Ann Thorac Surg 2002;74:2126-2131
© 2002 The Society of Thoracic Surgeons
Original article: cardiovascular
Comparative study of functional responses and morphometric state of distal radial arteries in male and female
Kam Mong, MDa,b,
Jennifer A. Duggan, BSa,b,
Reza Tabrizchi, PhDb*
a Discipline of Surgery, Health Care Corporation of St. John’s, St. John’s, Newfoundland, Canada
b Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada
Accepted for publication July 8, 2002.
* Address reprint requests to Dr Tabrizchi, Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada, A1B 3V6
e-mail: rtabrizc{at}mun.ca
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Abstract
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BACKGROUND: Differences can exist in terms of physiology and morphology of blood vessels on the basis of gender. Radial artery is now considered to be the second choice for coronary artery bypass grafting. However, there is a lack of comparative studies on the function and morphometery of radial arteries in female and male patients.
METHODS: Radial arteries from 9 female and 9 male patients undergoing coronary artery bypass grafting were used to compare the effects of vasoconstrictors, noradrenaline and 5-hydroxytryptamine, as well as the influence of endothelium-dependent (with methacholine) and endothelium-independent (with sodium nitroprusside) relaxations. Furthermore, morphomteric measurements of smooth muscle thickness, lumen perimeter, lumen area, and intima area (including plaque) of distal radial arteries from female and male patients were also made.
RESULTS: Radial arteries from female patients when compared to male patients were significantly more sensitive to the actions of noradrenaline, and somewhat more sensitive towards the actions of 5-hydroxytryptamine. However, no significant differences were found between the relaxant effects of methacholine in radial arteries of female and male patients. In contrast, radial arteries from female patients when compared to male patients were significantly less sensitive to the relaxant effects of sodium nitroprusside. Morphometric measurements of blood vessels from female and male patients revealed that vessels obtained from female patients had a smaller lumen area and perimeter than vessels from male patients. In contrast, there were no significant differences between tunica intima area (including plaque area) or smooth muscle thickness in radial arteries of female patients when compared to male patients. However, the radial arteries from female patients had a significantly greater ratio of tunica intima area (including plaque) to lumen area when compared with radial arteries from male patients.
CONCLUSIONS: Differences exist between the functional behavior and morphometery of radial arteries of female and male patients. It is possible that postbypass, radial artery graft may show different characteristics in female versus male patients.
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Introduction
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Historically, the use of radial artery as a conduit for coronary artery bypass grafting was abandoned shortly after its introduction by Carpentier and associates in 1973 [1] because of an unacceptable patency rate. However, its use has been revived over the last decade, in part, due to use of new technique of surgical harvesting but also as result of use of antispastic drugs [2–4]. In fact, it is recognized that the combination of verapamil and nitroglycerin can effectively prevent radial artery spasm and produce long-lasting vasorelaxant effects [5, 6].
The propensity of the radial artery to develop spasm may be due to greater muscle mass as compared to other arterial conduits [7, 8]. The increased reactivity of the radial artery may also explain propensity to spasm [9, 10]. In addition, it has also been reported that there is an increased prevalence in medial calcification and sclerosis, as well as, mild intimal thickening in radial arteries in comparison to internal mammary arteries and saphenous vein in patients that undergo coronary artery bypass graft [11]. However, the long-term implications of these histologic and morphologic observations in radial artery in terms of graft patency remains uncertain [11]. Regardless of these facts, the radial artery appears to be gaining favor, and is being more often used as a conduit in both male and female patients undergoing coronary artery bypass surgery [12, 13].
Differences may exist between the behavior of blood vessels of female and male to physiologic and pharmacological stimuli, and this present study has compared the effects of vasoconstrictors/vasodilators or morphometery of radial arteries of female and male patients undergoing coronary artery bypass grafting. Thus, we undertook the present investigation to compare the effects of vasoconstrictors, noradrenaline (NA), 5-hydroxytryptamine (5-HT), and the influence of endothelium-dependent (with methacholine) and endothelium-independent (with sodium nitroprusside) relaxation in isolated distal radial arteries from female and male patients undergoing coronary artery bypass graft. In addition, we compared the morphometry (smooth muscle thickness, lumen perimeter, lumen area, and tunica intima + plaque area) of distal radial arteries of female and male patients.
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Material and methods
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Experiments conducted for this investigation conform with Canadian Institute of Health and Research Guidelines on Research Involving Human Patients. The protocol for these experiments were approved by the Memorial University of Newfoundland Research Board of Ethics.
