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Cardiovascular Research 2004 61(2):317-324; doi:10.1016/j.cardiores.2003.11.022
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Copyright © 2004, European Society of Cardiology

Estrogen replacement therapy reverses changes in intramural coronary resistance arteries caused by female sex hormone depletion

Metin Mericlia,*, György L. Nádasyb, Maria Szekeresa, Szabolcs Várbíróc, Zoltan Vajoc, Máté Mátraib, Nándor Ácsa, Emil Monosb and Béla Székácsd

aSecond Department of Obstetrics and Gynecology, Faculty of Medicine, Semmelweis University, H-1082 Budapest, Üllõi u. 78, Hungary
bInstitute of Human Physiology and Clinical Experimental Research, Faculty of Medicine, Semmelweis University, Budapest, Hungary
cDepartment of Geriatrics, Faculty of Medicine, Semmelweis University, Budapest, Hungary
dSecond Department of Internal Medicine, Faculty of Medicine, Semmelweis University, Budapest, Hungary

* Corresponding author. Tel.: +36-1-2100290/3284; fax: +36-1-3334934. mmericli{at}hotmail.com

Received 24 July 2003; revised 11 November 2003; accepted 21 November 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: We tested the hypothesis that female sex hormone depletion and estradiol replacement therapy significantly influences the biomechanical properties of intramural coronary resistance arteries. Design: Female rats (n = 30) were divided into three groups. In group O, rats were subjected to bilateral ovariectomy. Group HRT was subjected to bilateral ovariectomy and estradiol replacement therapy. Rats in group C served as controls. One month after ovariectomy, intramural coronary arteries (approximately 200 µm in diameter) branching from the left anterior descending coronary were isolated, cannulated and studied by microarteriography. Intraluminal pressure was increased in steps between 0 and 90 mm Hg. The steady state diameter at each step was measured. These measurements were repeated in the presence of U46619 [GenBank] , a thromboxane (TX) A2 receptor agonist (at a concentration of 10–6 M), and bradykinin (BK; at 10–6 M). Finally, Ca2+-free Krebs-induced passive diameter (PD) was measured in each group. Results: Ovariectomy increased spontaneous myogenic tone of coronary arteries (p<0.05), which was normalized by estrogen replacement. Ovariectomy decreased distensibility observed at low pressure, although passive diameter was not changed. Estrogen replacement decreased wall stress and elastic modulus (p<0.05). The thromboxane A2 agonist induced the largest contraction in the ovariectomized group, whereas bradykinin-induced relaxation was the largest in the estrogen replacement group (p<0.05). Conclusion: Estradiol hormone replacement therapy (HRT) may exert a beneficial effect on myocardial perfusion in menopause by opposing the deterioration of biomechanical properties of intramural coronary resistance vessels induced by female sex hormone depletion.

KEYWORDS Coronary artery; Contractility; Vessel wall; Endothelium; Estrogen


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
It is a well-established fact that the postmenopausal state is an age-independent risk factor for cardiovascular disease [1]. Estrogen deficiency or replacement affects the cardiovascular system at many points of action. Coronary heart disease is the leading cause of death of females in all industrialized countries. Due partly to its adverse effects on factors of the blood clotting system, the coronary protective effect of postmenopausal hormone replacement therapy (HRT) has become questionable [2,3] despite several well-studied beneficial vascular effects [4,5]. Advantageous effects of female sex hormones on the mechanical properties of arteries [6–12] may partly oppose the unfavorable hemostatic influence [2,3,13].

Intramural coronary arteries have never been studied before in this respect, which is due to methodical difficulties [14]. The importance of studying intramural coronary segments lies in the fact that pharmacological and myogenic reactivity as well as flow conditions might be markedly different from those in subepicardiac or subendocardiac locations.

In this study, we tested the hypothesis that either female sex hormone depletion or estradiol replacement therapy can significantly influence the biomechanical characteristics of the intramural coronary resistance arteries, which are hardly preparable small vessels and represent a special coronary microvascular region because of the presence of the surrounding thick and contracting cardiac muscle [14].


