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Departments of Biomedical Sciences and Physiology and The Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri
Submitted 15 April 2004 ; accepted in final form 28 October 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-nitro-L-arginine to inhibit NOS, ACh-induced dilation was inhibited in old HF SFA such that the response was no longer greater than that of old NF SFA. These results indicate that increased intraluminal shear stress induces eNOS mRNA expression and improves endothelium-dependent dilation in senescent SFA by increasing NO production. endothelium; gene expression; real-time polymerase chain reaction; endothelial dysfunction
Endurance exercise training improves endothelial function in some arteries and arterioles in young healthy humans and animals (12, 16, 18, 19, 30, 31, 34). In addition, exercise training can attenuate, or reverse, age-associated decrements in endothelial function (10, 27, 28). Although the mechanism for the beneficial effects of training on endothelial function is not fully understood, evidence indicates that exercise training increases endothelial NO synthase (NOS; eNOS) gene expression, leading to improved NO-mediated, endothelium-dependent dilation (18, 19, 29–31, 34, 36).
The signal accounting for increased eNOS expression and enhanced NO-mediated dilation following endurance exercise training has not been determined; however, elevated levels of intraluminal shear stress during individual bouts of exercise may play an integral role. Indeed, shear stress is known to modulate eNOS expression in cultured endothelial cells and in intact arteries (22–24, 26, 33, 37). Although shear stress has been shown to modulate eNOS expression in some arteries (22, 37), the importance of this signal in regulating eNOS expression in soleus muscle feed arteries (SFA) is not known. In addition, the impact of aging on the efficacy of shear stress to induce eNOS expression has not been studied. Therefore, this study was designed to test the hypothesis that increased intraluminal shear stress induces eNOS mRNA expression and improves endothelium-dependent dilation in senescent SFA by increasing NO production. SFA were studied because they play an essential role in the control of soleus muscle blood flow during exercise (35) and because aging is associated with impaired NO-mediated, endothelium-dependent dilation in these arteries (38, 39).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Before initiation of this study, approval was received from the Institutional Animal Care and Use Committee at the University of Missouri. Male Fischer 344 rats (age 4 and 24 mo; n = 28/age group) were purchased from a commercial dealer (Harlan Sprague Dawley, Indianapolis, IN) and housed in the College of Veterinary Medicine’s Animal Care Facility. The facility was maintained at 24°C with a 12:12-h light-dark cycle. Animals were provided food and water ad libitum, and the rats were examined daily by the investigators and by veterinarians affiliated with the College of Veterinary Medicine.
Isolation of Feed Arteries
Procedures used to isolate SFA have been published previously (38, 39). Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt ip). Soleus muscles from the left and right hindlimb were removed and placed in MOPS-buffered physiological saline solution (PSS) containing (in mM) 145.0 NaCl, 4.7 KCl, 2.0 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, and 25.0 MOPS, at pH 7.4. SFA were dissected free of paired veins and connective tissue under a dissection microscope, cut on both ends, and transferred to a Lucite chamber containing MOPS-PSS (4°C) for cannulation.
Experimental Series 1: eNOS mRNA Expression
Eighteen rats were used in experimental series 1 (9 young, 9 old). Old rats weighed significantly more than the young rats (442 ± 15 vs. 374 ± 6 g). SFA were cannulated with two resistance-matched glass micropipettes and secured with 11-0 surgical silk, as described previously (38, 39). The micropipettes were subsequently attached to separate hydrostatic pressure reservoirs filled with MOPS-PSS supplemented with albumin (1 g/100 ml). Initially, all SFAs were pressurized to 60 cmH2O (1 mmHg = 1.36 cmH2O) and checked for leaks by verifying that intraluminal pressure remained constant when the valves to the reservoirs were closed. When a SFA was determined to be leak free, intraluminal pressure was raised to 90 cmH2O to approximate in vivo intraluminal pressure (35). Intraluminal flow was immediately established in the artery by changing proximal and distal pressures in equal but opposite directions to establish a pressure gradient across the artery while maintaining mean pressure at the midpoint of the artery at 90 cmH2O (17). A total of four SFA were studied in parallel from each rat. SFA 1 was exposed to no flow [NF; pressure change (
P) = 0 cmH2O] for 4 h. SFA 2 was exposed to low flow (LF; P = 2 cmH2O) for 4 h. SFA 3 was exposed to intermediate flow (IF; P = 30 cmH2O) for 4 h. SFA 4 was exposed to high flow (HF; P = 60 cmH2O) for 4 h. Intraluminal flow in each artery was measured throughout the protocol (Table 1) with a ball flowmeter (Omega Engineering) calibrated using a Razel perfusion pump (model A99). The LF conditions were selected based on previously published data indicating that flow rates of this magnitude elicited one-half maximal flow-induced dilation in SFA from young Fischer 344 rats (39). The IF conditions were selected based on data indicating that flow rates of this magnitude elicited maximal flow-induced dilation in young SFA (39). The HF conditions were set at two times the IF conditions.
