon before and after exercise. Values are based on average concentrations observed in paired dialysate samples (from right and left leg) obtained minimum 2 h after insertion of microdialysis probes (n = 6). Error bars represent SE.

 


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  		Fig. 4. Concentration of TGF-{beta}1 in dialysate samples obtained during 6 h of postexercise recovery (n = 6). *P < 0.05. Error bars represent SE.

 


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  		Fig. 5. Concentration of TGF-{beta}1 in dialysate obtained at different times after insertion of microdialysis catheters in resting subjects (control experiment) (n = 6). *P < 0.05. Error bars represent SE.

 


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  		Fig. 6. Relative changes over time in the concentration TGF-{beta}1 in dialysate sampled in the postexercise period of the exercise experiment (solid line) and in the control experiment (dotted line).

 

Changes in Type I Collagen Turnover

The concentration of PICP in dialysate was below the detection level (1.2 µg/l) in samples obtained before, immediately after, and 20 h after exercise, whereas a tissue level of 52 ± 12.6 µg/l was observed at 68 h postexercise (P < 0.05) (Fig. 7A). Tissue ICTP levels remained unchanged (P > 0.05) (Fig. 7B). In serum, PICP levels were significantly decreased 6 h after exercise (P < 0.05), whereas serum ICTP levels were significantly elevated immediately after exercise (P < 0.05) (Table 1).



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  		Fig. 7. Levels of procollagen I COOH-terminal propeptide (PICP; A) and COOH-terminal telopeptide of type I collagen (ICTP; B) in peritendinous tissue in response to uphill running. Values are based on the averages of paired observations (from right and left leg) corrected for relative recovery (n = 6). Levels of PICP were lower than the detection level in dialysate obtained before and at 3 and 20 h postexercise. *P < 0.05. Error bars represent SE.

 

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  		Table 1. Serum levels of type I collagen metabolism markers

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
The present study is to our knowledge the first to report detection of tissue TGF-{beta} by use of the microdialysis method. Employing high-molecular-mass cutoff microdialysis probes, we measured levels of TGF-{beta}1 in dialysate obtained from connective tissue in the peritendinous region of the human Achilles tendon before and after exercise, involving mechanical loading of the tendon. The magnitude of the changes in dialysate concentrations of TGF-{beta}1 after exercise, compared with the results obtained in control studies, indicates a possible exercise-induced increase in local TGF-{beta}1. This could play a role for the increase in local peritendinous type I collagen production observed 3 days after exercise. Furthermore, a postexercise increase in systemic TGF-{beta}1 concentrations indicates a release of the cytokine in response to exercise and may further support a role for TGF-{beta}1 in relation to exercise-induced collagen synthesis in regions that are subjected to mechanical loading.

The increase in formation of procollagen propeptides for type I collagen (PICP) indicates an increased accumulation of collagen in response to exercise and fits with the general view that mechanical loading influences ECM gene expression (7). Mechanically induced ECM remodeling has been observed in several tissue types, including bone (35) and cardiac tissue (52), and experiments with cell cultures and in vivo animal studies indicate that the tissue adaptations to mechanical loading consist in part of an increased type I collagen expression and/or production (3, 6, 19, 49, 53). A number of animal studies show an increase in total collagen content or collagen synthesis in tendon tissue in response to training (10, 51), whereas immobilization evidently results in a decreased collagen synthesis (23, 43) and loss of total collagen (20). Furthermore, human subjects performing frequent weight-bearing exercise have been found to have Achilles tendons with larger cross-sectional areas than normal control subjects (41, 54).

