Journal of Applied Physiology Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 100: 178-185, 2006. First published September 15, 2005; doi:10.1152/japplphysiol.00827.2005
8750-7587/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
100/1/178    most recent
00827.2005v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ryan, N. A.
Right arrow Articles by Gavin, T. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ryan, N. A.
Right arrow Articles by Gavin, T. P.

Lower skeletal muscle capillarization and VEGF expression in aged vs. young men

Nicholas A. Ryan,1,4 Kevin A. Zwetsloot,1,4 Lenna M. Westerkamp,1,4 Robert C. Hickner,1,2,4 Walter E. Pofahl,3 and Timothy P. Gavin1,2,4

Departments of 1Exercise and Sport Science, 2Physiology, and 3Surgery, and 4Human Performance Laboratory, East Carolina University, Greenville, North Carolina

Submitted 11 July 2005 ; accepted in final form 12 September 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Recently, we observed that muscle capillarization, vascular endothelial growth factor (VEGF) protein, and the VEGF mRNA response to acute exercise were lower in aged compared with young women (Croley AN, Zwetsloot KA, Westerkamp LM, Ryan NA, Pendergast aged men, Hickner RC, Pofahl WE, and Gavin TP. J Appl Physiol 99: 1875–1882, 2005). We hypothesized that similar age-related differences in muscle capillarization and VEGF expression would exist between young and aged men. Skeletal muscle biopsies were obtained from the vastus lateralis before and at 4 h after a submaximal exercise bout for the measurement of morphometry, capillarization, VEGF, KDR, and Flt-1 in seven aged (mean age 65 yr) and eight young (mean age 21 yr) sedentary men. In aged compared with young men, muscle capillary contacts and capillary-to-fiber perimeter exchange index were lower regardless of fiber type. Muscle VEGF mRNA and protein were lower in aged men both at rest and 4 h postexercise. Exercise increased muscle VEGF mRNA and protein and KDR mRNA independent of age group. There were no effects of exercise or age on muscle Flt-1 mRNA or protein or KDR protein. These results confirm that skeletal muscle capillarization and VEGF expression are lower in aged compared with young men.

KDR; Flt-1; fiber cross-sectional area

IN MEN, AGING LOWERS THE OVERALL number of capillaries surrounding skeletal muscle fibers (5, 8, 27, 30). Recently, we have observed that muscle capillary contacts (CC) surrounding type II, but not type I, fibers were lower in aged compared with young women (6). In normal fit and endurance-trained young and aged men, Proctor et al. (30) reported no difference in muscle capillarization of type I fibers but lower CC of type IIA and IIB fibers of aged compared with young men. However, the findings of Proctor et al. must be viewed with some caution because both sedentary and trained men were included in the statistical analysis. When examining the results of Proctor et al. exclusively from sedentary men (30), muscle CC was 18, 30, and 38% lower surrounding type I, IIA, and IIB fibers, respectively, in aged compared with young men. Thus the effect of aging on muscle fiber type-specific capillarization in sedentary men remains equivocal. Reductions in capillarization may be detrimental in aged muscle because skeletal muscle capillarization is an important determinant of maximal oxygen consumption (VO2 max), insulin sensitivity, and muscle fiber cross-sectional area (FCSA) (18, 19).

Vascular endothelial growth factor (VEGF) is a predominantly endothelial cell-specific, heparin-binding, 45-kDa homodimeric glycoprotein mitogen and is an important regulator of basal skeletal muscle capillarization as well as exercise-induced angiogenesis (1, 36). In women, aging lowers resting skeletal muscle VEGF protein and lowers the exercise-induced increase in VEGF mRNA in women (6). However, aged men may demonstrate greater maintenance of muscle capillarization than aged women (5, 30); therefore, VEGF expression may not be different between young and aged men. Consistent with this, the muscle VEGF mRNA response to acute resistance exercise is similar between young and aged men (21). Accordingly, the present study was designed to investigate whether fiber type-specific muscle capillarization is lower and whether muscle VEGF and VEGF receptor expression are lower at rest or in response to acute aerobic exercise in aged compared with young men.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Subjects.   Eight sedentary young men (range 19–25 yr) and seven sedentary aged men (range 62–72 yr) volunteered to participate in the study after receiving written and verbal explanations of the content and intent of the study in accordance with the University and Medical Center Institutional Review Board. All subjects were healthy nonsmokers, with no history of cardiopulmonary disease. Subject characteristics are listed in Table 1. Subjects were carefully prescreened to preclude participation by individuals with overt cardiovascular disease. Subjects taking medications for cardiovascular disease were excluded. Sedentary subjects were defined as participating in <1 h of strenuous physical activity per week.