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is the viscosity at 37°C (0.71 cP), Q is the volumetric flow rate, and r is the average vessel radius recorded during the 4-h experiment. It is important to note that flow was initiated immediately after setting intraluminal pressure was set to 90 cmH2O. Consequently, SFA did not have tone at the onset of flow, and diameter did not change significantly during the 4-h protocol. Experimental Series 2: Vasodilator Responses
Pretreatment of arteries with flow.
Eighteen rats were used in experimental series 2 (9 young, 9 old). Old rats weighed more than the young rats (441 ± 15 vs. 356 ± 13 g). All SFA were cannulated, pressurized to 60 cmH2O, and checked for leaks. When a SFA was determined to be leak free, intraluminal pressure was raised to 90 cmH2O. A total of four SFA were studied in parallel from each rat. SFA 1 and SFA 2 were exposed to NF (P = 0 cmH2O) for 4 h. SFA 3 and SFA 4 were exposed to HF (P = 60 cmH2O) for 4 h. At the end of the 4-h flow protocol, HF SFA were reset to NF conditions and allowed to develop tone. SFA that did not develop at least 25% spontaneous tone were constricted with phenylephrine.
Endothelium-dependent dilation. Endothelium-dependent dilation was assessed in NF and HF SFA by adding increasing doses of ACh to the bath solution in cumulative doses over the range of 10–9–10–4 M in whole log increments.
Endothelium-independent dilation. Endothelium-independent dilation was assessed in NF and HF SFA by adding increasing doses of SNP to the bath solution in cumulative doses over the range of 10–9–10–4 M in whole log increments.
Passive diameter. At the end of each experiment, SFA were incubated for 30 min in Ca2+-free PSS to determine passive diameter at an intraluminal pressure of 90 cmH2O.
Experimental Series 3: Role of NO
Twenty rats were used in experimental series 3 (10 young, 10 old). Old rats weighed more than the young rats (418 ± 21 vs. 342 ± 9 g). All SFA were cannulated, pressurized to 60 cmH2O, and checked for leaks. When a SFA was determined to be leak free, intraluminal pressure was raised to 90 cmH2O. A total of four SFA were studied in parallel from each rat. SFA 1 was exposed to NF (P = 0 cmH2O) for 4 h, and ACh-induced dilation was assessed in the absence of enzyme inhibitors. SFA 2 was pretreated with HF (P = 60 cmH2O) for 4 h and reset to NF conditions, and ACh-induced dilation was assessed in the absence of enzyme inhibitors. SFA 3 was pretreated with HF for 4 h and reset to NF conditions, and ACh-induced dilation was assessed in the presence of N-nitro-L-arginine (L-NNA; 300 µM) to inhibit NOS. SFA 4 was pretreated with HF for 4 h and reset to NF conditions, and ACh-induced dilation was assessed in the presence of L-NNA + indomethacin (Indo; 5 µM) to inhibit NOS and cyclooxygenase (COX). At the end of each experiment, SFA were incubated for 30 min in Ca2+-free PSS to determine passive diameter at an intraluminal pressure of 90 cmH2O.
Quantification of eNOS Expression
Relative differences in eNOS mRNA content in single SFA were assessed using a real-time reverse transcription polymerase chain reaction (RT-PCR). First-strand cDNA synthesis was performed as described previously (36). The reverse transcribed cDNA was used in an RT-PCR reaction using Brilliant SYBRgreen QPCR Master Mix (Stratagene). The PCR for eNOS was performed in a 25-µl volume containing 4-µl cDNA and 21-µl master mix containing primers specific for eNOS (100 nM). The primer sequences for eNOS were as follows: sense 5'-CCA CAA TCC TGG TGC GTC-3'; antisense 5'-GCC TTT TTC CAG TTG TTC CA-3'. GAPDH was amplified in a separate RT-PCR reaction using 2 µl of cDNA and primers specific for GAPDH. The primer sequences for GAPDH were as follows: sense 5'-ACT CTA CCC ACG GCA AGT TC-3'; antisense 5'-TAC TCA GCA CCA GCA TCA CC-3'. PCR reactions for eNOS and GAPDH were initiated with a denaturing step at 95° (10 min), followed by 40 cycles at 95° (30 s), 60° (60 s), and 75° (60 s). A melt curve, ramping from 60 to 95°, was performed following each RT-PCR to test for the presence of primer dimers. When primer dimer formation was detected, the PCR was rerun using a separate aliquot of cDNA. The threshold cycle at which PCR amplification became detectable by fluorescence was determined for eNOS and GAPDH, and copy number was determined from a standard curve.
