Flow-mediated vasodilation is suggested as one of the mechanisms involved in arterial expansive remodelling, which is thought to be a defence mechanism in atherogenesis. In the present study, we tested the hypothesis that lumen obstructive plaque formation is associated with failure of NO (nitric oxide)-dependent vasodilation in conduit vessels. Cardiac function and aortic root flow velocities were assessed using high-resolution echocardiography and two-dimensional-guided pulsed Doppler in ApoE−/− (apolipoprotein E-deficient) mice fed a standard or high-cholesterol diet. Endothelial function in the proximal and mid-descending aortic regions was studied using a myograph technique. Flow velocity at the aortic root of cholesterol-fed ApoE−/− mice was significantly increased as a result of lumen narrowing, detected via histological analysis. NO-dependent vasodilatory responses were selectively impaired in the atherosclerosis-prone vascular regions in cholesterol-fed ApoE−/− mice. In conclusion, consumption of a high-cholesterol diet results in lumen obstructive plaque formation in ApoE−/− mice, which significantly alters aortic root haemodynamics. This phenomenon is associated with impaired NO-dependent vasodilation in vessel segments known to be prone to atherosclerosis.
- ApoE−/− mice
- endothelial function
Expansive arterial remodelling has been suggested to be a defence mechanism in atherosclerosis aimed at delaying the development of lumen narrowing. In previous studies, Glagov and co-workers  have shown that coronary arteries underwent expansive remodelling in response to plaque formation in order to maintain sufficient lumen area. Failure of this mechanism leads to inward growth of atherosclerotic lesions, an independent predictor for future coronary events . Furthermore, lumen obstructive lesions may give rise to locally increased shear stress, known to be an extrinsic biomechanical factor, which may trigger plaque rupture, resulting in atherothrombotic events [3,4].
Thus it appears that the time point at which the vessel does not respond to plaque formation with expansive remodelling is crucial for the progression of this chronic vascular disease. Failure of expansive remodelling may mark the transition into a more advanced stage of atherosclerosis with severe clinical cardiovascular manifestations. However, the mechanisms underlying the transition from expansive remodelling to lumen obstruction still remain unclear. It has been suggested that factors such as smoking, age  and fibrocalcific plaque  may influence the remodelling process.
Endothelial dysfunction, usually linked to a reduced bioavailability of NO (nitric oxide), has been shown to be an independent risk factor for future cardiovascular events [7–9]. Furthermore, it has been suggested that a shear-induced release of NO may influence the remodelling process. According to this hypothesis, increased shear might give rise to chronic vasodilation, counteracting the lumen narrowing which occurs following inward growth of atherosclerotic lesions . This hypothesis is supported by studies in which blockade of NO production impaired expansive remodelling in a rabbit model of atherosclerosis . Thus plaque growth combined with endothelial dysfunction may cause lumen occlusive lesion progression, leading to locally altered haemodynamics in vivo. Using high-resolution Doppler ultrasound, local haemodynamic consequences of lumen occlusive lesions can be studied by means of 2D (two-dimensional)-guided Doppler measurement. Furthermore, this technique also facilitates detailed evaluation of cardiac function, which may influence local flow velocities in the aortic root. Hence using this non-invasive method to detect initiation of lumen occlusive lesions may be of value to increase our understanding of the atherosclerotic remodelling process.
The ApoE−/− (apolipoprotein E-deficient) mouse is a widely used animal model, which spontaneously develops atherosclerotic lesions similar to those observed in humans. In addition, dietary manipulations of ApoE−/− mice, using a high-cholesterol diet, can greatly accelerate lesion progression . Aortic root atherosclerosis is commonly used for plaque quantification and histological evaluation , and has been suggested as a model for human coronary plaques . Recently, Bentzon et al.  demonstrated that arterial expansive remodelling also occurred in ApoE−/− mice.
In the present study, we aimed to affect the plaque formation process in ApoE−/− mice by using an established dietary protocol of elevated cholesterol  to test the hypothesis that potential lumen obstructive plaque remodelling is associated with impaired NO-dependent vasodilation. In vivo haemodynamic consequences of lumen occlusive lesions were studied using a novel 2D-guided Doppler ultrasound technique.
Female ApoE−/− mice were purchased from Taconic M&B Breeding and Research Centre (Bomholtgaard, Denmark) at 7 weeks of age. Water and food were available ad libitum. Animals were housed in a room with a constant temperature (21–25 °C) and a 12 h light/dark cycle. All procedures involving animals were approved by the Regional Animal Ethics Committee at Göteborg University.
