Exercise training improves aging-induced downregulation of VEGF angiogenic signaling cascade in hearts
Abstract
Exercise training improves aging-induced deterioration of angiogenesis in the heart. However, the mechanisms underlying exercise-induced improvement of capillary density in the aged heart are unclear. Vascular endothelial growth factor (VEGF) is implicated in angiogenesis, which activated angiogenic signaling cascade through Akt and endothelial nitric oxide synthase (eNOS)-related pathway. We hypothesized that VEGF angiogenic signaling cascade in the heart contributes to a molecular mechanism of exercise training-induced improvement of capillary density in old age. With the use of hearts of sedentary young rats (4 mo old), sedentary aged rats (23 mo old), and exercise-trained aged rats (23 mo old, swim training for 8 wk), the present study investigated whether VEGF and VEGF-related angiogenic molecular expression in the aged heart is affected by exercise training. Total capillary density in the heart was significantly lower in the sedentary aged rats compared with the sedentary young rats, whereas that in the exercise-trained rat was significantly higher than the sedentary aged rats. The mRNA and protein expressions of VEGF and of fms-like tyrosine kinase-1 (Flt-1) and fetal liver kinase-1 (Flk-1), which are main VEGF receptors, in the heart were significantly lower in the sedentary aged rats compared with the sedentary young rats, whereas those in the exercise-trained rats were significantly higher than those in the sedentary aged rats. The phosphorylation of Akt protein and eNOS protein in the heart corresponded to the changes in the VEGF protein levels. These findings suggest that exercise training improves aging-induced downregulation of cardiac VEGF angiogenic signaling cascade, thereby contributing to the exercise training-induced improvement of angiogenesis in old age.
advanced age is a risk factor for cardiovascular disease. Aging-dependent decline in cardiac function, such as myocardial contraction and relaxation, causes an increased risk of cardiovascular morbidity (5, 30). Aging induces deterioration of energy supply with the suppression of angiogenic capacity in the heart (5, 14, 30). On the other hand, exercise training improves the aging-induced decrease in capillary density in the heart (4, 42). The exercise training-induced increase of capillary density may be a beneficial adaptation for the aged heart because capillary network participates in maintaining the supply of oxygen and energy substances in the heart (1). However, the molecular mechanisms underlying the exercise-induced improvement of angiogenesis in the aged heart are largely unknown.
Vascular endothelial growth factor (VEGF) is a potent mitogen of endothelial cells and a major stimulating factor in angiogenesis of animals and humans (33, 45). VEGF plays a critical role in both physiological and pathological angiogenesis (6). The biological actions of VEGF are mainly mediated by two structurally related receptor tyrosine kinases: fms-like tyrosine kinase (Flt)-1 and fetal liver kinase (Flk)-1 (12, 40). Flt-1 and Flk-1 receptors bind VEGF with high affinity. Flk-1 has a more important role in VEGF-mediated endothelial cell proliferation (38). In cardiac myocytes, the levels of VEGF and Flk-1 proteins are reduced by aging (13). On the other hand, exercise training in young rats increases mRNA and protein expressions of VEGF in the whole heart (21). VEGF activates an angiogenic signaling cascade, which promotes angiogenesis in association with activation of Akt and endothelial nitric oxide (NO) synthase (eNOS)-related pathway (10, 18, 46). Akt is a serine-threonine protein kinase and is activated by phosphoinositide-dependent kinases (9, 41). Activation of Akt is induced by phosphorylation and participates in the regulation of apoptosis suppression, endothelial cell migration, and proliferation (11). An angiogenic signaling pathway through Akt activates eNOS, leading to the production of NO (10, 18). Moreover, NO also has been shown to regulate VEGF gene expression (32). The angiogenic response to VEGF is partly mediated by eNOS because VEGF-induced angiogenesis is defective in eNOS−/− mice (17).