Tissue isolation
Experiments on discarded distal radial arteries for all the studies described were performed on the same day as they were obtained from patients. Distal radial arteries were obtained from 9 male patients (51 to 74 years old) and 9 female patients (56 to 76 years old) undergoing coronary artery bypass surgery. The tissues were placed in ice-cold physiologic salt solution immediately after they left the operating room. The composition of the physiologic salt solution was of the following (in mM), NaCl, 132, KCl, 1.8, MgCl2, 1.2, CaCl2, 2.5, KH2PO4, 1.2, NaHCO3, 12.5, EDTA 0.1, glucose 11.0, and pH 7.4 when saturated with a 95% O2-5% CO2 gas mixture. The arteries were cleaned of connective and adipose tissues in ice-cold physiologic buffer. Radial artery rings (2-mm length) were then mounted in 15-mL organ baths at 37°C continuously gassed with a 95% O2-5% CO2 gas and were placed under a force of 19.61 mN. The tissues were equilibrated for 90 minutes (washed once at 45 minutes with physiologic salt solution). Isometric tension was measured using force displacement transducers (Model FT03, Grass Instruments Co, Quincy, MA) connected to polygraph (Model 7PCB, Grass Instruments). Radial arterial rings were initially contracted with high potassium buffer (80 mmol/L with sodium being replaced by potassium) under three different tensions (19.6 mN, 29.41 mN, and 39.22 mN) at 35 minutes intervals, and the developed tension at the three different base line tensions for male were 28.6 ± 2.9 mN, 36.1 ± 2.9 mN, and 35.30 ± 2.9 mN, and for females 26.0 ± 1.7 mN, 31.0 ± 1.96 mN, and 29.0 ± 2.3 mN (mean ± standard error of mean), respectively. The base line tension was then adjusted to 29.41 mN (a tension that is on the upper phase of the tension-response curve which was maintained throughout the experiment), and tissues were allowed to equilibrate for an additional 60 minutes (washed once at 30 minutes with physiologic salt solution).
Contraction–response curves were then obtained by adding increasing concentration of vasoconstrictor, NA (1 x 10-9 to 3 x 10-5 M) and 5-HT (1 x 10-9 to 1 x 10-5 M) to the organ bath. As well, blood vessels were contracted with a single concentration of vasoconstrictor agent, 5-HT (1 x 10-6 M), and then relaxation–responses curves were obtained by adding increasing concentrations of methacholine (1 x 10-9 to 3 x 10-5 M) or sodium nitroprusside (1 x 10-9 to 3 x 10-6 M) to the organ bath. Only a single concentration–response curve to each vasoconstrictor or vasodilator was obtained in any given tissue.
Morphometric measurements
A segment of radial artery (2 mm in length) from each patient was placed in 10% buffered formaldehyde solution for 24 hours before processing for routine histology (the segment of blood vessel processed for histology was not used for functional studies in isolated tissue baths). The processing of arteries was done over night where tissues were exposed to a series of solutions of buffered formalin, ethanol, xylene, and surgipath blue paraffin bath using Tissue-Tek V.I.P. vacuum infiltration process (Sakura Finietek USA, Inc, Torrance, CA). Each tissue was then sectioned at 8 µm by a microtome, and mounted on positively charged slides. The tissues were finally stained with Weigerts van Geison’s dyes [14]. Arterial rings were then examined and morphometric measurements were made of smooth muscle thickness, lumen perimeter, lumen area, and an area which included tunica intima, plaque, and lumen using Neurolucide software (Micro Brightfield Inc, VT) with Microvid microscope Leitz Diaplan (Model 020 to 437.035, Ernst Leitz Wetzlar GMBH, Germany). The area of the tunica intima (including plaque) was obtained by subtracting the lumen area from the total area (which was tunica intima, plaque, and lumen area).
Data and statistical analysis
Results from the contraction studies were calculated as a percentage of the maximum contraction induced by that agonist. Results from the relaxation studies were calculated as a percentage of the maximum relaxation induced by methacholine or sodium nitroprusside following contraction with 5-HT. Percent maximum (Emax), Hill coefficient (nH) and PD2 values were obtained for individual curves using a program executed on an IBM compatible microcomputer [15]. These values were determined by fitting the percent relaxant response at increasing concentrations of agonist ([A]) by nonlinear least squares to the relation Y = a + bX, where Y = response and X = [A]n/([A]n + [EC50]n) with n (nH) fixed at "floating" integral values to obtain the best fit. Student’s unpaired t-test was used to compare between means of functional (PD2, nH and Emax values) and morphometric (smooth muscle thickness, lumen perimeter, lumen area, and tunica intima + plaque area) parameters of blood vessels of female and male patients. For all cases, a probability of error of less than 0.05 was selected as the criterion for statistical significance.