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Chemicals
Nembutal (Phylaxia-Sanofi, Budapest, Hungary) was used for anesthesia. For prevention of infections after the chronic surgical intervention, 100,000 IU of penicillin (TEVA-Biogal, Debrecen, Hungary) was administered intramuscularly. The protocol for HRT is described in detail elsewhere [9–11]. Stock solutions of estradiol propionate (Richter, Budapest, Hungary) were freshly prepared in sunflower oil (0.9 mg/ml). The composition of the normal Krebs–Ringer solution (nKR) used in these in vitro studies was (in mM/l): 119 NaCl, 4.7 KCl, 1.2 NaH2PO4, 1.17 MgSO4, 24 NaHCO3, 2.5 CaCl2, 5.5 glucose, and 0.034 EDTA. Ca2+-free Krebs solution containing (in mmol/l): 92 NaCl, 4.7 KCl, 1.18 NaH2PO4, 20 MgCl2, 1.17 MgSO4, 24 NaHCO3, 5.5 glucose, 2 EGTA and 0.025 EDTA was used. The temperature of the solution was kept at 37 °C, and it was bubbled with 5% CO2, 20% O2, 75% N2, which stabilized the pH at 7.4. U46619 [GenBank] , a thromboxane (TX) A2 receptor agonist and bradykinin were obtained from Sigma-Aldrich (St. Louis, MO, USA and Budapest, Hungary). Drugs were freshly prepared on the day of the experiment in nKR solution.

2.2. Animals
A total of 30 sexually mature, virgin female Sprague–Dawley rats (Charles River Laboratories, USA/Germany), weighing 250–270 g at the beginning of the study, were studied. Twenty of these rats were subjected to bilateral ovariectomy (group O) performed under anesthesia (by Nembutal 40 mg/kg i.m.) and sterile conditions. Ten from the ovariectomized animals were concurrently given estrogen replacement therapy (group HRT): 450 µg/kg estradiol propionate every 7 days i.m. [9–11]. This protocol produces physiological levels of estrogen [9–11]. The remaining 10 animals received matched treatments with the inactive solvent of the estradiol injections only and thus, served as controls (group C). No medical or surgical complications were observed. Conventional rat chow and tap water were provided ad libitum. The investigation conforms with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23 revised 1966), and with the Hungarian Law on Animal Care (Permission Number 36/1999).

2.3. In vitro mechanical and pharmacological reactivity of the intramural coronary artery
After 4 weeks of treatment, the animals were re-anesthetized (Nembutal 45 mg/kg i.m.). After opening the chest, heart was removed and placed into cold, oxygenized normal Krebs–Ringer (nKR) solution. Intramural coronary arteries (approximately 200 µm in diameter at preparation, secondary branches from the left anterior descending coronary artery) were isolated as previously described [14,15]. Then the segment was removed and placed in a vessel chamber filled with nKR, was cannulated at both ends with plastic microcannulas and extended to its in vivo length. Both cannulas were connected to pressure-servo systems (Living Systems, Burlington, VT, USA) and arteries were pressurized in a no-flow condition.

The outer diameter of the arteries was measured by microangiometry. The microangiometer setup was constructed in our Department [14–18]. In this setup, the glass-bottomed tissue bath was positioned in the light path of a microscope. A magnified picture of the vessel was formed with the aid of a video camera and a monitor (Philips). A specifically developed microcomputer evaluated the signals coming from the camera and automatically positioned two light markers adjusted to the contours of the vessel. The distance between the two light spots (which means the outer diameter of the segment) was continuously measured. Intraluminal pressure was measured at both sides of the segments using Gould pressure transducers, calibrated with a mercury manometer. A continuous superfusion was applied at a rate of 2.4 ml/min.

Pressure and diameter signals were digitized by an A/D converter (PCL 7/8, Adventech) and transmitted into an IBM Pentium PC for data storage and further processing.

Coronary arteries were allowed to equilibrate for 30 min at 50 mm Hg intraluminal pressure without intraluminal flow in nKR solution. Then the pressure was decreased to 2 mm Hg and then increased first to 30 mm Hg, then up to 90 mm Hg in 20 mm Hg steps. The steady state diameter at each step was measured after 5 min. The pressure load was repeated with U46619 [GenBank] , a thromboxane (TX) A2 receptor agonist (at a concentration of 10–6 M), and then with bradykinin (BK) at a concentration of 10–6 M, both administered into the superfusion with continuous flow, separately, one after another, allowing 10 min of incubation with each administration. Then, together with extraluminal application, BK (10–6 M) was also administered intraluminally through a side cannula—glued into the perfusion cannula—with continuous flow and at 50 mm Hg intraluminal pressure by setting the servo control system to 40 and 60 mm Hg on the two sides of the segment. Finally, passive diameter (PD) was obtained in Ca2+-free Krebs solution. The segments were incubated for 20 min then step increases in intraluminal pressure were repeated as above, and the passive diameter of arteries at each pressure step was obtained.