View this table:
[in this window]
[in a new window]
  		Table 1. Subject characteristics

 
VO2 max and body composition.   VO2 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 125 W for young men and 50 W for aged men. After the warm-up, the workload was increased 25 W for young men or 20 W for aged men 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 VO2 max test, subjects completed 45 min of cycle ergometer exercise (25 min of exercise, 5 min of rest, and 20 min of exercise) at 50% of VO2 max. Before the commencement of exercise and at 4 h postexercise, muscle biopsies were obtained from the vastus lateralis. 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 optimum cutting temperature-tragacanth mixture, frozen in liquid nitrogen-cooled isopentane, and stored at –80°C until processing for the measurement of muscle morphometry and capillarization.

RNA isolation and real-time PCR.   Approximately 10 mg of muscle were homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA). After addition of chloroform and phase separation, RNA was isolated from the aqueous phase by use of an RNeasy fibrous tissue mini kit (Qiagen, Valencia, CA). RNA was quantified fluorometrically using RiboGreen RNA quantitation kit (Molecular Probes, Eugene, OR), and 150 ng were reverse transcribed into first-strand cDNA using MultiScribe RT in the high-capacity cDNA archive kit [Applied Biosystems (AB), Foster City, CA]. Real-time PCR was conducted on 25 ng of cDNA per reaction in 50-µl reaction volumes using TaqMan Universal PCR Master Mix with commercially available (AB) primer and probe sets for human VEGF (product no. Hs00173626_m1) and human KDR (product no. Hs00176676_m1) by use of FAM/TAMRA-labeled dye on an AB PRISM 7000 sequence detection system instrument and software. The Flt-1 primer and probe set was designed using Primer Express software (AB) that selects primer and probes optimized for use with AB system products (forward primer: 5'-AAGGCAAGAAACCAAGACTAGATAGC-3'; reverse primer: 5'-CCTCAACATCACTCAGACTTTTATCTTC-3'; probe: 5'-AAAGCTTTGCGAGCTCCGGCTTTC-3'). Real-time PCR was run for 1 cycle (50°C for 2 min, 95°C for 10 min) immediately followed by 40 cycles (95°C for 15 s, 60°C for 1 min). Fluorescence was measured after each of the repeated cycles. RNA samples were normalized to 18S rRNA (eukaryotic 18S PDAR primer-limited VIC/TAMARA, AB, product no. 4310893E) multiplexed during the analysis of each specific gene.

Protein isolation and analysis.   A 10-mg portion of the muscle biopsy sample was homogenized with a Dounce homogenizer in RIPA (1x PBS, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS with protease inhibitors). Total protein was measured by bicinchoninic acid (Bio-Rad Laboratories, Hercules, CA). Commercial VEGF (from 50 µg of total protein), KDR (from 125 µg of total protein), and Flt-1 (from 50 µg of total protein) ELISA kits were used according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN) as previously described (14).

Morphometry, morphology, and immunohistochemistry.   Muscle tissue from the resting biopsy was sectioned to a thickness of 10 µm on a cryostat, mounted on slides, and kept at –20°C until fixation. Serial sections were stained for capillaries using the double-stain technique (31) as modified by Porter et al. (29) and for fiber type by use of a myosin ATPase stain (3). The myosin ATPase stain identifies muscle fibers as either type I, IIA, or IIB fibers (3). Despite the fact that human skeletal muscle identified as type IIB by histochemical analysis actually express type IIx (35), we have identified fibers types using the myosin ATPase stain nomenclature used in the original work.

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 (13). Capillaries were quantified manually from the digital image on individual fibers. The following indexes were measured (19): 1) the number of capillaries around a fiber (CC), 2) the capillary-to-fiber ratio on an individual-fiber basis (C/Fi), and 3) the number of fibers sharing each capillary [sharing factor (SF)]. Capillary density (CD) was calculated by using the fiber as the reference space. Capillary-to-fiber perimeter exchange index (CFPE) was calculated as an estimate of the capillary-to-fiber surface area. Quantification of the capillary supply was performed on at least 50 fibers by randomly selecting a fiber in an artifact-free region. Fiber cross-sectional area (FCSA) and perimeter (FP) 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 from an image of the myosin ATPase stain.