Recent microdialysis experiments, along with the present study, support the existence of an adaptation response in human tendon tissue in vivo. In response to both long-term strength training and acute long distance running (36 km), Langberg and coworkers (27, 29) found a local PICP increase in Achilles peritendon tissue independent of changes in circulating PICP levels, and, given that no changes were found in local ICTP levels, these results point toward an exercise-induced net increase in the local tendinous production of type I collagen. In the present study we saw a similar reaction, although the increase in tissue PICP in response to 1 h of uphill running was moderate compared with the response found to 36 km of running by Langberg et al. (29). This could indicate a doseresponse relationship in the exercise-induced local tendinous collagen production. The mechanisms linking mechanical loading to type I collagen synthesis remain to be explained in detail, and in the present study it was investigated whether TGF-{beta}1 is involved in exercise-induced type I collagen production in human tendon tissue in vivo. In vitro studies with human cell cultures show stretch-induced TGF-{beta}1 expression in tendon, ligament, and cardiac fibroblasts (24, 42, 45), smooth muscle cells (18, 38), and osteoblast-like cells (8); also in vivo and in vitro animal studies support a connection between mechanical strain and TGF-{beta} expression (9, 30, 32, 49, 55). Moreover, studies indicate that mechanically induced type I collagen synthesis can be ablated by inhibiting TGF-{beta}1 activity (18, 24, 33). In conflict with the hypothesis of TGF-{beta}1 as a mediator of collagen synthesis in tendon tissue, Fenwick and coworkers (13) were not able to detect the TGF-{beta} receptor I or TGF-{beta}1 in human Achilles tendon tissue. However, the normal tendon specimens analyzed by Fenwick et al. were from cadavers of very elderly people and from nondiseased parts of tendon from patients with chronic Achilles tendinopathy, and thus it must be assumed that such tendon tissue has not been subjected to a normal degree of mechanical loading. This may influence the presence of both TGF-{beta}1 and receptor I, because human and animal ex vivo and/or in vitro studies indicate that TGF-{beta}1 and its receptors are in fact present in tendon tissue and that their degree of expression is highly variable and inducible (15, 34, 37). Furthermore, in vitro studies with animal tenocytes show a marked response to stimulation with TGF-{beta}1, {beta}2, and {beta}3, indicating that the TGF-{beta} signaling is successful and that the TGF-{beta} receptors are present in these cells (2, 25, 40).

On the basis of the results obtained in the present study we cannot be fully conclusive with regard to TGF-{beta}1's role in exercise-induced type I collagen production in tendon tissue, because several methodological complications are related to microdialysis measurement of local TGF-{beta}1. Especially the large fluctuations in basal dialysate levels of TGF-{beta}1, presumably caused by initial trauma-induced platelet release of TGF-{beta}1, represent a problem. When analyzing the exercise-induced changes in local TGF-{beta}1, it is clear that these could be masked by changes caused exclusively by the microdialysis intervention itself. In view of the ongoing decrease in dialysate levels of TGF-{beta}1 seen at rest after insertion of the catheters, the comparatively stable level of dialysate TGF-{beta}1 observed in the postexercise recovery period could well indicate a relative exercise-induced increase in the local TGF-{beta}1 concentration as expressed by the difference in values between the two curves shown in Fig. 6.

The fact that no estimation was made of the actual tissue concentration of TGF-{beta}1, owing to the lack of a suitable tracer for estimation of the in vivo recovery, could represent an additional bias in the investigation of exercise-induced changes in local TGF-{beta}1. However, all microdialysis measurements were done on subjects in the resting state, and therefore it would be expected that the recovery of TGF-{beta}1 was relatively constant. This is so because relative recovery of several other substances, e.g., PGE2, glucose, and lactate, when determined in relation to exercise, has been found to remain constant during rest periods before and after exercise, whereas changes in recovery are seen when measurements are performed during exercise (28).

The increase found in circulating TGF-{beta}1 levels in response to exercise could, along with the microdialysis findings, support a role for the cytokine in relation to exercise-induced changes in type I collagen synthesis. Meanwhile, the time lapse between the increase in TGF-{beta}1 and the increase in local PICP may indicate alternative signaling pathways, possibly involving other growth factors, or at least the contribution of a downstream mediator of TGF-{beta}1. Studies indicate that connective tissue growth factor, acting downstream of TGF-{beta}1, is largely responsible for TGF-{beta}1-mediated type I collagen expression in fibroblasts (12, 17), and such a two-step signaling cascade could explain the latency in collagen production. In line with this, type I collagen mRNA levels have been shown to reach maximum as late as 24 h after stimulation with TGF-{beta}1 in human fibroblasts (14). Considering this, the latency in activation of type I collagen metabolism does not rule out the involvement of TGF-{beta}1. Moreover, it should be considered that potential changes in the expression of, e.g., TGF-{beta} receptors and procollagen C-proteinase, which cleaves PICP from procollagen (31) could influence the timing of type I collagen expression and accumulation.