Preparation of DNA Standards
Preparation of DNA standards was performed as described previously (6). Specifically, separate aliquots of reverse-transcribed cDNA from SFA were used in a conventional PCR using the eNOS or GAPDH-specific real-time PCR primer sets. The PCR-amplified products were electrophoresed on a 1.5% agarose gel and visualized with ethidium bromide staining. The eNOS and GAPDH PCR fragments were identified by size, and their identity was confirmed by direct nucleotide sequencing of gel-purified (Qiagen) material (2). eNOS and GAPDH PCR products were cloned into plasmid pCR 2.1 using the TOPO TA cloning kit (Invitrogen). eNOS and GAPDH clones were linearized before PCR by a single cut with Nco I (New England Biolaboratories). Linearized plasmid was quantified using a Hitachi U2000 spectrophotometer. Cloned eNOS and GAPDH PCR standards were then diluted to 106, 105, 104, 103, 102, and 101 molecules per 2-µl volume. Real-time PCR was performed using 5 µl of each standard and 100 nM eNOS or GAPDH primers in separate reaction vessels. A standard curve (threshold cycle vs. copy number of single-stranded DNA) was constructed for eNOS and GAPDH. All eNOS mRNA data are expressed as a ratio relative to GAPDH.
Statistical Analysis
All values are means ± SE. Between-group differences in eNOS-to-GAPDH mRNA ratios, flow rates, shear stress, percent tone, and passive diameter were assessed using one-way ANOVA. Two-way ANOVA with repeated measures on one factor (dose) was used to determine whether dilation to ACh or SNP differed by group. Concentration-response data were expressed as a percentage of maximal possible dilation. Percent possible dilation was calculated as (Ddose – DB/DP – DB) x 100, where Ddose is measured diameter for a given dose, DB is baseline diameter before a given dose, and DP is maximal passive diameter. When a significant F value was obtained, post hoc analyses were performed with Duncan’s multiple-range test. Statistical significance was set at the P 
0.05 probability level.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Intraluminal flow rates and calculated shear stress for SFA used in experimental series 1 are shown in Table 1. Statistical analysis revealed no between-group differences in intraluminal flow rate or shear stress. RT-PCR analysis revealed that eNOS mRNA content was significantly lower in old NF SFA than young NF SFA. In young SFA, eNOS mRNA content was not significantly altered by exposure to LF, IF, or HF (Fig. 1A; P = 0.46). In old SFA, eNOS mRNA content tended to be increased by IF (154%, P = 0.10) and was significantly increased by HF (136%, P = 0.04), resulting in a level of expression that was not different from that of young SFA (Fig. 1B).
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Intraluminal flow and calculated shear stress for the HF pretreatment period of experimental series 2 are shown in Table 2. Statistical analysis did not reveal a significant between-group difference in intraluminal flow (P = 0.21) or shear stress (P = 0.11) during the HF treatment. Before ACh dose-response curves were initiated, percent tone was similar among young NF (46 ± 2%), young HF (44 ± 3%), old NF (50 ± 2%), and old HF (46 ± 4%) arteries. ACh elicited a dose-dependent dilation of young SFA; however, ACh-induced dilation was not enhanced by pretreatment with HF (Fig. 2A). ACh-induced dilation in old NF arteries was significantly less than that in young NF and young HF arteries. Pretreatment of old SFA with HF for 4 h significantly improved ACh-induced dilation such that the response was not different from that of young NF or young HF arteries (Fig. 2B).