The alteration of molecular expression of VEGF and its receptors and the angiogenic signaling cascade via Akt and eNOS pathway in the heart by exercise training in old age are unclear. Because VEGF angiogenic signaling cascade participates in the molecular mechanism underlying regulation in capillary network, we hypothesized that exercise training during old age improves aging-induced downregulation of VEGF signaling cascade in the heart with change of cardiac capillary density. We investigated whether the aging-induced change in mRNA and protein levels of VEGF and its receptors Flt-1 and Flk-1 is improved by exercise training. We also studied the phosphorylation of Akt protein and eNOS protein, which is a VEGF angiogenic signaling cascade, in the aged heart by exercise training. Finally, we confirmed whether exercise training improved the aging-induced reduction of cardiac capillary density. In the present study, we tested our hypothesis by using sedentary young rats (sedentary-young group, 4 mo old), sedentary aged rats (sedentary-aged group, 23 mo old), and swim-trained aged rats (trained-aged group, 23 mo old, swim training for 8 wk, 5 days/wk, 90 min/day). Thus it is of great interest and importance to investigate whether exercise training upregulates VEGF angiogenic signaling cascade in the aged heart with the improvement of coronary capillary density.
METHODS
Animals and protocol.
The experimental protocols were approved by the Committee on Animal Research at the University of Tsukuba. Male 21-mo-old and 2-mo-old Wistar rats were obtained from the Institute for Animal Reproduction (Ibaraki, Japan) and cared for according to the “Guiding Principles for the Care and Use of Animals” based on the Helsinki Declaration of 1964. These rats were maintained on a 12:12-h light-dark cycle and received food and water ad libitum. Eight 21-mo-old rats were exercised by swimming in a tank of water at 35–37°C for 5 days/wk (trained-aged group) with a surface area of 2,830 cm2 and a depth of 60 cm. The rats swam for 15 min/day for the first 2 days, and then the swimming time was gradually increased by 1-wk periods from 15 to 90 min/day. Thereafter, the trained-aged group continued swim training for 7 wk. Therefore, the trained-aged group received 8 wk of swim training. Eight 21-mo-old rats (sedentary-aged group) and eight 2-mo-old rats (sedentary-young group) were confined to their cages for 8 wk but were handled daily. Trained-aged rats in the present study received exercise training for 8 wk because exercise training improves cardiac angiogenesis and energy metabolism in the aged rats (4, 23, 42), and in the young rats, exercise training for 8 wk increases mRNA and protein expression of cardiac VEGF (21). Measurements of body weight and hemodynamic parameters after 8 wk of swim training were performed after the animals rested for 24 h. Therefore, it was considered that there was no acute effect from the most recent bout of exercise. Systolic and diastolic blood pressures (SBP and DBP) and heart rate (HR) of the animals were measured using a tail-cuff sphygmomanometer (model MK-1030, Muromachi Kikai, Tokyo, Japan). After these measurements, the rats were anesthetized with pentobarbital sodium (40 mg/kg body wt ip). After the rats were anesthetized, the heart and epitrochlearis muscle were removed and rinsed in ice-cold saline to remove contaminating blood. The left ventricle was then separated from the right ventricle and atria, weighed, and frozen in liquid nitrogen. The left ventricle samples were stored at −80°C for staining capillaries and determining the mRNA expression of VEGF, Flt-1, and Flk-1 by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis, protein levels of VEGF, Flt-1, Flk-1, phospho-Akt, Akt, phospho-eNOS, and eNOS by Western blotting analysis and/or enzyme immunoassay. The epitrochlearis muscle was chosen for measurement of citrate synthase activity because a previous study (20) reported that swim training caused the increase in citrate synthase activity in the epitrochlearis muscle. Therefore, the epitrochlearis muscle samples were also stored at −80°C. Sedentary-young rats and sedentary-aged rats were killed at the same time point as the trained-aged rats (sedentary-young rats: 4 mo old, sedentary-aged rats: 23 mo old, and trained-aged rats: 23 mo old).
Muscle oxidative enzyme activity.
Citrate synthase activity, a marker of mitochondrial content, was measured in the whole epitrochlearis muscle homogenate by using the spectrophotometric method according to the method described in our previous paper (22).
Staining of capillary morphology.