Chemicals
Stock solutions of all drugs were made in twice distilled water. NA, 5-HT, methacholine, and sodium nitroprusside were purchased from Sigma/RBI Chemical Company (Ontario, Canada).
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Results
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Functional studies
Radial arteries were obtained from patients that were taking one or more of the following medications, ß-blockers, ASA, angiotensin converting enzyme inhibitors, calcium channel antagonists, nitrates, HMG-CO A reductase, diuretics, H+/K+ ATPase inhibitor, cardiac glycoside, 
-adrenoceptor antagonist, antiplatelet agent. The only notable difference between the type of medications that were being taken by female versus male patients was that 9 females in comparison to 3 males were on nitrates.
It was clear that addition of either NA or 5-HT to isolated distal radial arteries produced concentration-dependent contractions in both groups of patients. The PD2 values of the concentration-response curves for both vasoconstrictor agonists were modestly but insignificantly greater in radial arteries o
f female when compared to male patients. (Fig 1; Table 1).
Contractile responses to NA in radial arteries of females when compared to male patients were more sensitive to the actions of the vasoconstrictor, NA. This was found to be significant at concentration levels of 3 x 10-8, 10-7, 3 x 10-7, 10-6, and 3 x 10-6 M (Fig 1). The radial arteries obtained from female patients exhibited significantly higher sensitivity to the effects of 5-HT at two concentrations which were 10-7 and 3 x 10-7 M (Fig 1).
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Fig 1. Concentration-response curves to contractions elicited by (A) noradrenaline and (B) 5-hydroxytryptamine in distal radial arteries obtained from female or male patients undergoing coronary artery bypass grafting. Each value represents the mean of 9 ± SEM. *Significantly different from corresponding response in males. p< 0.05.
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Table 1. PD2, Hill Coefficient (nH), and Percent Maximum Response (Emax) Values Obtained From Individual Concentration-response Curves for Agonists in Isolated Human Distal Radial Arteries Obtained From Female and Male Patients
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Both methacholine and sodium nitroprusside produced concentration-dependent relaxation in tissues contracted with 5-HT (Fig 2; Table 1).
However, it was clear that there were no apparent differences between relaxation produced by methacholine in radial arteries from male and female patients. In contrast, we found that there was a significant difference in pD2 values obtained for relaxant responses to sodium nitroprusside in radial arteries from male versus female patients (Fig 2; Table 1). Thus indicating that male blood vessels were significantly more sensitive to relaxant effects of sodium nitroprusside when compared to female blood vessels (Table 1).
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Fig 2. Concentration-response curves to relaxation elicited by (A) methacholine and (B) sodium nitroprusside in distal radial arteries obtained from female or male patients undergoing coronary artery bypass grafting following contractions induced by 5-hydroxytryptamine (maximal tension induced by 5-hydroxytryptamine: 24.1 ± 2.9 mN and 24.5 ± 2.9 mN [mean ± SEM] in tissue from females and males, respectively). Each value represents the mean of 9 ± SEM.
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Morphometric studies
Morphometric measurements of blood vessels from male and female patients revealed that vessels obtained from male patients had larger lumen area and perimeter than vessels from female patients (Table 2).
In contrast, there were no significant differences between tunica intima area (including plaque) or smooth muscle thickness in radial arteries of female when compared to male patients. However, the ratio of lumen area to tunica intima area (including plaque) of the radial artery was significantly greater in male when compared to female patients (Table 2).
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Comment
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Based on morphologic evidence, it is apparent that the radial artery is quite muscular, in comparison to such arteries as internal mammary or gastroepiploic arteries [8], and more prone to spasm [7]. As well, physiologic studies have revealed that radial arteries when compared with internal mammary or gastroepiploic arteries show greater reactivity towards vasoconstrictors [9, 10]. In the present investigation, we find that smooth muscle thickness of the distal radial arteries are similar in female and male patients. However, the lumen area of distal radial arteries are significantly smaller in female when compared to male blood vessels. This indicates that similar shortening of smooth muscle length in radial arteries of female and males may result in greater reduction in lumen diameter in female arteries. In addition, there appears to be no significant difference between tunica intima area (including plaque) in radial arteries of male and female patients. This observation would further imply that lumen perimeter and lumen area in blood vessels of female versus male patients could be significantly smaller following similar shortening of smooth muscle cells during the excitation-contraction coupling process. This view is further supported by the fact that a significant difference exist between the ratio of tunica intima area (including plaque) to lumen area in the radial arteries of female in comparison to male patients. It is certainly possible that the morphologic differences observed in male and female blood vessels may have a significant physiologic difference in terms of blood flow. Based on Poiseuille’s law, the rate of blood flow through a vessel is directly proportional to its radius4. Here, the evidence from our present investigation has revealed that the lumen area of radial artery from female patients to be significantly smaller than the male patients, and as such the rate of blood flow would be expected to be much less in female radial artery than the male radial artery under normal physiologic conditions. It is possible that with any degree of vasoconstriction, and based on Poiseuille’s law, the degree of increase in resistance and the extent of reduction in rate of blood flow would be greater in radial arteries of females compared to males. This may have clinical implications following coronary artery bypass operation as we have also demonstrated that female radial artery appears more responsive to the actions of vasoconstrictor NA, and less responsive to vasorelaxant effects of sodium nitroprusside than the male radial artery.
