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Departments of 1Exercise and Sport Science, 2Physiology, and 3Surgery, and 4Human Performance Laboratory, East Carolina University, Greenville, North Carolina
Submitted 29 April 2005 ; accepted in final form 11 July 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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kinase insert domain-containing receptor; Flt-1; fiber cross-sectional area; vascular endothelial growth factor
O2 max), insulin sensitivity, and FCSA in humans (27, 28). To our knowledge, in aged women (AW) compared with young women (YW), there is only a single report demonstrating lower overall skeletal muscle (gastrocnemius) capillarization (13) and no report on fiber type-specific effects of aging on muscle capillarization. Thus the effects of aging on capillarization in muscles other than the gastrocnemius and on fiber type-specific capillarization in women are unknown.
It is well known that endurance exercise training promotes skeletal muscle angiogenesis in humans (5, 7, 12, 28). Endurance exercise training can also increase muscle capillarization in aged humans (12, 28, 42). However, debate exists on whether the angiogenic response to endurance exercise training is similar between young and aged humans. In response to a 20-wk endurance-training program (1 h/day; 4 days/wk at 70–80% O2 max), young men (22 yr) demonstrated a significant increase in the number of capillaries in contact with both type I and IIa fibers (14, 42). In contrast, aged men (62 yr) did not demonstrate an increase in capillary contacts (CC) with any fiber type (14), suggesting that the angiogenic response to exercise training may be at least attenuated in aged muscle. This study by Denis et al. (14) included aged men who were fairly active at the beginning of the study; thus results from this study must be interpreted with caution. A subsequent study by this same group did demonstrate significant increases in individual C/F of type I and IIa fibers, but not type IId/x fibers, in response to 14 wk of endurance training in aged men (8). Proctor et al. (42) demonstrated similar C/F surrounding type I but lower C/F surrounding type IIA and IIB fibers in aged compared with young untrained and trained men.
In young mouse skeletal muscle, inhibition of endogenous vascular endothelial growth factor (VEGF) production reduces the skeletal muscle capillary supply by 64% and promotes skeletal muscle cell apoptosis demonstrating that VEGF is important in basal skeletal muscle capillary regulation, which in turn is important in the basal maintenance of skeletal muscle mass (51). In human skeletal muscle, acute exercise increases VEGF mRNA (20, 21, 26, 44), whereas exercise training increases VEGF protein levels (25, 26). In rats, inhibition of VEGF prevents increases in vessel density in response to exercise training, demonstrating that VEGF is important in skeletal muscle angiogenesis (3). Together, these data demonstrate that VEGF is important for the maintenance and expansion of skeletal muscle capillarization.
Aging lowers VEGF mRNA expression in breast tissue (22) and fibroblasts (37) in humans, whereas aging lowers VEGF protein in the kidney (36) and carotids bodies (16) in rats. In skeletal muscle, the VEGF mRNA response to the same relative intensity of acute resistance exercise is similar in young and aged men (35), whereas the VEGF mRNA and protein responses to severe ischemia are lower in aged mice (46). The VEGF mRNA response to the same relative intensity of acute aerobic exercise is preserved in 24- vs. 6-mo-old F344 rats (48). Whether muscle VEGF expression is lower at rest and in response to acute aerobic exercise in AW is unknown. Compared with YW, we hypothesized that AW would demonstrate 1) lower muscle capillarization in a fiber type-specific manner and 2) lower VEGF and VEGF receptor expression at rest and in response to acute exercise.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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O2 max and body composition.
O2 max was measured on an electronically braked cycle ergometer (Lode, Excaliber Sport, Groningen, The Netherlands) by open-circuit spirometry (True Max 2400, Parvo Medics, Salt Lake City, UT). The test began with a 5-min warm-up at 50 W for YW and 20 W for AW. After the warm-up, the workload was increased 25 W for YW and 20 W for AW every 2 min until volitional fatigue. Body density was determined via hydrostatic weighing, and body fat percent was determined from body density based on a two-compartment model.