Statistical treatment.   For muscle fiber characteristics, mRNA, and protein, a two-way mixed-plot factorial analysis of variance (age x fiber type or age x rest/exercise) 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 young men and aged men. Multiple linear regression was performed to identify relationships between variables. There was inadequate sample for muscle morphology in two young men. Significance was established at P ≤ 0.05 for all statistical sets, and data reported are means (SD).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Subject characteristics.   Aged men were ~45 yr older than young men (Table 1). Aged men had greater body mass and a higher percentage of body fat. As anticipated, aged men demonstrated a significantly lower VO2 max, even when expressed per kilogram of fat-free mass.

Muscle morphology.   There were no statistically significant differences in FCSA, FP, or fiber-type percent between young men and aged men (Fig. 1). FCSA and FP were greater in type IIA fibers compared with IIB fibers.



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 1. Fiber cross-sectional area (A), fiber perimeter (B), and fiber type percent (C) for type I, IIA, and IIB fibers in young and aged men. Values are means (SD); n = 6 for young and 7 for aged. *Significantly different from type IIB fibers, P ≤ 0.05.

 
CC and C/Fi of type IIA fibers were greater than CC and C/Fi of type I or IIB fibers, respectively (Fig. 2). In aged men compared with young men, CC, C/Fi, and CFPE were lower regardless of fiber type. There was no difference in SF between groups (data not shown). A significant relationship was identified between VO2 max and type I CFPE in aged but not young men (Fig. 3).



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 2. Capillary contacts (CC; A), individual capillary-to-fiber ratio (C/Fi; B), capillary-to-fiber perimeter exchange index (CFPE; C), and capillary density (CD; D) in young and aged men. CC, C/Fi, and CFPE were lower in aged compared with young men independent of fiber type. Values are means (SD); n = 6 for young and 7 for aged. *Significantly different from type I and IIB fibers, P ≤ 0.05.

 


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 3. Linear regression between maximal oxygen consumption (VO2 max) [per kg fat-free mass (FFM)] and type I CFPE for all subjects (A), young men only (B), and aged men only (C). VO2 max is significantly related to CFPE in aged men.

 
VEGF and VEGF receptor expression.   In Fig. 4, mRNA at rest and 4 h postexercise for VEGF, KDR, and Flt-1 are shown normalized to 18S rRNA. Muscle VEGF mRNA was lower in aged men regardless of rest/exercise and exercise increased muscle VEGF mRNA independent of age group. Systemic exercise increased muscle KDR mRNA similarly between young men and aged men, whereas no effect of acute exercise or age was observed for muscle Flt-1 mRNA.



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 4. Vascular endothelial growth factor (VEGF; A), KDR (B), and Flt-1 (C) mRNA in skeletal muscle of young and aged men at rest and 4 h postexercise. Young rest data were normalized to 1.0. All other data were normalized to young + rest to allow for comparisons. Exercise increased VEGF and KDR mRNA independent of group. VEGF mRNA was lower in aged men independent of rest/exercise. Values are means (SD); n = 8 for young and 7 for aged.

 
Muscle VEGF (A), KDR (B), and Flt-1 (C) proteins at rest and 4 h postexercise are shown in Fig. 5. Muscle VEGF protein was lower in aged men regardless of rest/exercise, and it was increased by exercise independent of age. There was no difference in muscle KDR or Flt-1 protein between groups or rest/exercise.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 5. Skeletal muscle protein for VEGF (A), KDR (B), and Flt-1 (C) at rest and 4 h postexercise. Exercise increased VEGF protein independent of group. VEGF protein was lower in aged men independent of rest/exercise. KDR and Flt-1 were unaffected by age or exercise. Values are means (SD); n = 8 for young and 7 for aged.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The principal findings of the present study from the vastus lateralis muscle of men are as follows: 1) CC, C/Fi, and CFPE are lower in aged compared with young regardless of fiber type; 2) VEGF mRNA and protein are lower in aged compared with young; and 3) VEGF protein is increased by acute exercise. To our knowledge, these are the first results demonstrating lower CC and CFPE of all three muscle fiber types in aged compared with young men, lower muscle VEGF expression in aged compared with young men, and an increase in muscle VEGF protein after a single acute exercise bout.