2.4. Biomechanical calculations
From the original calibrated pressure–diameter plots, the following geometrical and biomechanical parameters were computed for each intraluminal pressure level [11,15–18]. Tangential stress was computed according to the Laplace equation: {sigma}{theta}=pri/h, where {sigma}{theta} is the tangential (circumferential) wall stress, p is the intraluminal pressure, ri is the inner radius and h is the wall thickness (h = ro–ri), where ro is the outer radius.

Incremental distensibility was calculated as follows: Dinc={Delta}V/V{Delta}P, where Dinc is the incremental distensibility, {Delta}V is the change in vessel lumen volume in relation to the initial volume V in response to pressure change ({Delta}P).

The circumferential incremental elastic modulus was computed from the following equation: Einc=({Delta}p/{Delta}ro)2ri2ro/(ro2–ri2), where Einc is the incremental elastic modulus, ri is the inner, ro is the outer radius, {Delta}ro is the change in outer radius in response to intraluminal pressure change of {Delta}p.

The active strain expresses the percent contraction of passive radius at each intraluminal pressure level. Thus, the vasoreactivity is expressed in percent changes independently of the original vascular lumen. Active strain for nKR (also called "in vitro spontaneous tone") was quantitated for each intraluminal pressure level: TnKR=(ri Ca-free–ri nKR)/riCa-free, where ri Ca-free, and ri nKR are the inner radii measured in calcium-free nKR solution and in normal Krebs–Ringer solution, respectively. Active strain for TXA2 agonist was quantitated for each intraluminal pressure level as follows: TTXA2=(ri Ca-free–ri TXA2)/ri Ca-free, where ri Ca-free, and ri TXA are the inner radii measured in calcium-free nKR solution and with U46619 [GenBank] (a TXA2-agonist), respectively. Thus the size of the vascular lumen does not influence the measure (%) of vascular reactivity. Active strain for bradykinin was quantitated for each intraluminal pressure level as follows: TBK=(ri Ca-free–ri BK)/ri Ca-free, where ri Ca-free, and ri BK are the inner radii measured in calcium-free nKR solution and bradykinin, respectively. Thus the size of the vascular lumen does not influence the measure (%) of vascular reactivity.

For statistical analysis of data measured at certain intraluminal pressures, groups with different treatments were compared by one way ANOVA. In vitro parameters plotted as a function of intraluminal pressure were compared by multiple comparison ANOVA (Sigma Stat). Paired comparisons for treatment groups were made either for the whole curve or for a selected pressure interval. As a post hoc test, Tukey's test was used. P<0.05 was uniformly accepted as significant difference. Data are presented as mean±S.E.M.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
Body weights on the day of the experiment were 259±10.7, 333±6.0, 261.5±9.8 g, respectively, in the C, O and HRT groups, respectively. Heart weights were: control: 0.811±0.028 g, ovariectomy: 0.887±0.043 g, and ovariectomy+HRT: 0.817±0.042 g, p: n.s.

3.1. Effects of ovariectomy and sex hormone replacement therapy on biomechanical characteristics of rat coronary resistance arteries
Outer radius of rat coronary resistance arteries measured in vitro in spontaneous contraction (in nKR) at 50 mm Hg were 130±8, 119±9 and 130±13 µm in C, O and HRT groups, respectively (Fig. 1a). In spontaneous contraction, radii of coronary arteries were significantly less in the ovariectomized group at all pressures compared to the other two groups (p<0.05) (Fig. 1a). On the other hand, values of inner radii were not significantly different in the three groups in control (nKR) conditions (Fig. 1b). Minor outer radius in ovariectomized group in control condition (nKR) resulted in significantly smaller wall thickness (at 50 mm Hg in spontaneous contraction were 32.5±2.4, 25.4±2.3 and 35.2±2.3 µm in C, O and HRT groups, respectively; p<0.05). Passive outer and inner radius values (measured in calcium-free solution) were not different in the three groups (data not shown).