Serial sections of 8-μm thickness were cut from the frozen left ventricle and were stained with lectin Griffonia simplicifolia (GSA-B4, Sigma Chemical, St. Louis, MO), and the capillary morphology was analyzed in the subendocardial region (25). The sections were fixed with acetone, air-dried, and placed in phosphate-buffered saline (PBS). After being treated with 3% H2O2 in methanol and washed in PBS, the sections were incubated with GSA-B4 (1:100 dilution in PBS) overnight at 4°C, followed by reaction with streptoavidin conjugated to peroxidase (Nichirei, Tokyo, Japan) and throughly rinsed in PBS. For visualization diaminobenzidine-H2O2 as chromogen was used. To enhance the diaminobenzidine reaction, the sections were rinsed with 0.05 M NaHCO3 (pH 9.6) and then incubated in diaminobenzidine enhancing solution (Vector Laboratories, Burlingame, CA). Vascular endothelium was stained by the lectin with capillaries appearing as black-dark brown dots. Capillary density and capillary-to-myocyte ratio were determined as previously described in other papers (25, 26). Serial sections of 16-μm thickness were cut from the frozen left ventricle, and double staining of frozen tissue sections was carried out to discriminate arteriolar and venular capillaries according to previous papers (25, 26). Arteriolar capillaries were stained blue because they contained alkaline phosphatase, whereas venular capillaries were stained red because they contained dipeptidylpeptidase IV. Intermediate capillaries were stained violet because they contain both enzymes. The number of each type of capillaries was counted in a given microscopic field. Sections were examined with an Olympus microscope, and counts of capillaries were made in cross sections in 30 fields (117,617 mm2/field) per sample at a final magnification of ×400. In addition, myocyte scan area was calculated by using ImageJ 1.33 software (National Institutes of Health) as previously described (26).
Quantitative PCR to determine levels of mRNA expression in the heart.
Total tissue RNA was isolated with Isogen reagent (Nippon Gene, Toyama, Japan) according to the method described in our previous paper (22). Briefly, the tissue was homogenized in Isogen (50 mg tissue/1 ml Isogen) with a Polytron tissue homogenizer (model PT10SK/35, Kinematica, Lucerne, Switzerland). Total RNA was extracted with chloroform, precipitated with isopropanol, and washed with 75% (vol/vol) ethanol. Total RNA was treated with an Rnase-free DNAse kit (QIAGEN, Tokyo, Japan) and further purified with an Rneasy minikit (QIAGEN). Single-strand cDNA from prepared RNA (2 μg) was synthesized with omniscript reverse transcriptase (QIAGEN) using an oligo (dT) primer at 37°C for 60 min.
The mRNA expression levels of VEGF, Flt-1, Flk-1, and β-actin in the left ventricle were analyzed by real-time quantitative PCR with TaqMan probe (FAM) using an ABI Prism 7700 Sequence Detector (Perkin-Elmer Applied Biosystems, Foster, CA). Real-time quantitative PCR was performed according to the method described in our previous paper with minor modification (22). Gene-specific primers and TaqMan probes were synthesized using Primer Express version 1.5 software (Perkin-Elmer Applied Biosystems) according to the published cDNA sequences for each of the following: VEGF (8), Flt-1 (44), Flk-1 (43), and β-actin (34) mRNA. The sequences of the oligonucleotides were as follows: VEGF forward: 5′-TGAGACCCTGGTGGACATCTT-3′; VEGF reverse: 5′-CACACAGGACGGCTTGAAGA-3′; VEGF probe: 5′-CCCCGATGAGATAGAGTAT-3′; Flt-1 forward: 5′-TCGGCTGTCCATGAAAGTGAAGT-3′; Flt-1 reverse: 5′-GCGGGTACGCCATCTTTTAAC-3′; Flt-1 probe: 5′-CCTCGCCAGAAGTCGTATG-3′; Flk-1 forward: 5′-GAAACTGAATGGCACCGTGTT-3′; Flk-1 reverse: 5′-GCAGGGAGGCATTCTGGAAT-3′; Flk-1 probe: 5′-CTAACAGCACAAACGACATCT-3′; β-actin forward: 5′-GGCCGGGACCTGACA-3′; β-actin reverse: 5′-GCTGTGGTGGTGAAGCTGTAG-3′; and β-actin probe: 5′-ACTACCTCATGAAGATCC-3′.
The expression of β-actin mRNA was determined as an internal control. The PCR mixture (25 μl total volume) consisted of 450 nM of both forward and reverse primers for VEGF, Flt-1, Flk-1, and β-actin, 200 nM of FAM-labeled primer probes (Perkin-Elmer Applied Biosystems), and TaqMan Universal PCR Master Mix (Perkin-Elmer Applied Biosystems). Each PCR amplification was performed in triplicate by using the following profile: 1 cycle of 95°C for 10 min, 40 cycles of 94°C for 15 s, and 60°C for 1 min.