Aging and skeletal muscle capillarization.   There are several different measures of muscle capillarization. In the present report, we have performed different measurements in an effort to understand whether absolute capillary numbers (CC and C/Fi) and relative measures of capillarization (CD and CFPE) are different between young and aged sedentary men. In terms of absolute capillary numbers, muscle CC and C/Fi were ~25% lower in aged compared with young men independent of fiber type. Proctor et al. (30) reported no difference in CC surrounding type I fibers (9%) but statistically lower CC of both IIA (23%) and IIB (30%) fibers in aged compared with young men. The difference between the present findings and those of Proctor et al. likely results from differences in the populations studied. The present report analyzed only sedentary young and aged men, whereas Proctor et al. concurrently analyzed groups of sedentary and trained young and aged men. As a result, low power may have prevented finding a statistical difference in type I CC of the sedentary men (18% lower in aged sedentary men but similar between young and aged trained men) (30).

An interesting observation with respect to habitual exercise training is that training increases the number of capillaries surrounding type I, IIA, and IIB muscle fibers regardless of age (30). However, although exercise training may prevent the age-associated loss of muscle capillaries surrounding type I fibers, it does not prevent the loss of capillaries surrounding either type IIA or IIB fibers, suggesting that aging does lower angiogenic potential of type II muscle fibers (30). In elderly men, endurance exercise training promotes a greater relative increase in microvessel tortuosity than the increase in the number of capillaries surrounding muscle fibers (4). The greater relative increase in microvessel tortuosity in elderly men may be different from the response observed in young muscle (28) and may represent a compensatory mechanism with aging to improve muscle diffusive capacity (4).

Because aging results in the loss of capillaries, it should be questioned whether the magnitude of the loss in capillaries is similar to the magnitude of the loss in FCSA and FP such that relative measures of muscle capillarization are unchanged with aging. In the present report, CD was similar between young and aged men, whereas CFPE was lower. Results from previous reports are equivocal, with lower CD (5) and the maintenance of CD (27, 30) in aged compared with young men. To our knowledge, the present report is the first to demonstrate lower CFPE in aged men, suggesting that overall capillary loss is greater than the loss in muscle fiber perimeter, which would have detrimental effects on muscle diffusive capacity. Current evidence suggests that the size of the capillary-to-fiber interface is a major determinant of O2 flux (19, 24). CFPE is an estimate of the capillary-to-fiber interface (21). Consistent with this, we observed that type I CFPE was significantly related to VO2 max in aged men.

In our previous report, we observed that capillary contacts of type II, but not type I, fibers were lower in aged compared with young women (6). In an attempt to identify whether age-associated changes in muscle are different between men and women, we measured muscle fiber types and capillarization using the double-stain and myosin ATPase methods employed in the present report where possible in the women from our previous study to make direct comparisons between young and aged men and women (6). Six young and six aged women had sufficient sample remaining to perform the double stain and myosin ATPase stain. When young and aged men and women were analyzed by a two-way analysis of variance for each individual variable, there were no age x gender interactions identified (P > 0.200) except for a trend in type IIB CD where the interaction effect was P = 0.095. As a result, we analyzed the women separately by performing the same two-way analysis of variance (age x fiber type) as performed in the men. Aged women demonstrated smaller FCSA of type IIA and IIB fibers and smaller FP of type IIB fibers compared with young women (Fig. 6), which is consistent with the type II fiber atrophy observed in these women using the Rosenblatt stain but is in contrast to our results in aged compared with young men. Similar to differences observed between aged and young men, muscle CC, C/Fi, and CFPE were lower and CD was not different in aged compared with young women (Fig. 7). These results clearly demonstrate that muscle capillarization is lower in aged compared with young men and women whether muscle capillarization is reported by either absolute (CC and C/Fi) or relative (CFPE) measurements.



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 6. Fiber cross-sectional area (A), fiber perimeter (B), and fiber type percent (C) for type I, IIA, and IIB fibers from selected young and aged women from Croley et al. (6) using the myosin ATPase stain. Values are means (SD); n = 6 for young and aged. *Significantly different from all other values, P ≤ 0.05. # Significantly different from young type IIA, P ≤ 0.05.

 


View larger version (34K):
[in this window]
[in a new window]
  		Fig. 7. CC (A), C/Fi (B), CFPE (C), and CD (D) from selected young and aged women from Croley et al. (6) using the double stain. CC, C/Fi, and CFPE were lower in aged compared with young men independent of fiber type. Values are means (SD); n = 6 for young and aged. *Significantly different from type I and IIA fibers, P ≤ 0.05.