Figure 1
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  		Fig. 1 Outer and inner radius of rat intramural coronary resistance arteries as a function of intraluminal pressure (n = 30) in control solution (panels a and b, respectively). Values of control group (closed circles), ovariectomized group (OV, closed squares) and estrogen replaced group (OV+E, open squares) are shown. Mean±S.E.M. values. Asterisk indicates statistically significant difference (p<0.05) between control and OV groups, and cross indicates statistically significant difference (p<0.05) between OV and OV+E groups.

 
All segments developed significant spontaneous myogenic tone: 7.6±1.6%, 12.8±2.6% and 7.9±1.5% contraction from the passive radius in C, O and HRT groups at 50 mm Hg, respectively; p<0.05. Pressure-dependence of myogenic tone is visualized as active strain (Fig. 2). Active strain was significantly larger in the ovariectomized group compared to the other groups on pressures above 30 mm Hg (p<0.05, Fig. 2).


Figure 2
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  		Fig. 2 Active strain of rat intramural coronary resistance arteries as a function of intraluminal pressure (n = 30) in control solution (in Krebs) expressed as percent contraction of outer radius (ro) from fully passive (calcium-free solution). Values of control group (closed circles), ovariectomized group (OV, closed squares) and estrogen replaced group (OV+E, open squares) are shown. Mean±S.E.M. values. Asterisk indicates statistically significant difference (p<0.05) between control and OV groups, and cross indicates statistically significant difference (p<0.05) between OV and OV+E groups.

 
Tangential (circumferential) wall stress gradually increased with increasing pressure (Fig. 3). Interestingly, pressure-dependent wall stress was significantly less in the HRT group compared to other groups at higher pressures (above 50 mm Hg) both in control (nKR) and in passive conditions (p<0.05). Values of wall stress were 20.9±2.4, 23.5±2.8 and 17.6±1.6 kPa in C, O and HRT groups, respectively, at 50 mm Hg in nKR.


Figure 3
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  		Fig. 3 Tangential wall stress of rat intramural coronary resistance arteries as a function of intraluminal pressure (n = 30) in control solution (in Krebs, panel a) and in passive condition (with calcium-free solution, panel b). Values of control group (closed circles), ovariectomized group (OV, closed squares) and estrogen replaced group (OV+E, open squares) are shown. Mean±S.E.M. values. "+" indicates statistically significant difference (p<0.05) between control and OV+E groups, and the cross indicates statistically significant difference (p<0.05) between OV and OV+E groups.

 
Incremental distensibility decreased with increasing pressure (Fig. 4). Distensibility of O and HRT groups at 2 mm Hg in nKR were significantly less compared to control group (p<0.05); however, there was no difference in the distensibility between groups in passive condition. Values at 50 mm Hg were 0.04±0.01, 0.02±0.01 and 0.03±0.006 1/kPa in C, O and HRT groups, respectively, in nKR.


Figure 4
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  		Fig. 4 Incremental distensibility of rat intramural coronary resistance arteries as a function of intraluminal pressure (n = 30) in control solution (in Krebs, panel a) and in passive condition (with calcium-free solution, panel b). Values of control group (closed circles), ovariectomized group (OV, closed squares) and estrogen replaced group (OV+E, open squares) are shown. Mean±S.E.M. values. Asterisk indicates statistically significant difference (p<0.05) between control and OV groups, and "+" indicates statistically significant difference (p<0.05) between control and OV+E groups.

 
Incremental elastic modulus increased with increasing pressure (Fig. 5). Elastic modulus of HRT group was significantly less than in control animals at high pressures (above 50 mm Hg) in nKR (p<0.05). Values of group O were not significantly different from controls. However, elastic modulus in passive condition was significantly less both in O and HRT groups compared to control at 70 mm Hg. HRT group values were also significantly less than in O group at the same pressure (p<0.05). Values of elastic modulus in the passive state and at 50 mm Hg were 482.4±146.6, 469.9±100.8, 302.2±58.5 kPa in C, O and HRT groups, respectively, in nKR, not significantly different.