In the present study analysis, the slope of the standard curve was between −3.0 and −4.0, and the correlation coefficient was ≥0.95. The amount of target cDNA product in the myocardial tissue sample was calculated automatically by plotting each sample on the standard curve. The quantitative values of VEGF, Flt-1, and Flk-1 mRNA were normalized by that of β-actin mRNA expression as the passive reference.
Electrophoresis and immunoblot analysis in the heart.
Western blot analysis of VEGF, Flt-1, Flk-1, phospho-Akt, Akt, phospho-eNOS, and eNOS proteins was performed according to previous papers with minor modification (24, 27). Briefly, each sample was separated on SDS-polyacrylamide gel (10%) and then transferred to a polyvinylidene difluoride (Millipore, Tokyo, Japan) membrane at 3 mA/cm2 for 60 min. The membrane was treated with blocking buffer 5% skim milk (VEGF, phospho-Akt, Akt, phospho-eNOS) or 1% skim milk (eNOS) in phosphate-buffered saline contained 0.1% Tween 20 (PBS-T) for 12 h at 4°C. The membrane was probed with polyclonal anti-VEGF (1:500 dilution with blocking buffer, IBL, Gunma, Japan), Flt-1 (1:500 dilution with blocking buffer, Santa Cruz Biotechnology, Santa Cruz, CA), Flk-1 (1:500 dilution with blocking buffer, Santa Cruz Biotechnology), Ser473-phospho-Akt (1:1,000 dilution with blocking buffer, Cell Signaling, Beverly, MA), Akt antibody (1:1,000 dilution with blocking buffer, Cell Signaling), and Ser1177-phospho-eNOS (1:250 dilution with blocking buffer, Santa Cruz Biotechnology), and monoclonal anti-eNOS antibody (1:800 dilution with blocking buffer, Transduction Laboratories, Lexington, KY) for 1 h at room temperature. The membrane was washed three times with PBS-T and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody, which was an anti-rabbit immunoglobulin (VEGF, Flt-1, Flk-1, and phospho-eNOS; 1:5,000 dilution with blocking buffer, Amersham Biosciences Piscataway, NJ), an anti-rabbit immunoglobulin (phospho-Akt and Akt; 1:2,000 dilution with blocking buffer, Cell Signaling), and an anti-mouse immunoglobulin (eNOS; 1:2,000 dilution with blocking buffer, Amersham Biosciences) for 1 h at room temperature. After this reaction, the membrane was washed three times with PBS-T. Finally, VEGF, phospho-Akt, Akt, phospho-eNOS, and eNOS were detected by ECL plus system (Amersham Life Science) and exposed to Hyperfilm (Amersham Biosciences).
Sandwich-enzyme immunoassay.
Concentrations of VEGF, Flt-1, and Flk-1 in heart tissue extracts were determined using a sandwich-enzyme immunoassay Kit (R&D systems, Minneapolis, MN) (21). All techniques and materials used in this analysis were in accordance with the manufacturer's protocol. The immobilized antibodies were monoclonal against VEGF121 and VEGF165, polyclonal against recombinant and natural mouse Flt-1, and monoclonal against recombinant and natural mouse Flk-1, whereas each secondary HRP-coupled antibody was polyclonal. Optical density was quantified on a microplate reader by using BioLumin 960 (Molecular Dynamics, Tokyo, Japan). All samples were assayed in duplicate.
Statistical analysis.
Values are expressed as means ± SE. Statistical analysis was carried out by analysis of variance followed by Scheffé's F-test for multiple comparisons. P < 0.05 was accepted as significant.