 
Aging, VEGF, and VEGF receptor expression.   In the present report, muscle VEGF mRNA and protein were lower in aged compared with young men both at rest and postexercise. Recently, we observed that resting muscle VEGF protein was lower in aged compared with young women, whereas resting muscle VEGF mRNA was similar between groups (6). From our present and previous work (6), it is clear that muscle VEGF protein is lower in aged men and women. We are unaware of any report of an age-related gender-based difference in VEGF mRNA in any tissue.

It is well established that acute exercise can increase VEGF mRNA (13, 14, 16, 32); however, this response is lower in aged compared with young women (6). The present finding of lower VEGF mRNA after acute aerobic exercise in men is consistent with our previous finding in women (6), but it is inconsistent with a similar VEGF mRNA response to acute resistance exercise in aged compared with young men (21). Jozsi et al. (21) reported a similar 2.7-fold resistance exercise-induced increase in VEGF mRNA in young and aged men. As speculated previously (6), the difference in our data and that of Jozsi et al. most likely reflects different intracellular signaling events between aerobic and resistance exercise. However, it should be noted that the data reported by Joszi et al. reflect fold changes and not absolute levels of VEGF mRNA, which may reflect less muscle VEGF mRNA in aged men.

In attempt to identify whether the age-associated differences in resting and exercise-induced muscle VEGF mRNA between the present findings and our previous report in women were the result of different techniques for measuring VEGF mRNA (Northern blot vs. real-time PCR), we measured VEGF mRNA using real-time PCR where possible from women in our previous study (6). Three young and three aged women had sufficient sample remaining to perform real-time PCR (Fig. 8). Although the relative increase in muscle VEGF mRNA with acute exercise measured using real-time PCR was approximately half as great as previously observed using Northern blot, our previous results of similar resting muscle VEGF mRNA and an attenuated muscle VEGF mRNA response to acute exercise in aged compared with young women are consistent between techniques. Therefore, the greatest difference in VEGF mRNA observed with aging between men and women is that aged men demonstrate lower resting muscle VEGF compared with young men, whereas VEGF mRNA postexercise in aged men and women is very similar compared with their respective young counterparts.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 8. VEGF mRNA in skeletal muscle of young and aged women men at rest and 4 h postexercise from our previous report (6). Young rest data were normalized to 1.0. All other data were normalized to young + rest to allow for comparisons. VEGF was significantly increased by exercise 4.1-fold in young women. In aged women, the VEGF mRNA response to acute exercise was significantly lower (2.3-fold increase). Values are means (SD); n = 3 for young and aged. *Significantly different from all other conditions, P ≤ 0.05. #Significantly different from young + rest and aged + rest, P ≤ 0.05.

 
In addition to an increase in muscle VEGF mRNA, we also observed a 15% increase in muscle VEGF protein in response to a single acute exercise bout. Previously, our laboratory reported a decrease in muscle VEGF protein immediately after the conclusion of acute exercise, with a return to baseline at 2 and 4 h postexercise (13). In additional reports, our laboratory did not observe an increase in muscle VEGF protein at either 2 h post-aerobic exercise in lean and obese young men (14) or 4 h postexercise in young and aged women (6). In these individuals, exercise increased muscle VEGF protein in ~60% of cases (6, 14). In the present report, muscle VEGF protein was increased in 11 of 15 (73%) individuals, which may explain our finding of a significant increase in muscle VEGF protein with acute exercise in the present report.

In similar fashion to our comparisons of muscle morphometry and VEGF mRNA between young and aged men and women, we wished to compare VEGF protein levels between the present report and our previous findings (6). However, VEGF protein is ~50% lower in samples homogenized using a Dounce compared with a Polytron homogenizer (unpublished observations), making direct comparisons of VEGF impossible between the two data sets. We have recently shown that, when isolated in a similar manner, VEGF protein is ~20% lower in young women compared with young men and that muscle capillarization is strongly related to muscle VEGF protein (6). Therefore, muscle VEGF is greatest in young men and lowest in aged women with muscle VEGF in young women and aged men in between these groups. It must be highlighted that muscle VEGF protein was significantly lower (~25%) in aged compared young men, which is consistent with our previous report of lower (~30%) muscle VEGF in aged compared with young women (6).

Aging lowers VEGF mRNA expression in breast tissue (15) and fibroblasts (23) in humans, lowers VEGF protein in the kidney (22) and carotid bodies (7) in rats, and lowers hypoxia-induced VEGF mRNA and protein expression in rabbit and mouse skeletal muscle and in rabbit smooth muscle cells (33, 34). The reduction in hypoxia-induced VEGF expression in aged smooth muscle cells results from lower binding activity of hypoxia-inducible factor-1 (34), a well-known transcriptional regulator of VEGF. It has been proposed that hypoxia regulates in part exercise-induced increases in muscle VEGF expression (37), and recent data suggest that acute exercise with restricted blood flow increases muscle hypoxia-inducible factor-1 binding activity in humans (2). Whether lower skeletal muscle VEGF expression in aged compared with young humans is due to lower hypoxia signaling remains to be elucidated.