Figure 5
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  		Fig. 5 Incremental elastic modulus of rat intramural coronary resistance arteries as a function of intraluminal pressure (n = 30) in control solution (in Krebs, panel a) and in passive condition (with calcium-free solution, panel b). Values of control group (closed circles), ovariectomized group (OV, closed squares) and estrogen replaced group (OV+E, open squares) are shown. Mean±S.E.M. values. Asterisk indicates statistically significant difference (p<0.05) between control and OV groups, cross indicates statistically significant difference (p<0.05) between OV and OV+E groups, and "+" indicates statistically significant difference (p<0.05) between control and OV+E groups.

 
3.2. Effects of ovariectomy and sex hormone replacement therapy on vascular contractility and on endothelial function of rat coronary resistance arteries
In the control group, U46619 [GenBank] increased and bradykinin decreased the tone of coronary arteries (p<0.05 at 2 mm Hg to both agents). In the O group, TXA2 agonist U46619 [GenBank] induced large, significant contraction (increased vascular tone) of coronary arteries at all pressures (p<0.05), whereas BK-dilation was absent in this group. In the HRT group, contraction to U46619 [GenBank] was unpronounced, whereas BK induced significant vasodilation (decreased vascular tone) of the coronary arteries (p<0.05, data not shown).

Ovariectomy significantly increased contractility of coronary resistance arteries to U46619. [GenBank] As expressed in active strain, values of group O increased 2–2.5 times compared to the control group (Fig. 6a). The increased contractility seen in the ovariectomy group to TXA2 agonist was depressed in the HRT group back to the control level. With bradykinin, coronary arterial tone was still significantly larger in the O group compared to other groups (Fig. 6b, p<0.05); however, in the HRT group arterial tone was decreased, even lower than the level of the control group (p<0.05 at 50 and 70 mm Hg in comparison between HRT and control groups).


Figure 6
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  		Fig. 6 Active strain of rat intramural coronary resistance arteries as a function of intraluminal pressure (n = 30) expressed as percent contraction from outer radius (ro) with thromboxane A2 agonist U46619 (1 µM, panel a) and with bradykinin (1 µM, panel b). Values of control group (closed circles), ovariectomized group (OV, closed squares) and estrogen replaced group (OV+E, open squares) are shown. Mean±S.E.M. values. Asterisk indicates statistically significant difference (p<0.05) between control and OV groups as well as between OV and OV+E groups, and "+" indicates significant difference (p<0.05) between control and OV+E groups.

 
Intraluminal administration of bradykinin in addition to extraluminal administration induced slight, but significant vasodilation of coronary arteries in all groups (Fig. 7).


Figure 7
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  		Fig. 7 Outer diameter of rat intramural coronary resistance arteries as a function of intraluminal pressure (n = 30) in Krebs with bradykinin administration. Demonstrated are values with extraluminal administration of bradykinin (1 µM, closed bars) and with the simultaneous administration of extraluminal and intraluminal bradykinin (1 µM, open bars) in control group, ovariectomized group (OV) and estrogen replaced group (OV+E). Mean±S.E.M. values. Asterisk indicates statistically significant difference (p<0.05) between the two treatments.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Some recent randomized-controlled trials of HRT were disappointing in terms of the vascular, especially coronary protective effects of combined hormone replacement [2,3]. Besides the fact that in both studies hormone replacement was started many years after the menopause, this could probably be explained to some degree with the unfavorable effects of HRT on some factors of the blood clotting system [13,19]. Despite the lack of protective effect in the randomized-controlled clinical trials, there has been evidence for beneficial effects of estrogen on the biomechanical properties of the vascular system in both animal and human studies [7,9,20].

Both estrogen- and combined hormone replacement therapy showed favorable effects on the biomechanical characteristics of small peripheral arteries [9,11]. Majority of the hormone replacement studies were performed on aorta and carotid artery segments, and the obviously very important coronary arteries have only been studied once by Zhang et al. [21]. However, their study used extramural coronary segments. Intramural coronary arteries have never been studied before in this respect, which is due to the difficulties associated with the preparation of these arteries [14,15,22]. The importance of studying intramural coronary segments lies in the fact that pharmacological reactivity and flow conditions are markedly different in different serially connected segments of the network [22]. Mechanical properties of the intramural segments are of special importance, since as a consequence of cardiac pumping function, these vessels are subjected to chronic cyclic mechanical compression.