RESULTS
Body weight was significantly lower in the trained-aged group than in the sedentary-aged group (Table 1). Left ventricular weight and cardiomyocyte surface area in the sedentary-aged group and trained-aged group were significantly higher than those in the sedentary-young group, and there were no significant differences in left ventricular weight and myocyte surface area between the sedentary-aged and trained-aged groups (Table 1). There was no significant difference in SBP and DBP among the sedentary-young, sedentary-aged, and trained-aged groups (Table 1). There was no significant difference in resting HR between the sedentary-young and sedentary-aged groups, and that in the trained-aged group was significantly lower than in the sedentary-young and sedentary-aged groups (Table 1). Citrate synthase activity in the epitrochlearis muscle was significantly lower in the sedentary-aged group than in the sedentary-young group and was significantly higher in the trained-aged group than in the sedentary-aged group (Table 1). These results suggest that the trained-aged rats exhibited physiological effects of exercise training.
| Sedentary-Young Group | Sedentary-Aged Group | Trained-Aged Group | |
|---|---|---|---|
| Body weight, g | 423.3±4.7 | 721.3±26.4* | 570.8±18.4*† |
| Left ventricular weight, g | 0.80±0.03 | 1.27±0.03* | 1.26±0.01* |
| Myocyte surface area, μm2 | 515.4±15.9 | 758.2±4.9* | 753.4±7.0* |
| Systolic blood pressure, mmHg | 135.0±3.2 | 133.5±5.3 | 138.1±3.4 |
| Diastolic blood pressure, mmHg | 87.3±3.6 | 93.9±6.1 | 90.4±4.8 |
| Heart rate, beats/min | 368.3±5.9 | 370.9±6.1 | 317.1±4.8*† |
| Citrate synthase activity | |||
| Epitrochlearis muscle, μmol·min−1·g tissue−1 | 18.3±0.5 | 12.3±1.7* | 16.5±1.6† |
Figure 1A shows representative micrographs coronary capillaries of the heart (left ventricular sections) in sedentary-young, sedentary-aged, and trained-aged rats obtained by the lectin staining method. Total capillary density in the heart and capillary-to-myocyte ratio were significantly lower in the sedentary-aged rats compared with the sedentary-young rats and were significantly higher in the trained-aged rats compared with the sedentary-aged rats (Fig. 1B). Furthermore, the capillary domain area, which shows an area supplied by a single capillary and is defined as the area where one capillary provides oxygen, was significantly higher in the sedentary-aged rats compared with the sedentary-young rats and was significantly lower in the trained-aged rats compared with the sedentary-aged rats (Fig. 1C). Figure 2A \. shows representative micrographs of coronary capillaries of the heart (left ventricular sections) in sedentary-young, sedentary-aged, and trained-aged rats obtained by the double-staining method. In the heart, the arteriolar capillary portion, which was stained blue, was significantly higher in the sedentary-aged and trained-aged rats compared with the sedentary-young rats, and there is no significant difference between the sedentary-aged and trained-aged rats (Fig. 2B). The venular capillary portion, which was stained red, was significantly lower in the hearts of sedentary-aged rats compared with the sedentary-young rats and was significantly higher in the trained-aged rats compared with the sedentary aged rats (Fig. 2B). In the heart, the intermediate capillary portion, which was stained violet, was significantly higher in the sedentary-aged rats compared with the sedentary-young rats and was significantly lower in the trained-aged rats compared with the sedentary-aged rats (Fig. 2B).
Fig. 1.Total capillaries density, capillary-to-myocyte ratio, and capillary domain area in cross sections of the heart of subendocardium from sedentary-young (n = 8), sedentary-aged (n = 8), and trained-aged (n = 8, swim trained for 8 wk) rats. A: photomicrographs showing capillaries of cross sections of left ventricular (LV) subendocardium detected by staining the endothelium with lectin. Horizontal bar represents 100 μm. Magnification ×100. B: results of statistical analysis of total capillaries density, capillary-to-myocyte ratio, and capillary domain area in cross sections of LV subendocardium, obtained by the lectin staining method. Total capillary density is expressed as the number per squared millimeter. Values are expressed as means ± SE.
Fig. 2.Proportions of arteriolar, intermediate, and venular capillaries obtained by double-staining method in cross sections of heart of LV subendocardium from sedentary-young (n = 8), sedentary-aged (n = 8), and trained-aged (n = 8, swim trained for 8 wk) rats. A: photomicrographs of cross sections of LV subendocardium. Arteriolar, intermediate, and venular capillaries were stained blue, violet, and red, respectively (see arrows). Magnification ×400. B: results of statistical analysis of proportions of arteriolar, intermediate, and venular capillaries in cross sections of LV subendocardium as a percentage of total capillaries. Values are expressed as means ± SE.