There was no effect of age on either KDR or Flt-1 mRNA or protein. Given that capillary density is similar between young and aged, this result is not surprising and suggests that KDR and Flt-1 content within endothelial cells is similar in young and aged men and is consistent with findings in young and aged women (6). KDR mRNA was increased by acute exercise, whereas Flt-1 mRNA was not. Our laboratory (6, 13, 14) and others (17) have previously observed increases in both KDR and Flt-1 mRNA in response to acute exercise in humans. Consistent with this, several reports have shown an increase in Flt-1 mRNA with acute exercise in rats (9–12, 26), but an increase in Flt-1 mRNA with acute exercise has not always been observed in rats (27).

In summary, we have demonstrated that muscle capillarization is lower and that muscle VEGF expression is lower in aged compared with young men both at rest and after acute aerobic exercise. These results are consistent with lower muscle capillarization and muscle VEGF expression recently reported in aged compared with young women (6). Our data suggest that lower VEGF expression may play an important role in the lower muscle capillarization observed in aged compared with young men.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was supported by National Institute on Aging Grant AG-021891 (T. P. Gavin). R. C. Hickner was supported in part by National Institute on Aging Grants AG-18407 and AG-19209.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors thank Kathryn M. Verbanac, PhD, for assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. P. Gavin, 363 Ward Sports Medicine Bldg., East Carolina Univ., Greenville, NC 27858 (e-mail: gavint{at}mail.ecu.edu)