Submaximal exercise and muscle biopsies.
At least 1 wk after the O2 max test, subjects completed 45 min of cycle ergometer exercise (25 min exercise, 5 min rest, and 20 min of exercise) at 50% O2 max. Before commencement of exercise and at 4 h postexercise, a muscle biopsy was obtained from the vastus lateralis for the measurement of mRNA and protein. The resting and postexercise muscle biopsy samples were obtained from alternate legs. Samples were stored at –80°C until analysis. A section of the resting biopsy sample was oriented in an optimal cutting temperature (OCT)-tragacanth mixture, frozen in liquid nitrogen-cooled isopentane, and stored at –80°C until processing for the measurement of muscle morphometry and capillarization.
The postexercise biopsy was obtained at 4 h postexercise, as we have previously shown that VEGF mRNA is increased to a similar level at 2 and 4 h after the completion of a single acute systemic cycle ergometer exercise bout in young men (20). Because it is possible that the VEGF mRNA response to acute exercise may be delayed in aged compared with young humans, we chose to obtain a single biopsy at 4 h and not 2 h postexercise.
Estradiol. Estradiol has been shown to modulate VEGF expression in human vascular smooth muscle cells (6). To minimize the potential for estradiol influences in the present study, YW were studied during the menses phase of the menstrual cycle. Six YW were taking an oral contraceptive and were studied during the low-estradiol phase of their oral contraceptive treatment. All AW were postmenopausal. Plasma estradiol was measured from a venous sample obtained before the submaximal exercise bout by a chemiluminescent enzyme immunoassay (Access, Beckman Coulter, Brea, CA).
Morphometric and morphological analysis. At –20°C, muscle tissue mounted in OCT-tragacanth was sectioned to a thickness of 10 µm on a cryostat, mounted on slides, and kept until fixation. Sections were stained for capillaries using the Rosenblatt method (47), which simultaneously provides fiber typing (type I and II) and capillary visualization. There is no difference in the number of capillaries visualized with frozen biopsy samples using the Rosenblatt technique and the number visualized in tissue sections prepared from perfusion-fixed muscle (29). In human skeletal muscle, type I fibers stain more intensely than type II fibers (10).
Muscle sections were viewed under a light microscope (Nikon 400), and a digital image was taken of the section (Nikon Coolpix 990), as previously described (20). Capillaries were quantified manually from the digital image on individual fibers in an artifact-free region. The following indexes were measured (28): 1) the number of capillaries around a fiber (CC), 2) the C/F on an individual-fiber basis, and 3) the number of fibers sharing each capillary (sharing factor). Capillary density (CD) was calculated by using the fiber as the reference space. Capillary-to-fiber perimeter exchange index was calculated as an estimate of the capillary-to-fiber surface area. Quantification of the capillaries was performed on at least 50 fibers by randomly selecting a fiber in an artifact-free region. FCSA and fiber perimeter were measured with the image-analysis system and commercial software (SigmaScan, Jandel Scientific) calibrated to transform the number of pixels (viewed on a computer monitor) into micrometers.
RNA isolation and Northern analysis.
Total cellular RNA was isolated from each sample, and standard Northern blot analysis was performed for VEGF, KDR, and Flt-1 mRNA as previously described (20). Blots were exposed to XAR-5 X-ray film (Eastman Kodak, New Haven, CT) by use of a Cronex Lightning Plus screen at –80°C. Autoradiographs were quantitated by densitometry within the linear range of signals and normalized to 
-actin mRNA levels.
Protein isolation and analysis. A portion of the muscle biopsy sample was homogenized in RIPA (1x PBS, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS with protease inhibitors) as previously described (20). Total protein was measured by bicinchoninic acid (Bio-Rad, Hercules, CA). For each sample, VEGF (from 50 µg of total protein), KDR (from 125 µg of total protein), and Flt-1 (from 50 µg of total protein) were measured in duplicate. Commercial VEGF, KDR, and Flt-1 ELISA kits were used according to the manufacturer's instructions (R&D Systems, Minneapolis, MN).