In view of the strong relationship between mechanical loading and TGF-{beta}1 synthesis seen in various cell types (3, 8, 9, 38, 45, 53), the increase in circulating levels of TGF-{beta}1 may indicate a release of TGF-{beta}1 from tissues that are mechanically loaded during exercise, including bone, muscle, tendon, and perhaps cardiac and vascular tissues. Considering the tendency to an increase in dialysate-TGF-{beta}1 after exercise in the present study, the exercise-induced increase in systemic TGF-{beta}1 could well be a reflection of locally synthesized TGF-{beta}1 acting in an autocrine manner to induce collagen synthesis. Alternatively, it is possible that the increased plasma TGF-{beta}1 concentration is caused by exercise-induced platelet release of TGF-{beta}1, because prolonged exercise has been shown to induce platelet activation (11). Such a platelet activation could be linked to local exercise-induced tissue damage, and in this manner TGF-{beta}1 may function to induce local type I collagen synthesis in relation to mechanical loading during exercise. This hypothesis is supported by in vivo studies that show immediate increases in levels of TGF-{beta}1 in rat muscle in response to endurance exercise (4, 16). On the basis of the present study it cannot be concluded whether the increase in plasma TGF-{beta}1, seen in response to exercise, reflects a release of the cytokine from platelet {alpha}-granules or whether it originates from an increased production in tissues subjected to mechanical loading during exercise. The present study is the first to report an increase in plasma-TGF-{beta}1 in response to acute exercise, although pre- and postexercise levels of TGF-{beta}1 have been measured earlier. Suzuki et al. (46) found a small rise in TGF-{beta}1 in response to a marathon race, but the change did not obtain statistical significance. The high basal level of TGF-{beta}1 observed by Suzuki and coworkers (~13 ng/ml) could indicate that plasma samples were contaminated with TGF-{beta}1 from platelet granules because of inefficient inhibition of platelet activation (50). Such a contamination could well mask a significant increase in response to a marathon race, because a moderate increase in plasma-TGF-{beta}1, like the one observed in the present study of 0.3 ng/ml, could easily be overlooked in samples containing amounts of TGF-{beta}1 in the range of 13 ng/ml. Results reported by Hering et al. (21) support the possibility of an exercise-induced increase in TGF-{beta}1, because they showed an increase in plasma-TGF-{beta}1 after 14 and 21 days of strength training.

Taken together, the present study shows an increase in circulating levels of TGF-{beta}1 in response to acute exercise and indicates a local elevation of TGF-{beta}1 in the peritendinous tissue of the Achilles tendon. The TGF-{beta}1 response is followed by an increase in indicators of local type I collagen formation. Although microdialysis measurement of TGF-{beta}1 is novel and associated with technical difficulties, these findings suggest that TGF-{beta}1 may play a role in initiation of local peritendinous collagen formation after mechanical loading of tendon structures in humans.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
ACKNOWLEDGMENTS
 REFERENCES
 
This study was supported by grants from the Danish Medical Research Council (22010154), Team Denmark, Ministry of Culture (Sports Research Foundation), and the Danish National Research Foundation (504-14).


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Annie Hoej and Birgitte Lillethorup for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Heinemeier, Sports Medicine Research Unit, Bispebjerg hospital-Bldg. 8, 1st floor, 23 Bispebjerg Bakke, DK-2400 Copenhagen NV, Denmark (E-mail: kh08{at}bbh.hosp.dk).