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
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Amaral SL, Papanek PE, and Greene AS. Angiotensin II and VEGF are involved in angiogenesis induced by short-term exercise training. Am J Physiol Heart Circ Physiol 281: H1163–H1169, 2001.[Abstract/Free Full Text]
  2. Ameln H, Gustafsson T, Sundberg CJ, Okamoto K, Jansson E, Poellinger L, and Makino Y. Physiological activation of hypoxia inducible factor-1 in human skeletal muscle. FASEB J. 19: 1009–1011, 2005.[Abstract/Free Full Text]
  3. Brooke MH and Kaiser KK. Three "myosin adenosine triphosphate" systems: the nature of their pH lability and sulfhydryl dependence. J Histochem Cytochem 18: 670–672, 1970.[Web of Science][Medline]
  4. Charifi N, Kadi F, Feasson L, Costes F, Geyssant A, and Denis C. Enhancement of microvessel tortuosity in the vastus lateralis muscle of old men in response to endurance training. J Physiol 554: 559–569, 2004.[Abstract/Free Full Text]
  5. Coggan AR, Spina RJ, King DS, Rogers MA, Brown M, Nemeth PM, and Holloszy JO. Histochemical and enzymatic comparison of the gastrocnemius muscle of young and elderly men and women. J Gerontol 47: B71–B76, 1992.
  6. Croley AN, Zwetsloot KA, Westerkamp LM, Ryan NA, Pendergast AM, Hickner RC, Pofahl WE, and Gavin TP. Lower capillarization, VEGF protein, and VEGF mRNA response to acute exercise in the vastus lateralis muscle of aged vs. young women. J Appl Physiol 99: 1875–1882, 2005.
  7. Di Giulio C, Bianchi G, Cacchio M, Macri MA, Ferrero G, Rapino C, Verratti V, Piccirilli M, and Artese L. Carotid body HIF-1alpha, VEGF and NOS expression during aging and hypoxia. Adv Exp Med Biol 536: 603–610, 2003.[Web of Science][Medline]
  8. Frontera WR, Hughes VA, Fielding RA, Fiatarone MA, Evans WJ, and Roubenoff R. Aging of skeletal muscle: a 12-yr longitudinal study. J Appl Physiol 88: 1321–1326, 2000.[Abstract/Free Full Text]
  9. Gavin TP, Spector DA, Wagner H, Breen EC, and Wagner PD. Effect of captopril on skeletal muscle angiogenic growth factor responses to exercise. J Appl Physiol 88: 1690–1697, 2000.[Abstract/Free Full Text]
  10. 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]
  11. Gavin TP and Wagner PD. Attenuation of the exercise-induced increase in skeletal muscle Flt-1 mRNA by nitric oxide synthase inhibition. Acta Physiol Scand 175: 201–209, 2002.[CrossRef][Web of Science][Medline]
  12. Gavin TP and Wagner PD. Acute ethanol increases angiogenic growth factor gene expression in rat skeletal muscle. J Appl Physiol 92: 1176–1182, 2002.[Abstract/Free Full Text]
  13. Gavin TP, Robinson CB, Yeager RC, England JA, Nifong LW, and Hickner RC. Angiogenic growth factor response to acute systemic exercise in human skeletal muscle. J Appl Physiol 96: 19–24, 2004.[Abstract/Free Full Text]
  14. Gavin TP, Stallings HW III, Zwetsloot KA, Westerkamp LM, Ryan NA, Moore RA, Pofahl WE, and Hickner RC. Lower capillary density but no difference in VEGF expression in obese vs. lean young skeletal muscle in humans. J Appl Physiol 98: 315–321, 2005.[Abstract/Free Full Text]
  15. Greb R, Maier I, Wallwiener D, and Kiesel L. Vascular endothelial growth factor A (VEGF-A) mRNA expression levels decrease after menopause in normal breast tissue but not in breast cancer lesions. Br J Cancer 81: 225–231, 1999.[CrossRef][Web of Science][Medline]
  16. Gustafsson T, Puntschart A, Kaijser L, Jansson E, and Sundberg CJ. Exercise-induced expression of angiogenesis-related transcription and growth factors in human skeletal muscle. Am J Physiol Heart Circ Physiol 276: H679–H685, 1999.[Abstract/Free Full Text]
  17. Gustafsson T, Ameln H, Fischer H, Sundberg CJ, Timmons JA, and Jansson E. VEGF-A splice variants and related receptor expression in human skeletal muscle following submaximal exercise. J Appl Physiol 98: 2137–2146, 2005.[Abstract/Free Full Text]
  18. Hedman A, Berglund L, Essen-Gustavsson B, Reneland R, and Lithell H. Relationships between muscle morphology and insulin sensitivity are improved after adjustment for intra-individual variability in 70-year-old men. Acta Physiol Scand 169: 125–132, 2000.[CrossRef][Web of Science][Medline]
  19. Hepple RT, Mackinnon SL, Goodman JM, Thomas SG, and Plyley MJ. Resistance and aerobic training in older men: effects on VO2 peak and the capillary supply to skeletal muscle. J Appl Physiol 82: 1305–1310, 1997.[Abstract/Free Full Text]
  20. Hepple RT and Mathieu-Costello O. Estimating the size of the capillary-to-fiber interface in skeletal muscle: a comparison of methods. J Appl Physiol 91: 2150–2156, 2001.[Abstract/Free Full Text]
  21. Jozsi AC, Dupont-Versteegden EE, Taylor-Jones JM, Evans WJ, Trappe TA, Campbell WW, and Peterson CA. Aged human muscle demonstrates an altered gene expression profile consistent with an impaired response to exercise. Mech Ageing Dev 120: 45–56, 2000.[CrossRef][Web of Science][Medline]
  22. Kang DH, Anderson S, Kim YG, Mazzalli M, Suga S, Jefferson JA, Gordon KL, Oyama TT, Hughes J, Hugo C, Kerjaschki D, Schreiner GF, and Johnson RJ. Impaired angiogenesis in the aging kidney: vascular endothelial growth factor and thrombospondin-1 in renal disease. Am J Kidney Dis 37: 601–611, 2001.[Web of Science][Medline]