Statistical treatment.
For mRNA and protein, a two-way mixed-plot factorial analysis of variance (age x exercise level) was used. Following a significant F ratio, a Bonferroni post hoc analysis was used. Unpaired Student's t-tests were used to compare differences in all other variables between YW and AW. Due to technical difficulties, muscle morphometry was performed from only 10 YW and 8 AW. In three YW, both rest and 4-h postexercise mRNA samples were lost during isolation. Significance was established at P 
0.05 for all statistical sets, and data are reported are means + SD.
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RESULTS |
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50 yr older than YW (Table 1). Despite the difference in age, groups were well matched for height, weight, and body composition. As anticipated, O2 max was lower in AW compared with YW. Estradiol was not different between groups. Muscle morphology. There was no difference in type I FCSA between groups, but type II FCSA was smaller in AW compared with YW (Table 2). There was no difference in type I fiber percentage, but the area percentage of type I fibers was greater in AW compared with YW.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Subjects.
Groups were well matched for height, weight, and body composition. Consistent with previous reports, aerobic capacity was lower in AW (13). As groups, both YW and AW were in the lowest 10th percentile in aerobic capacity (1). These age-adjusted values are based on treadmill exercise, whereas cycle ergometry was used in the current report. O2 max is 3–5 ml O2·kg–1·min–1 lower during cycle ergometry compared with treadmill exercise (19). When adjusting for this difference, both YW and AW groups were in the 20th percentile for their age, respectively.
Aging and skeletal muscle capillarization.
Our finding of a loss of capillaries surrounding muscle fibers in the vastus lateralis of AW compared with YW is consistent with a previous finding in gastrocnemius muscle (13). It could be hypothesized that a greater loss of capillaries would be present in the vastus lateralis than the gastrocnemius since the gastrocnemius is used frequently during common everyday activities such as walking. However, citrate synthase activity is lowered by advanced age in gastrocnemius, but not the vastus lateralis, suggesting that more distal muscles may be more greatly affected by aging than more proximal muscles (33). In the present report, C/F on an individual-fiber basis and CC were 12% lower in AW, whereas Coggan et al. (13) reported an 35% reduction in C/F and CC. Thus differences in the magnitude of the loss of muscle capillaries between the present report and Coggan et al. could possibly be due to greater age-associated loss of muscle capillaries in more distal muscles
To our knowledge, the present findings are the first report demonstrating a fiber type-specific loss of muscle capillaries in AW. In men, aging results in an 25% loss of muscle capillaries surrounding type II fibers, with no significant loss surrounding type I fibers (42). Combined, these data suggest that type II fibers may be more susceptible to the age-associated loss of muscle capillaries than type I fibers. It should noted that, because of the nature of human muscle biopsies, our results could be influenced by fiber-type grouping in aged muscle (15).
Recent data suggests that muscle capillarization is well preserved in F344 x Brown Norway (31, 40) and F344 (48) rats with aging. Animal models are attractive in aging research in that genetic differences between animals are minimized and successful subject recruitment issues are not relevant. Several factors likely influence group results from human aging studies and make definitive statements more complex. These factors include differences in physical activity levels (9, 14, 34), differences in the number of men and women studied in each group (34), and the lack of statistical analyses (4, 23). Except for a single report (14), a review of the human aging literature suggests that muscle C/F is lower in aged compared with young subjects whether statistical differences were present or not (4, 9, 13, 23, 34, 41, 42). We are aware of only three reports that have adequately controlled participant enrollment to study sedentary young and aged individuals (13, 41, 42). In each of these reports, muscle C/F is statistically lower in aged compared with young subjects. In addition, Frontera et al. (18) demonstrated that, from the age of 65 to 77 yr, there is a 22% reduction in muscle capillarization without a loss of muscle FCSA. Therefore, the vast majority of data in humans suggests that aging lowers muscle capillarization, which is in contrast to recent reports in rats (31, 40, 48).