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Aastrand P and Rodahl K. Textbook of Work Physiology: Physiological Bases of Exercise. New York: McGraw-Hill, 1986.
  2. Arai K, Kasashima Y, Kobayashi A, Kuwano A, and Yoshihara T. TGF-beta alters collagen XII and XIV mRNA levels in cultured equine tenocytes. Matrix Biol 21: 243–250, 2002.[ISI][Medline]
  3. Breen EC. Mechanical strain increases type I collagen expression in pulmonary fibroblasts in vitro. J Appl Physiol 88: 203–209, 2000.[Abstract/Free Full Text]
  4. Breen EC, Johnson EC, Wagner H, Tseng HM, Sung LA, and Wagner PD. Angiogenic growth factor mRNA responses in muscle to a single bout of exercise. J Appl Physiol 81: 355–361, 1996.[Abstract/Free Full Text]
  5. Brown JC and Timpl R. The collagen superfamily. Int Arch Allergy Immunol 107: 484–490, 1995.[ISI][Medline]
  6. Butt RP and Bishop JE. Mechanical load enhances the stimulatory effect of serum growth factors on cardiac fibroblast procollagen synthesis. J Mol Cell Cardiol 29: 1141–1151, 1997.[ISI][Medline]
  7. Chiquet M. Regulation of extracellular matrix gene expression by mechanical stress. Matrix Biol 18: 417–426, 1999.[ISI][Medline]
  8. Cillo JE Jr, Gassner R, Koepsel RR, and Buckley MJ. Growth factor and cytokine gene expression in mechanically strained human osteoblast-like cells: implications for distraction osteogenesis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 90: 147–154, 2000.[ISI][Medline]
  9. Cucina A, Sterpetti AV, Borrelli V, Pagliei S, Cavallaro A, and D'Angelo LS. Shear stress induces transforming growth factor-beta 1 release by arterial endothelial cells. Surgery 123: 212–217, 1998.[ISI][Medline]
  10. Curwin SL, Vailas AC, and Wood J. Immature tendon adaptation to strenuous exercise. J Appl Physiol 65: 2297–2301, 1988.[Abstract/Free Full Text]
  11. Drygas WK. Changes in blood platelet function, coagulation, and fibrinolytic activity in response to moderate, exhaustive, and prolonged exercise. Int J Sports Med 9: 67–72, 1988.[ISI][Medline]
  12. Duncan MR, Frazier KS, Abramson S, Williams S, Klapper H, Huang X, and Grotendorst GR. Connective tissue growth factor mediates transforming growth factor beta-induced collagen synthesis: down-regulation by cAMP. FASEB J 13: 1774–1786, 1999.[Abstract/Free Full Text]
  13. Fenwick SA, Curry V, Harrall RL, Hazleman BL, Hackney R, and Riley GP. Expression of transforming growth factor-beta isoforms and their receptors in chronic tendinosis. J Anat 199: 231–240, 2001.[ISI][Medline]
  14. Fine A and Goldstein RH. Regulation of type I collagen mRNA translation by TGF-beta. Reg Immunol 5: 218–224, 1993.[Medline]
  15. Fu SC, Wang W, Pau HM, Wong YP, Chan KM, and Rolf CG. Increased expression of transforming growth factor-beta1 in patellar tendinosis. Clin Orthop 400: 174–183, 2002.
  16. Gavin TP and Wagner PD. Effect of short-term exercise training on angiogenic growth factor gene responses in rats. J Appl Physiol 90: 1219–1226, 2001.[Abstract/Free Full Text]
  17. Grotendorst GR. Connective tissue growth factor: a mediator of TGF-beta action on fibroblasts. Cytokine Growth Factor Rev 8: 171–179, 1997.[Medline]
  18. Gutierrez JA and Perr HA. Mechanical stretch modulates TGF-{beta}1 and {alpha}1(I) collagen expression in fetal human intestinal smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 277: G1074–G1080, 1999.[Abstract/Free Full Text]
  19. Harter LV, Hruska KA, and Duncan RL. Human osteoblast-like cells respond to mechanical strain with increased bone matrix protein production independent of hormonal regulation. Endocrinology 136: 528–535, 1995.[Abstract]
  20. Harwood FL and Amiel D. Differential metabolic responses of periarticular ligaments and tendon to joint immobilization. J Appl Physiol 72: 1687–1691, 1992.[Abstract/Free Full Text]
  21. Hering S, Jost C, Schulz H, Hellmich B, Schatz H, and Pfeiffer H. Circulating transforming growth factor beta1 (TGFbeta1) is elevated by extensive exercise. Eur J Appl Physiol 86: 406–410, 2002.[ISI][Medline]