  23. Kim H, Lee DK, Choi JW, Kim JS, Park SC, and Youn HD. Analysis of the effect of aging on the response to hypoxia by cDNA microarray. Mech Ageing Dev 124: 941–949, 2003.[CrossRef][Web of Science][Medline]
  24. Mathieu-Costello O, Ellis CG, Potter RF, MacDonald IC, and Groom AC. Muscle capillary-to-fiber perimeter ratio: morphometry. Am J Physiol Heart Circ Physiol 261: H1617–H1625, 1991.[Abstract/Free Full Text]
  25. Olfert IM, Breen EC, Mathieu-Costello O, and Wagner PD. Chronic hypoxia attenuates resting and exercise-induced VEGF, flt-1, and flk-1 mRNA levels in skeletal muscle. J Appl Physiol 90: 1532–1538, 2001.[Abstract/Free Full Text]
  26. Olfert IM, Breen EC, Mathieu-Costello O, and Wagner PD. Skeletal muscle capillarity and angiogenic mRNA levels after training in normoxia and chronic hypoxia. J Appl Physiol 91: 1176–1184, 2001.[Abstract/Free Full Text]
  27. Parizkova J, Eiselt E, Sprynarova S, and Wachtlova M. Body composition, aerobic capacity, and density of muscle capillaries in young and old men. J Appl Physiol 31: 323–325, 1971.[Free Full Text]
  28. Poole DC and Mathieu-Costello O. Relationship between fiber capillarization and mitochondrial volume density in control and trained rat soleus and plantaris muscles. Microcirculation 3: 175–186, 1996.[Medline]
  29. Porter MM, Stuart S, Boij M, and Lexell J. Capillary supply of the tibialis anterior muscle in young, healthy, and moderately active men and women. J Appl Physiol 92: 1451–1457, 2002.[Abstract/Free Full Text]
  30. Proctor DN, Sinning WE, Walro JM, Sieck GC, and Lemon PW. Oxidative capacity of human muscle fiber types: effects of age and training status. J Appl Physiol 78: 2033–2038, 1995.[Abstract/Free Full Text]
  31. Qu Z, Andersen JL, and Zhou S. Visualisation of capillaries in human skeletal muscle. Histochem Cell Biol 107: 169–174, 1997.[CrossRef][Web of Science][Medline]
  32. Richardson RS, Wagner H, Mudaliar SR, Henry R, Noyszewski EA, and Wagner PD. Human VEGF gene expression in skeletal muscle: effect of acute normoxic and hypoxic exercise. Am J Physiol Heart Circ Physiol 277: H2247–H2252, 1999.[Abstract/Free Full Text]
  33. Rivard A, Fabre JE, Silver M, Chen D, Murohara T, Kearney M, Magner M, Asahara T, and Isner JM. Age-dependent impairment of angiogenesis. Circulation 99: 111–120, 1999.[Abstract/Free Full Text]
  34. Rivard A, Berthou-Soulie L, Principe N, Kearney M, Curry C, Branellec D, Semenza GL, and Isner JM. Age-dependent defect in vascular endothelial growth factor expression is associated with reduced hypoxia-inducible factor 1 activity. J Biol Chem 275: 29643–29647, 2000.[Abstract/Free Full Text]
  35. Smerdu V, Karsch-Mizrachi I, Campione M, Leinwand L, and Schiaffino S. Type IIx myosin heavy chain transcripts are expressed in type IIb fibers of human skeletal muscle. Am J Physiol Cell Physiol 267: C1723–C1728, 1994.[Abstract/Free Full Text]
  36. Tang K, Breen EC, Gerber HP, Ferrara NM, and Wagner PD. Capillary regression in vascular endothelial growth factor-deficient skeletal muscle. Physiol Genomics 18: 63–69, 2004.[Abstract/Free Full Text]
  37. Wagner PD. Skeletal muscle angiogenesis. A possible role for hypoxia. Adv Exp Med Biol 502: 21–38, 2001.[Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. W. Ji, F. Mac Gabhann, and A. S. Popel
Skeletal muscle VEGF gradients in peripheral arterial disease: simulations of rest and exercise
Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3740 - H3749.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
T. P. Gavin, R. S. Ruster, J. A. Carrithers, K. A. Zwetsloot, R. M. Kraus, C. A. Evans, D. J. Knapp, J. L. Drew, J. S. McCartney, J. P. Garry, et al.
No difference in the skeletal muscle angiogenic response to aerobic exercise training between young and aged men
J. Physiol., November 15, 2007; 585(1): 231 - 239.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
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]

Home page
 Journals of Gerontology Series A: Biological Sciences and Medical SciencesHome page
D. Leosco, G. Rengo, G. Iaccarino, E. Sanzari, L. Golino, G. D. Lisa, C. Zincarelli, F. Fortunato, M. Ciccarelli, V. Cimini, et al.
Prior Exercise Improves Age-Dependent Vascular Endothelial Growth Factor Downregulation and Angiogenesis Responses to Hind-Limb Ischemia in Old Rats
J. Gerontol. A Biol. Sci. Med. Sci., May 1, 2007; 62(5): 471 - 480.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
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]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
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]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
100/1/178    most recent
00827.2005v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ryan, N. A.
Right arrow Articles by Gavin, T. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ryan, N. A.
Right arrow Articles by Gavin, T. P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.