Certainly, the responsiveness of the radial arteries to the vasoconstrictor, NA, were significantly greater in blood vessels of female patients when compared to male patients at a number of concentration levels. In addition, it was also apparent that there was a moderate increase in sensitivity to responses of 5-HT in radial arteries of female when compared to male patients. Therefore, these differences in the characteristic behavior of radial arteries from female versus male patients may also underline the morphologic differences that existed between these vessels. Based on our present observations it is possible that radial artery conduit grafts in female patients when compared with male patients may offer a lower perfusion of the myocardium immediately following bypass. On a long-term basis, this may also be of importance in terms of long-term patency rate of radial artery in female patients.
It is recognized that radial artery has this inherent ability to constrict, and thus spasms [7]. This physiologic characteristic of this vessel was the main reason that its use was initially abandoned as a conduit for coronary artery bypass grafting. However, the use of this vessel as a conduit has been revived over the last decade due to the use of a new technique of surgical harvesting as well as the use of antispastic drugs [3, 4]. It is now becoming increasingly evident that the radial artery is the second choice to the internal mammary artery as a conduit for coronary artery bypass grafting [12, 13, 16]. However, it is also clear that in comparison to the internal mammary artery, radial artery develops significantly higher tension to vasoconstrictors, NA, phenylephrine as well as high potassium solution [17]. Furthermore, it seems that radial artery when compared to internal mammary artery also displayed less sensitivity to relaxant effects of sodium nitroprusside [17]. Now if gender differences exist in terms of both morphology and physiology in radial artery as our study seems to suggest then it is possible that the long-term patency of this vessel may be lower in female patients when compared to male patients that undergo coronary artery bypass grafting. Perhaps a long-term clinical study is needed to investigate this issue.
Here, it is also clear that while endothelium-dependent relaxation (with methacholine) were of similar efficacy and sensitivity in radial arteries obtained from female and male patients, that of endothelium-independent relaxation (with sodium nitroprusside) were significantly greater in terms of sensitivity but not efficacy in radial arteries of male when compared to those of female patients. This difference is most likely not the result of tolerance to the actions of sodium nitroprusside even though a larger number of female patients were taking nitrates before surgery. In general, there is little cross tolerance between nitrates and sodium nitorprusside in isolated blood vessels [18]. Furthermore, He and Yang [19] have demonstrated that in the human radial arteries, sensitivity to relaxant responses of sodium nitroprusside was not affected while those of nitroglycerin was significantly impaired following prior exposure of radial arteries to nitroglycerin and verapamil. It has also been reported that while tolerance to the actions of glyceryl trinitrate exist in isolated human internal mammary arteries of patients treated with glyceryl trinitrate, no such tolerance exist to the vasorelaxant actions of sodium nitroprusside in the same vessel of these patients [20]. Based on these findings, it would be very unlikely that the lower sensitivity to the vasorelaxant effects of sodium nitroprusside in the radial arteries from female patients in comparison to those from male patients were due to tolerance to nitrates. This difference however could relate to the ability of sodium nitroprusside to generate substantially lower quantities of nitric oxide molecule in the radial arteries of female as opposed to vessels from male patients. Alternatively, it is possible that blood vessels from male patients are more sensitive to actions of nitric oxide. However, the latter explanation is tenuous as endothelium-dependent relaxations were found to be similar but again methacholine-mediated relaxations are not entirely due to the actions of nitric oxide, and other factors such as endothelium-dependent hyperpolarizing factor also contribute to the relaxant response that is initiated through the activation of muscarinic receptors located on endothelial cells [21].
In summary, our present investigation even with a modest sample size has identified certain differences in both physiologic and morphologic characteristics of the radial artery on the basis of gender. It is certainly possible that these differences may contribute to the differential behavior of this vessel in male versus female patients once it is used as conduit for coronary artery bypass grafting.
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Acknowledgments
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This work was supported by grant-in-aid from Heart and Stroke Foundation of Newfoundland & Labrador and New Brunswick. We are grateful to Judy Foote for histologic work, as well as Dr John McLean for allowing us to use his image analysis set-up for morphometric measurements.
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References
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Abstract
Introduction