By the successful standardization of the preparation method, our team was able to study the effects female sex hormone deficiency and estrogen replacement on the pharmacological reactivity and biomechanical properties. From our findings, we would like to highlight that ovariectomy decreased the outer radius and wall thickness of intramural coronary segments. This finding is similar to the results of studies on small peripheral arteries [9], but is different from the findings of Zhang et al. on subepicardial coronaries [21]. Our finding that female sex hormone deficiency decreased the radius and wall thickness was accompanied by an increased wall stress and vascular tone. On the other hand, estrogen replacement prevented geometrical changes of the vessel wall, vascular tone was identical to control and mechanical stress was reduced. In estrogen-treated animals coronary segments were more distensible in the higher physiological pressure range and above (above 50 mm Hg). This indicates changes in vascular conductance function: at higher pressures more distensible arteries with smaller mechanical stress are capable of providing better and more regulable tissue perfusion. This is of special importance in the intramural coronary circulatory system, where blood flow is intermittent.

To eliminate the confounding effects of weight gain in altered female sex hormone states [11,23,24], we analyzed active strain values. The importance of active strain is that it is independent of body weight. Since vessel wall thickness and vascular diameter are weight-dependent variables, differences in body weights may mask biological vascular changes. On the other hand, changes measured in percentage of passive vessel diameter are independent of body weight [25].

Based on our results, in our experimental model of menopause we detected early vascular changes. Wall distensibility was not (yet) diminished, but increased mechanical stress (which subsequently leads to wall stiffness) was already detectable. This seems to be related to the increased spontaneous vascular tone, which can be the cause of long term morphological remodeling (meaning the reduction of wall thickness and outer radius).

Bradykinin induced relaxation and U46619 [GenBank] induced contraction both were significant in the control group. In comparison with these values, ovariectomy induced an increased contraction response to TXA2 and decreased relaxation to bradykinin. Estrogen-treated animals showed contraction responses identical to control and even an elevated bradykinin induced relaxation.

Bradykinin is a potent vasodilator in coronary arteries acting mainly through endothelium-derived nitric oxide (NO) [26]. In the control group, the amount of the difference between maximal vascular contraction and relaxation was approximately 20% in our study. This means that the intramural coronary segments have approximately a 10% room in both directions to respond to any stimuli. Compared to control, ovariectomy significantly increased maximal (TXA2 induced) contractions and decreased endothelium dependent (bradykinin induced) relaxation. Lamping et al. found the same in epicardial coronary segments, which are completely different from the subendocardial coronary vessel segments we studied in terms of the mechanical conditions due to the characteristics of the surrounding tissues [27]. However, their study reports the effects of a 30 min in vitro preincubation with estradiol. Therefore, their data are only partially comparable with our in vivo, chronic administration, where animals were treated for 4 weeks with estradiol. This models long term HRT, while Lamping et al. studied the acute vascular effects 17β-estradiol [27].

We found that estrogen replacement resulted in changes in the opposite direction. TXA2 induced contractions were the same as in the control group and endothelium (bradykinin) dependent dilatation was even slightly increased compared to control. The latter is probably explained by our continuous, rather than cyclic estrogen replacement and by the lack of progesterone replacement [9]. Bradykinin induced significant vasodilation in all groups, which supports the presence of a functionally intact endothelium.

In summary, our menopause model showed significant pathological changes in vascular adaptation both in regards of biomechanics, regulation of spontaneous vessel tone and pharmacological response of intramural coronary artery segments. Estrogen replacement therapy protected against the unfavorable vascular changes related to female sex hormone deficiency. Our data are the first on the effects of HRT on intramural coronary segments, most likely because these vessels are extremely difficult to isolate. Maintaining optimal mechanical conditions in intramural coronaries is of obvious importance because of the constant mechanical challenge these vessels are subjected to by the contracting cardiac muscle, which surrounds them.


   Acknowledgements
 
The authors thank the expert technical assistance of Ms. Ildikó Oravecz. This work was supported by Hungarian National Grants OTKA T-032019, OTKA T-42670, T-037832, ETT 149/2000, BO/00080/03 and ETT 240/2003.


   Notes
 
Time for primary review 00 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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