The mRNA and protein levels of VEGF in the heart were significantly lower in the sedentary-aged rats compared with the sedentary-young rats and were significantly higher in the trained-aged rats compared with the sedentary-aged rats (Fig. 3, A and B). Figure 3C shows representative film of immunoblotting for VEGF protein expression in the hearts of sedentary-young, sedentary-aged, and trained-aged rats. We confirmed that VEGF protein in the heart decreased by aging, whereas exercise training reversed the aging-induced reduction of VEGF protein.
Fig. 3.Expression of vascular endothelial growth factor (VEGF) mRNA and protein in heart (left ventricle) of sedentary-young (n = 8), sedentary-aged (n = 8), and trained-aged (n = 8, swim trained for 8 wk) groups. A: expression of VEGF mRNA in the heart was analyzed by real-time quantitative PCR. Expression of β-actin mRNA was used as an internal control to normalize gene expression data. Data are expressed as means ± SE. B: level of VEGF protein in the heart was quantitatively determined by sandwich-enzyme immunoassay (EIA). Data are expressed as means ± SE. C: typical examples of Western blotting analysis are shown for levels of VEGF protein. Arrow indicates immunoblot band for VEGF protein.
The mRNA and protein levels of Flt-1 and Flk-1, which are VEGF receptors, in the heart were significantly lower in the sedentary-aged rats compared with the sedentary-young rats and were significantly higher in the trained-aged rats compared with the sedentary-aged rats (Fig. 4, A and B). Figure 4C shows a representative film of immunoblotting for Flt-1 and Flk-1 protein expressions in the hearts of sedentary-young, sedentary-aged, and trained-aged rats. We confirmed that Flt-1 and Flk-1 proteins in the heart decreased by aging, whereas exercise training reversed the aging-induced reduction of Flt-1 and Flk-1 proteins.
Fig. 4.Expression of Flt-1 and Flk-1 mRNA and protein in heart (left ventricle) of sedentary-young (n = 8), sedentary-aged (n = 8), and trained-aged (n = 8, swim trained for 8 wk) groups. A: expression of fms-like tyrosine kinase (Flt)-1 and fetal liver kinase (Flk)-1 mRNA in the heart was analyzed by real-time quantitative PCR. Expression of β-actin mRNA was used as an internal control to normalize the gene expression data. Data are expressed as means ± SE. B: level of Flt-1 and Flk-1 proteins in the heart was quantitatively determined by sandwich-EIA. Data are expressed as means ± SE. C: typical examples of Western blotting analysis are shown for the levels of Flt-1 and Flk-1 proteins. Arrow indicates the immunoblot band for Flt-1 protein and Flk-1 protein.
The protein level of Ser473-phospho-Akt in the heart was significantly lower in the sedentary-aged rats compared with the sedentary-young rats and was significantly higher in the trained-aged rats compared with the sedentary-aged rats (Fig. 5A). There was no significant difference in the protein level of Akt among the sedentary-young, sedentary-aged, and trained-aged groups (Fig. 5B). The phosphorylation of Akt, the ratio of phospho-Akt to Akt, in the heart was significantly lower in the sedentary-aged rats compared with the sedentary-young rats and was significantly higher in the trained-aged rats compared with the sedentary-aged rats (Fig. 5C). The protein level of Ser1177-phospho-eNOS and eNOS in the heart was significantly lower in the sedentary-aged rats compared with the sedentary-young rats and was significantly higher in the trained-aged rats compared with the sedentary-aged rats (Fig. 6, A and B). The phosphorylation of eNOS, the ratio of phospho-eNOS to eNOS, in the heart was significantly lower in the sedentary-aged rats compared with the sedentary-young rats and was significantly higher in the trained-aged rats compared with the sedentary-aged rats (Fig. 6C).
Fig. 5.Expression of Ser473-phospho-Akt protein (A) and Akt protein (B), and phosphorylation of Akt protein (C) in heart (left ventricle) of sedentary-young (n = 8), sedentary-aged (n = 8), and trained-aged (n = 8, swim trained for 8 wk) groups. Left, typical examples of Western blotting. Arrows, Immunoblot band for each protein. Right, results of statistical analysis of the levels of expression of phospho-Akt protein (A) and Akt protein (B) determined by a densitometer. Akt phosphorylation (C) in the heart was calculated by dividing Akt by phospho-Akt. Data are expressed as means ± SE.