  22. Huijing PA. Muscle as a collagen fiber reinforced composite: a review of force transmission in muscle and whole limb. J Biomech 32: 329–345, 1999.[ISI][Medline]
  23. Karpakka J, Vaananen K, Virtanen P, Savolainen J, Orava S, and Takala TE. The effects of remobilization and exercise on collagen biosynthesis in rat tendon. Acta Physiol Scand 139: 139–145, 1990.[ISI][Medline]
  24. Kim SG, Akaike T, Sasagaw T, Atomi Y, and Kurosawa H. Gene expression of type I and type III collagen by mechanical stretch in anterior cruciate ligament cells. Cell Struct Funct 27: 139–144, 2002.[ISI][Medline]
  25. Klein MB, Yalamanchi N, Pham H, Longaker MT, and Chang J. Flexor tendon healing in vitro: effects of TGF-beta on tendon cell collagen production. J Hand Surg [Am] 27: 615–620, 2002.[Medline]
  26. Langberg H, Olesen JL, Gemmer C, and Kjaer M. Substantial elevation of interleukin-6 concentration in peritendinous tissue, in contrast to muscle, following prolonged exercise in humans. J Physiol 542: 985–990, 2002.[Abstract/Free Full Text]
  27. Langberg H, Rosendal L, and Kjaer M. Training-induced changes in peritendinous type I collagen turnover determined by microdialysis in humans. J Physiol 534: 297–302, 2001.[Abstract/Free Full Text]
  28. Langberg H, Skovgaard D, Karamouzis M, Bulow J, and Kjaer M. Metabolism and inflammatory mediators in the peritendinous space measured by microdialysis during intermittent isometric exercise in humans. J Physiol 515: 919–927, 1999.[Abstract/Free Full Text]
  29. Langberg H, Skovgaard D, Petersen LJ, Bulow J, and Kjaer M. Type I collagen synthesis and degradation in peritendinous tissue after exercise determined by microdialysis in humans. J Physiol 521: 299–306, 1999.[Abstract/Free Full Text]
  30. Lee AA, Delhaas T, McCulloch AD, and Villarreal FJ. Differential responses of adult cardiac fibroblasts to in vitro biaxial strain patterns. J Mol Cell Cardiol 31: 1833–1843, 1999.[ISI][Medline]
  31. Lee S, Solow-Cordero DE, Kessler E, Takahara K, and Greenspan DS. Transforming growth factor-beta regulation of bone morphogenetic protein-1/procollagen C-proteinase and related proteins in fibrogenic cells and keratinocytes. J Biol Chem 272: 19059–19066, 1997.[Abstract/Free Full Text]
  32. Li Q, Muragaki Y, Hatamura I, Ueno H, and Ooshima A. Stretch-induced collagen synthesis in cultured smooth muscle cells from rabbit aortic media and a possible involvement of angiotensin II and transforming growth factor-beta. J Vasc Res 35: 93–103, 1998.[ISI][Medline]
  33. Lindahl GE, Chambers RC, Papakrivopoulou J, Dawson SJ, Jacobsen MC, Bishop JE, and Laurent GJ. Activation of fibroblast procollagen alpha 1(I) transcription by mechanical strain is TGF-beta dependent and involves increased binding of CCAAT-binding factor (CBF/NF-Y) at the proximal promoter. J Biol Chem 277: 6153–6161, 2001.
  34. Marchuk L, Sciore P, Reno C, Frank CB, and Hart DA. Postmortem stability of total RNA isolated from rabbit ligament, tendon and cartilage. Biochim Biophys Acta 1379: 171–177, 1998.[Medline]
  35. Meyer U, Wiesmann HP, Meyer T, Schulze-Osthoff D, Jasche J, Kruse-Losler B, and Joos U. Microstructural investigations of strain-related collagen mineralization. Br J Oral Maxillofac Surg 39: 381–389, 2001.[ISI][Medline]
  36. Michna H and Hartmann G. Adaptation of tendon collagen to exercise. Int Orthop 13: 161–165, 1989.[ISI][Medline]
  37. Ngo M, Pham H, Longaker MT, and Chang J. Differential expression of transforming growth factor-beta receptors in a rabbit zone II flexor tendon wound healing model. Plast Reconstr Surg 108: 1260–1267, 2001.[ISI][Medline]
  38. O'Callaghan CJ and Williams B. Mechanical strain-induced extracellular matrix production by human vascular smooth muscle cells: role of TGF-beta(1). Hypertension 36: 319–324, 2000.[Abstract/Free Full Text]
  39. Perrone CE, Fenwick-Smith D, and Vandenburgh HH. Collagen and stretch modulate autocrine secretion of insulin-like growth factor-1 and insulin-like growth factor binding proteins from differentiated skeletal muscle cells. J Biol Chem 270: 2099–2106, 1995.[Abstract/Free Full Text]