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Intraluminal flow and calculated shear stress for the HF pretreatment period of experimental series 3 are shown in Table 3. Statistical analysis did not reveal a significant between-group difference in intraluminal flow (P = 0.18) or shear stress (P = 0.65) during the HF treatment. Before ACh dose-response curves were initiated, percent tone was similar in all groups of arteries: young NF (45 ± 4%), young HF (40 ± 5%), young HF + L-NNA (42 ± 5%), young HF + L-NNA + Indo (45 ± 2%), old NF (43 ± 5%), old HF (39 ± 6%), old HF + L-NNA (42 ± 6%), and old HF + L-NNA + Indo (40 ± 4%). ACh elicited a dose-dependent dilation of young NF SFA (Fig. 3A). ACh-induced dilation was not enhanced by pretreatment with HF. ACh-induced dilation was not significantly inhibited by L-NNA in young HF SFA; however, in the presence of L-NNA + Indo, ACh-induced dilation was significantly inhibited in young HF SFA. ACh-induced dilation in old NF SFA was significantly less than that in young NF SFA (Fig. 3B). Pretreatment with HF for 4 h significantly improved ACh-induced dilation in old SFA such that the response was similar to that in young SFA. In the presence of L-NNA, or L-NNA + Indo, ACh-induced dilation was inhibited in old HF SFA, resulting in a response that was no longer greater than that in old NF SFA.
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Before SNP dose-response curves were initiated, percent tone was similar among young NF (34 ± 2%), young HF (35 ± 3%), old NF (36 ± 2%), and old HF (31 ± 1%) arteries. SNP elicited a dose-dependent dilation of young NF and young HF arteries; however, the vasodilator response was significantly attenuated in the young HF arteries (Fig. 4A). SNP-induced dilation was similar in old NF and old HF arteries (Fig. 4B).
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Maximal diameter was similar among young NF (182.9 ± 10.6 µm), young HF (188.8 ± 9.0 µm), old NF (190.3 ± 11.9 µm), and old HF (193.4 ± 11.9 µm) arteries.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Shear stress is an important signal regulating eNOS mRNA expression in cultured endothelial cells (23, 24, 26, 33) and in intact arteries (22, 37). Although shear stress has been shown to modulate eNOS expression in some arteries (22, 37), the importance of this signal in regulating eNOS expression in SFA is not known. In addition, it is not known whether aging influences the efficacy of shear stress to induce eNOS expression.
In the present study, basal eNOS mRNA content was lower in old NF arteries than in young NF SFA (Fig. 1). These data are in accord with previously published data indicating that eNOS mRNA expression declines with age in some arteries (1, 4, 8). Treatment with LF for 4 h did not alter eNOS mRNA content in old SFA; however, treatment with IF or HF increased eNOS mRNA expression. In addition, the results indicate that eNOS mRNA content in old SFA following the IF or HF treatment was not different from that in young SFA. Thus, in old SFA, we were able to demonstrate a significant effect of shear stress on eNOS mRNA content within a range of shear stress values shown to produce robust vasodilator responses in isolated SFA (38, 39). More importantly, the induction of eNOS mRNA expression occurred in response to intraluminal shear stress that was similar in magnitude to levels likely to be present in rat SFA during light-intensity exercise (14).
Interestingly, the lower basal eNOS mRNA levels in old SFA differed from a previous study that indicated that eNOS mRNA content is similar in young and old SFA (38). It is important to note, however, that, in the previous study, eNOS protein content was lower in SFA isolated from old rats (38). In addition, NO-mediated, endothelium-dependent vasodilation was impaired in these arteries (39). Consequently, increased eNOS mRNA content induced by HF could improve endothelial function.
To determine whether exposure to HF improved endothelium-dependent dilation, we pretreated SFA with 4-h HF, reset the arteries to NF conditions, and measured vasodilator responses to ACh (Fig. 2). The finding that ACh-induced dilation was less in old NF than in young NF SFA is in accord with previous studies that indicated that ACh-induced dilation is impaired by aging in these arteries (38, 39). Pretreatment with HF improved ACh-induced dilation in old SFA such that the response was not different from that in young SFA. Thus increasing intraluminal shear stress induced eNOS mRNA expression and improved endothelium-dependent dilation in old SFA. Importantly, the improvement in ACh-induced dilation could not be attributed to enhanced vascular smooth muscle responses to NO because SNP-induced dilation was similar in old NF and old HF arteries (Fig. 4). Collectively, these data suggest that increased intraluminal shear stress improved ACh-induced dilation by enhancing NO-mediated, endothelium-dependent dilation.