Fig. 6.Expression of Ser1177-phospho-endothelial nitric oxide synthase (eNOS) protein (A) and eNOS protein (B) and phosphorylation of eNOS protein (C) in heart (left ventricle) of sedentary-young (n = 8), sedentary-aged (n = 8), and trained-aged (n = 8, swim trained for 8 wk) groups. Left, typical examples of Western blotting analysis. Arrows, immunoblot band for each protein. Right, results of statistical analysis of the levels of expression of phospho-eNOS protein (A) and eNOS protein (B) determined by a densitometer. eNOS phosphorylation (C) in the heart was calculated by dividing eNOS by phospho-eNOS. Data are expressed as means ± SE.
DISCUSSION
We revealed that exercise training improved the aging-induced decrease in mRNA and protein expression of VEGF and its receptors Flt-1 and Flk-1 in the heart. The present study also demonstrated that exercise training improved the aging-induced reduction of phosphorylation of Akt protein and eNOS protein in the heart, and these corresponded to the changes in VEGF protein levels. These results suggest that exercise training improves aging-induced downregulation of VEGF angiogenic signaling cascade in the heart. Moreover, the present study showed that total capillary density of the heart and capillary-to-myocyte ratio were decreased by aging, and these reductions are reversed by exercise training during old age. Previous studies have also shown that swim-exercise training in the rat increased the capillary density of the heart and capillary-to-myocyte ratio (4, 42). Taken together, the present study suggests that exercise training during old age improves an aging-induced decrease in angiogenesis in the heart, and the underlying mechanism participates in the exercise training-induced upregulation in VEGF angiogenic signaling cascade in the aged heart.
VEGF signaling pathway is considered a major stimulating factor in both physiological and pathological angiogenesis (6). Several animal studies using gene knockout mice, which the genes encoding VEGF or its receptors have been mutated, have been reported (2, 7, 15, 16, 39). The lack of VEGF (VEGF-A−/− and VEGF-A+/− mice) were unable to survive due to an impairment of vessel formation in the early embryo (7, 15). VEGF-B knockout mice, which have sequence homology with VEGF-A, caused a coronary vascular dysfunction and a reduction of recovery from cardiac ischemia (2). On the other hand, Flt-1−/− mice died by defection of angiogenesis (16). Flk-1-deficient mice also died by an early defection of the development in hematopoietic and endothelial cells (39). Taken together, these observations suggest that VEGF and its receptors in the heart play critical roles in the regulation of embryonic and homeostatic angiogenesis. On the other hand, in various tissues, the decrease of angiogenesis by aging is associated with the levels of VEGF expression (6, 36, 37). Furthermore, it has been reported that exercise training for 8 wk induced an increase in expression of VEGF mRNA and protein in the heart of young rats (21). The present study demonstrated that exercise training during old age improved the aging-induced decrease in mRNA and protein expression of VEGF and its receptors Flt-1 and Flk-1 in the heart. Therefore, it is considered that VEGF and its receptors may be a responsible regulating factor for the physiological adaptation of cardiac angiogenesis by aging and exercise training.
VEGF-mediated activation of Flk-1 upregulates endothelial cell proliferation, migration, and differentiation (3). VEGF-dependent phosphorylation of Akt via Flk-1 receptor results in the activation of eNOS, which finally leads to enhanced NO production (10, 18). Furthermore, the inhibition of NO production causes suppression of angiogenesis and vascular permeability induced by VEGF in vivo (46). These findings indicate that VEGF-Flk-1 signaling promotes an angiogenic cascade through the eNOS-NO pathway via the activation of Akt. In the present study, exercise training caused an upregulation in the phospho-Akt and phospho-eNOS levels with the promotion of cardiac VEGF expression in the aged heart. Thus it might be reasonable to speculate that the exercise training-induced increase in VEGF expression in the aged heart might cause the increased binding of VEGF to its angiogenic receptors, causing an increase in cardiac eNOS phosphorylation state through the activation of Akt protein, which finally may lead to the increased cardiac angiogenesis during exercise training in old age. On the other hand, the upregulation of NO levels and NOS expression are also shown to enhance VEGF gene expression on coronary venular endothelium (32). Increased VEGF mRNA expression in the skeletal muscle in response to a bout of exercise was attenuated by NOS inhibition (19). Thus a reciprocal regulation between NO level and VEGF expression also exists (28). The present study cannot exclude the possibility that the upregulation of eNOS in the heart also may be an important transcriptional regulator of increased VEGF mRNA expression caused by exercise training in old age. To clarify this issue, specific VEGF and/or NOS inhibitor should be used in the future studies.