There are two prominent theories on the regulation of muscle capillarization. The first suggests that muscle capillarization is primarily scaled to fiber size (2), whereas the second suggests that muscle capillarization is scaled to metabolic demand (32). Although aging resulted in the loss of muscle capillaries, the magnitude of the loss was similar to the losses in FCSA and fiber perimeter such that there was no difference in either CD or capillary-to-fiber perimeter exchange index between YW and AW. It is impossible to determine from our data which mechanism predominates in the regulation of human muscle capillarization. It is interesting, however, that the relationship between muscle capillarization and muscle fiber size is maintained (no difference in CD). Many mechanisms have been proposed to contribute to age-associated sarcopenia, including motoneuron denervation, mitochondria-induced apoptosis, changes in anabolic hormones, and increased rates of proteolysis (recently reviewed in Refs. 15, 30, 39). Given that the loss of endogenous VEGF results in capillary rarefaction and, therefore, indirectly results in muscle nuclei apoptosis (a likely mechanism of muscle atrophy) in transgenic mice (51), an additional mechanism for age-associated sarcopenia could be that aging reduces VEGF, resulting in the loss of muscle capillaries, which in turn promotes muscle atrophy. This theory remains to be experimentally tested.
Aging, resting VEGF, and resting VEGF receptor expression. We had hypothesized that resting muscle VEGF expression would be lower in AW. In the present report, there was no difference in resting VEGF mRNA between young and aged muscle, but resting VEGF protein was 35% lower in aged muscle, suggesting that lower VEGF may contribute to lower muscle capillarization in AW. Lower VEGF protein and a concomitantly lower muscle capillarization are consistent with a recent report from Tang et al. (51) demonstrating that VEGF is important in basal maintenance of muscle capillarization, where inactivation of endogenous skeletal muscle VEGF production decreased capillarization by 64%. To further identify whether VEGF protein levels may in part regulate muscle capillarization, we analyzed the relationship between muscle capillarization and VEGF protein from individuals in the present report and our laboratory's previous report from lean and obese young men (21). Linear regression revealed a strong relationship between resting VEGF protein levels and overall CC, consistent with the suggestion that VEGF likely contributes to the regulation of muscle capillarization in humans (Fig. 4).
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The biological activity of VEGF occurs predominantly through binding of VEGF to the VEGF receptors KDR and Flt-1. It is currently hypothesized that most angiogenic activity occurs through KDR, whereas Flt-1 acts as a negative (angiostatic) regulator of angiogenesis (17). Lower capillarization in aged tissue therefore could be the result of either lower KDR or increased Flt-1 expression. There were no differences in KDR or Flt-1 mRNA and protein between young and aged muscle, suggesting that lower VEGF receptor expression does not play an important role in the lower capillarization observed in AW.
Aging, exercise, VEGF, and VEGF receptor expression. As hypothesized, the VEGF mRNA response to acute aerobic exercise was 50% lower in aged human muscle. It was also hypothesized that aging would reduce the VEGF receptor response to exercise; however, both KDR and Flt-1 mRNA were increased by exercise independent of age. Our finding that the VEGF mRNA response to acute exercise is lower in aged muscle is significant, as the angiogenic response to exercise training may be attenuated in aged compared with young human muscle (14, 42).
Exercise is a well-known stress to muscle that results in intracellular hypoxia (43, 45) and angiogenesis (5, 7, 12, 28). Consistent with this, acute exercise increases VEGF mRNA (20, 24, 44), whereas exercise training increases VEGF protein in human muscle (25, 26). Interestingly, the skeletal muscle VEGF mRNA response to resistance exercise is similar in young and aged men (35). The discrepancy between our data and those of Jozsi et al. (35) may likely reflect different VEGF regulatory mechanisms involved in resistance vs. aerobic exercise. A likely mediator of resistance exercise-induced VEGF mRNA expression may be activation of Akt by hypertrophic stimuli (50). Likewise, aerobic exercise-induced VEGF expression has been proposed to involve hypoxia (52). A lower VEGF mRNA response to ischemia-induced hypoxia has been observed in aged mouse muscle (46), whereas a similar muscle VEGF mRNA response to the same relative acute aerobic exercise intensity between young and aged F344 rats has been recently reported (48). Thus the age-attenuated VEGF mRNA response to the same relative aerobic exercise intensity appears to be unique to humans.