  40. Robbins JR, Evanko SP, and Vogel KG. Mechanical loading and TGF-beta regulate proteoglycan synthesis in tendon. Arch Biochem Biophys 342: 203–211, 1997.[ISI][Medline]
  41. Rosager S, Aagaard P, Dyhre-Poulsen P, Neergaard K, Kjaer M, and Magnusson SP. Load-displacement properties of the human triceps surae aponeurosis and tendon in runners and non-runners. Scand J Med Sci Sports 12: 90–98, 2002.[ISI][Medline]
  42. Ruwhof C, van Wamel AE, Egas JM, and van der Laarse A. Cyclic stretch induces the release of growth promoting factors from cultured neonatal cardiomyocytes and cardiac fibroblasts. Mol Cell Biochem 208: 89–98, 2000.[ISI][Medline]
  43. Savolainen J, Myllyla V, Myllyla R, Vihko V, Vaananen K, and Takala TE. Effects of denervation and immobilization on collagen synthesis in rat skeletal muscle and tendon. Am J Physiol Regul Integr Comp Physiol 254: R897–R902, 1988.[Abstract/Free Full Text]
  44. Scheller D and Kolb J. The internal reference technique in microdialysis: a practical approach to monitoring dialysis efficiency and to calculating tissue concentration from dialysate samples. J Neurosci Methods 40: 31–38, 1991.[ISI][Medline]
  45. Skutek M, van Griensven M, Zeichen J, Brauer N, and Bosch U. Cyclic mechanical stretching modulates secretion pattern of growth factors in human tendon fibroblasts. Eur J Appl Physiol 86: 48–52, 2001.[ISI][Medline]
  46. Suzuki K, Yamada M, Kurakake S, Okamura N, Yamaya K, Liu Q, Kudoh S, Kowatari K, Nakaji S, and Sugawara K. Circulating cytokines and hormones with immunosuppressive but neutrophil-priming potentials rise after endurance exercise in humans. Eur J Appl Physiol 81: 281–287, 2000.[ISI][Medline]
  47. Swartz MA, Tschumperlin DJ, Kamm RD, and Drazen JM. Mechanical stress is communicated between different cell types to elicit matrix remodeling. Proc Natl Acad Sci USA 98: 6180–6185, 2001.[Abstract/Free Full Text]
  48. Van Beaumont W, Strand JC, Petrofsky JS, Hipskind SG, and Greenleaf JE. Changes in total plasma content of electrolytes and proteins with maximal exercise. J Appl Physiol 34: 102–106, 1973.[Free Full Text]
  49. Villarreal FJ and Dillmann WH. Cardiac hypertrophy-induced changes in mRNA levels for TGF-beta 1, fibronectin, and collagen. Am J Physiol Heart Circ Physiol 262: H1861–H1866, 1992.[Abstract/Free Full Text]
  50. Wakefield LM, Letterio JJ, Chen T, Danielpour D, Allison RS, Pai LH, Denicoff AM, Noone MH, Cowan KH, O'Shaughnessy JA, and. Transforming growth factor-beta1 circulates in normal human plasma and is unchanged in advanced metastatic breast cancer. Clin Cancer Res 1: 129–136, 1995.[Abstract]
  51. Woo SL, Ritter MA, Amiel D, Sanders TM, Gomez MA, Kuei SC, Garfin SR, and Akeson WH. The biomechanical and biochemical properties of swine tendons—long term effects of exercise on the digital extensors. Connect Tissue Res 7: 177–183, 1980.[ISI][Medline]
  52. Yamamoto K, Masuyama T, Sakata Y, Nishikawa N, Mano T, Yoshida J, Miwa T, Sugawara M, Yamaguchi Y, Ookawara T, Suzuki K, and Hori M. Myocardial stiffness is determined by ventricular fibrosis, but not by compensatory or excessive hypertrophy in hypertensive heart. Cardiovasc Res 55: 76–82, 2002.[Abstract/Free Full Text]
  53. Yasuda T, Kondo S, Homma T, and Harris RC. Regulation of extracellular matrix by mechanical stress in rat glomerular mesangial cells. J Clin Invest 98: 1991–2000, 1996.[ISI][Medline]
  54. Ying M, Yeung E, Li B, Li W, Lui M, and Tsoi CW. Sonographic evaluation of the size of achilles tendon: the effect of exercise and dominance of the ankle. Ultrasound Med Biol 29: 637–642, 2003.[ISI][Medline]
  55. Zheng W, Seftor EA, Meininger CJ, Hendrix MJ, and Tomanek RJ. Mechanisms of coronary angiogenesis in response to stretch: role of VEGF and TGF-beta. Am J Physiol Heart Circ Physiol 280: H909–H917, 2001.[Abstract/Free Full Text]



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