The dilated cardiomypathic hamster heart exhibited a decrease in proportion of venular capillaries with a reduction of total capillary density (29), and an increase in arteriolar capillaries with the decrease in capillary density in the heart was observed in hypertensive rats with cardiac hypertrophy (31). Also further deterioration of these pathological cardiac diseases was associated with the reduction of total capillary density and an alteration in the proportion of venular and arteriolar capillaries in the heart. The present study showed that exercise training partly reversed the aging-induced decrease in venular capillary portion and greatly improved the decrease in total capillary density in the heart. Moreover, we demonstrated that both the arteriolar and intermediate capillary portions in the heart were increased by aging, and the increase in intermediate capillary type was reversed by exercise training. Impaired angiogenic capacity in the heart may affect the aging-dependent decline of cardiac function and viability of the heart because myocardial contraction involves a continuous supply of energy substances and oxygen from blood. Thus the exercise training-induced reversal of different capillary proportion with a recovery of total capillary density in the aged heart may contribute to the exercise training-induced beneficial effect in the aged heart, which ultimately improves cardiac function and energy metabolism. In addition, the exercise-induced amelioration of the downregulated VEGF signaling cascade in the heart in old age might play a role in the reversal of capillary proportions as well as the total coronary capillary density.
Although VEGF is a crucial and leading angiogenic growth factor, a number of other angiogenic factors such as transforming growth factor (TGF)-β1, basic fibroblast growth factor (bFGF), angiopoietin, platelet-derived growth factor, and hepatocyte growth factor (35) may also contribute to the exercise training-induced angiogenesis in the heart. In our preliminary experiments, there was no significant difference in cardiac TGF-β1 mRNA and protein expression among three groups, and expression of bFGF mRNA and protein in the heart was increased by aging, whereas there was no significant difference in bFGF expression between the trained-aged and sedentary-aged rats (data not shown). More detailed studies should be required to identify and elucidate other angiogenic growth factor with their signal transductions underlying the exercise training-induced improvement of angiogenesis in the aged heart.
The present study demonstrates an improvement of aging-induced downregulation of VEGF-angiogenic signaling cascade in the heart by exercise training in old age. We cannot rule out the possibility of the exercise training-induced improvement of myocardial VEGF-angiogenic signaling cascade in young adult rats. Indeed, exercise training-induced increase in expression of VEGF mRNA and protein in the heart of young adult rats has recently been stated (21). Thus it would be important to add a young adult training group in the present experimental setting. In addition, the body weight of sedentary-aged rats was greater than the trained-aged rats and the sedentary-young rats in the present study. Also, the myocyte surface area was greater in both sedentary-aged and trained-aged rats compared with the sedentary-young rats. Although the aging-induced increase in capillary domain area was ameliorated by exercise training, the increased myocardial fiber size with body weight may affect the capillary density in aged heart. Future studies using model rats where body weight can be kept unchanged even after exercise training may help to clarify the effect of myocardial fiber size in the current experimental setting.
In conclusion, we demonstrated that exercise training during old age improved the aging-induced reduction of capillary density in the heart. The present study also revealed that exercise training reversed the aging-induced decrease in mRNA and protein expression of VEGF and its receptors Flt-1 and Flk-1 in the heart. Furthermore, the present study showed that the phosphorylation levels of Akt protein and eNOS protein in the heart by aging and exercise training were changed in association with alterations of the VEGF protein levels. These findings suggest that exercise training improves aging-induced downregulation of VEGF angiogenic signaling cascade in the heart with the change of cardiac capillary density. Thus we propose that regulation of molecular level-mediated VEGF signaling cascade in the heart partly contributes to the mechanism of the angiogenic adaptation in the heart during aging and exercise training in old age.
FOOTNOTES
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.
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (15390077, 15650130, 16500391, and 17700484).
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