Several studies have demonstrated an angiogenic response to endurance exercise training in aged individuals (11, 12, 28, 42); however, these studies employed either a cross-sectional design of young and aged untrained and trained individuals (11, 42) or an experimental design utilizing aged individuals only (12, 28). In the only known report investigating the angiogenic response to the same relative intensity exercise training program in young and aged subjects, angiogenesis was observed in young but not aged individuals (14). These results must be viewed with some caution, because the aged individuals were fairly active before the initiation of the training. However, if the pattern of a lower VEGF mRNA response to acute exercise persists throughout an exercise-training program, our results of a lower VEGF mRNA response to the same relative exercise intensity are consistent with an aging-related impairment of the angiogenic response to exercise training.
In summary, we have demonstrated in the vastus lateralis muscle of AW that capillarization surrounding type II muscle fibers, resting VEGF protein, and the exercise-induced increase in VEGF mRNA are lower compared with YW. Whether a lower VEGF mRNA response to acute exercise contributes to a lower angiogenic response to exercise training in AW remains to be determined.
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GRANTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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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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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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O2 peak and the capillary supply to skeletal muscle. J Appl Physiol 82: 1305–1310, 1997.This article has been cited by other articles:
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  L. Leick, Y. Hellsten, J. Fentz, S. S. Lyngby, J. F. P. Wojtaszewski, J. Hidalgo, and H. Pilegaard PGC-1{alpha} mediates exercise-induced skeletal muscle VEGF expression in mice Am J Physiol Endocrinol Metab, July 1, 2009; 297(1): E92 - E103. [Abstract] [Full Text] [PDF]
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 J. Wong, L. Molyneaux, M. Constantino, S. M. Twigg, and D. K. Yue Timing Is Everything: Age of Onset Influences Long-Term Retinopathy Risk in Type 2 Diabetes, Independent of Traditional Risk Factors Diabetes Care, October 1, 2008; 31(10): 1985 - 1990. [Abstract] [Full Text] [PDF]
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 J. J. Steinle, S. Sharma, and V. C. Chin Normal Aging Involves Altered Expression of Growth Factors in the Rat Choroid J. Gerontol. A Biol. Sci. Med. Sci., February 1, 2008; 63(2): 135 - 140. [Abstract] [Full Text] [PDF]
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 M. Lindenberger and T. Lanne Decreased capillary filtration but maintained venous compliance in the lower limb of aging women Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3568 - H3574. [Abstract] [Full Text] [PDF]
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 M. J. Lyon, L. M. Steer, and L. T. Malmgren Stereological estimates indicate that aging does not alter the capillary length density in the human posterior cricoarytenoid muscle J Appl Physiol, November 1, 2007; 103(5): 1815 - 1823. [Abstract] [Full Text] [PDF]
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F. Mac Gabhann, J. W. Ji, and A. S. Popel VEGF gradients, receptor activation, and sprout guidance in resting and exercising skeletal muscle J Appl Physiol, February 1, 2007; 102(2): 722 - 734. [Abstract] [Full Text] [PDF]
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F. M. Gabhann and A. S. Popel Interactions of VEGF isoforms with VEGFR-1, VEGFR-2, and neuropilin in vivo: a computational model of human skeletal muscle Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H459 - H474. [Abstract] [Full Text] [PDF]
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 B. J. Behnke, R. D. Prisby, L. A. Lesniewski, A. J. Donato, H. M. Olin, and M. D. Delp Influence of ageing and physical activity on vascular morphology in rat skeletal muscle J. Physiol., September 1, 2006; 575(2): 617 - 626. [Abstract] [Full Text] [PDF]
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