To directly test the hypothesis that increased intraluminal shear stress improved ACh-induced dilation in senescent SFA by enhancing NO-mediated, endothelium-dependent dilation, ACh-induced dilation was assessed in the presence of L-NNA to inhibit NOS. The finding that L-NNA inhibited ACh-induced dilation in old HF SFA (Fig. 3), such that the response was no longer greater than in old NF SFA, is consistent with the interpretation that increased intraluminal shear stress improved ACh-induced dilation by enhancing NO production.
In the present study, increased intraluminal shear stress did not induce eNOS mRNA expression, or improve endothelium-dependent dilation, in young SFA. The mechanism accounting for the age-related difference in the response of SFA to increased intraluminal flow is not known. However, it is important to note that young NF SFA had higher levels of eNOS mRNA expression than did old NF SFA. In addition, young NF SFA exhibited robust vasodilator responses to ACh (93% possible dilation). Consequently, there was less room for improvement in young SFA than in old SFA. These results are consistent with previously published findings indicating that endurance exercise training does not enhance endothelium-dependent vasodilation in SFA from young adult rats (15). Because resting blood flow to the soleus muscle is greater than the flow rate that causes maximal flow-induced dilation in SFA, Jasperse and Laughlin (14) proposed that further increases in blood flow and shear stress associated with daily bouts of exercise do not increase eNOS expression or improve endothelium-dependent dilation in SFA from young rats. It is important to note, however, that resting soleus muscle blood flow is similar in young and old rats (9, 13, 21). In addition, eNOS mRNA expression was induced by shear stress in the old SFA used in the present study. Thus further studies are needed to determine the mechanism accounting for the age-related difference in the regulation of eNOS expression by shear stress in SFA.
It is interesting to note that SNP-induced vasodilation was attenuated in young SFA pretreated with HF for 4 h. The mechanism accounting for decreased SNP-induced dilation in young HF SFA is not known; however, it is possible that pretreatment with HF stimulated release of NO throughout the 4-h flow protocol, resulting in decreased smooth muscle sensitivity to NO. Despite decreased smooth muscle responsiveness to NO, maximal ACh-induced dilation of young HF arteries was similar to that of young NF arteries. These data suggest the possibility that PGI2 and/or endothelium-derived hyperpolarizing factor compensated for decreased smooth muscle responsiveness to NO. The finding that ACh-induced dilation in young HF SFA was attenuated (not eliminated) in the presence of L-NNA + Indo, whereas L-NNA alone had no effect, is consistent with the interpretation that PGI2 and endothelium-derived hyperpolarizing factor play an integral role in ACh-induced dilation in the young HF SFA. Importantly, experimental evidence indicates that COX and prostacyclin synthase mRNA expression are induced by shear stress in cultured endothelial cells (25). Further studies will be needed to determine whether COX and prostacyclin synthase expression are induced by shear stress in young or old SFA.
Physiological Implications
Previous studies indicate that exercise training can attenuate, or reverse, age-related decrements in endothelium-dependent dilation in human subjects (10, 27, 28). In addition, it has been proposed that increases in intraluminal shear stress during exercise upregulate eNOS gene expression, enhance NO production, and improve endothelial function (10, 27, 28). The results of the present study indicate that increased intraluminal shear stress induces eNOS mRNA expression and improves endothelium-dependent dilation in senescent SFA by increasing NO production. In addition, the beneficial effect of shear stress occurred at a level of shear stress likely to be present in these arteries during light-intensity exercise (14). Thus these data lend credibility to the hypothesis that increased intraluminal shear stress during exercise is an important signal contributing to improved NO-mediated, endothelium-dependent dilation in senescent arteries.
In conclusion, the results of this study indicate that increased intraluminal shear stress induces eNOS mRNA expression and improves endothelium-dependent dilation in senescent SFA by increasing NO production. These results suggest that shear stress induction of eNOS expression is one mechanism by which exercise training attenuates, or reverses, the detrimental effects of aging on NO-mediated, endothelium-dependent dilation.
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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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1 subunit generating the ouabain-resistant mutant L793P. J Biol Chem 271: 15879–15883, 1996.This article has been cited by other articles:
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  C. Yan, A. Huang, G. Kaley, and D. Sun Chronic high blood flow potentiates shear stress-induced release of NO in arteries of aged rats Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H3105 - H3110. [Abstract] [Full Text] [PDF]
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 C. R. Woodman, D. W. Trott, and M. H. Laughlin Short-term increases in intraluminal pressure reverse age-related decrements in endothelium-dependent dilation in soleus muscle feed arteries J Appl Physiol, October 1, 2007; 103(4): 1172 - 1179. [Abstract] [Full Text] [PDF]
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