Mice (9 weeks of age; n=16) were randomly divided into two equally sized groups and fed either a standard diet (control group) or high-cholesterol diet containing 1.25% cholesterol  (Laktamin, Stockholm, Sweden) for a 5 week period. Doppler echocardiographic measurements were carried out after 4 weeks on the diet, 1 week prior to the animals being killed. Invasive blood pressure measurements were performed on the day of termination. The aortic arch and the descending thoracic aorta were carefully dissected free for ex vivo study of endothelial function. The heart and ascending aorta were thereafter perfusion-fixed with formalin for subsequent histological analysis. Plaque burden at various vascular sites was studied in an additional group of cholesterol-fed ApoE−/− mice (n=7) of similar age. To address the potential vascular effects of hypercholesterolaemia, another group of C57Bl/6 mice (9 weeks old; n=16; M&B Breeding and Research Centre) underwent the same experimental protocol for ex vivo vascular function.
High-resolution Doppler ultrasound
Echocardiographic studies were performed on anaesthetized animals (1.0–1.8% isoflurane; Baxter Healthcare, Chicago, IL, U.S.A.). The anaesthetized animals were placed on a heating pad to maintain normothermia during the echocardiographic procedure. The chest area was shaved and ultrasound coupling gel was liberally applied to the left chest wall. All ultrasound scanning was performed using a high-frequency 15 MHz linear transducer (Sonos 5500; Agilent, Andover, MA, U.S.A.) connected to an ultrasound system (HDI 5000; ATL Ultrasound, Bothell, WA, U.S.A.) with a maximum frame rate of 230 frames/s. 2D echocardiographic loops of at least 20 cardiac cycles and M-mode tracings were stored digitally on a magneto-optical disk for off-line analysis. M-mode was recorded from the long-axis view for measurement of systolic and diastolic LV (left ventricular) internal diameters. Heart rate was derived from a simultaneous ECG trace using a lead II configuration. Off-line measurements were performed using an image analysis system (MedArch Viewer 2.1; Secure Archive, Indianapolis, IN, U.S.A.) according to the guidelines from the American Society of Echocardiography . FS (fractional shortening), EF (ejection fraction) and CO (cardiac output) were calculated according to formulas published previously , and SV (stroke volume) was calculated based on EDV (end-diastolic volume) and ESV (end-systolic volume) using the Teichholtz formula .
Flow velocities in the aortic root were measured with pulsed Doppler under guidance of 2D-echocardiography. Imaging of the aortic root was achieved by means of a tilted two-chamber view (Figure 1). The pulsed-Doppler measurements were performed using 6 MHz Doppler under 2D guidance. The gate size was 1 mm targeting the aortic root approx. 1 mm above the aortic valve. In this view, using a steered Doppler beam, the angle between the flow and the Doppler signal was always below 60°.
Invasive measurements of MAP (mean arterial pressure)
At the end of the experimental period, mice were anaesthetized with isoflurane (0.7–1.5%) and a catheter (heat-stretched PE50) was placed into the right femoral artery for direct measurement of arterial pressure. A computerized data acquisition software program (Pharmlab 3.0; AstraZeneca, Mölndal, Sweden) was used to collect the data. Following stabilization of the arterial pressure trace (for 20–30 min), data were averaged over a period of 2 min.
Ex vivo vascular function
Following arterial pressure measurements, the animals were killed by an overdose of pentobarbital (Apoteksbolaget, Uppsala, Sweden). The aortic arch was ligated between the left carotid artery and left subclavian artery branch. A vessel segment immediately distal to the left subclavian artery branch (proximal descending aorta) and a segment from the thoracic aorta at the level of the sixth intercostal branch (mid-thoracic aorta) was dissected free. The vessel strips, approx. 3 mm in length, were rinsed and placed in an ice-cold PSS [physiological salt solution; 119 mmol/l NaCl, 4.7 mmol/l KCl, 5.5 mmol/l glucose, 25 mmol/l NaHCO3, 1.18 mmol/l KH2PO4, 0.026 mmol/l EDTA, 2.5 mmol/lCaCl2, 1.17 mmol/l MgSO4, equilibrated with 5% CO2 (pH 7.4)].
The vessel strips were mounted on two stainless-steel hooks (100 μm diameter), one of which was connected to a force transducer. Vessel preparations were immersed in PSS-containing vessel chambers (40 ml). Chambers were gassed continuously (95% O2/5% CO2) and a constant temperature (37 °C) was achieved by means of an external circulatory heating system. Isometric tension forces were recorded and amplified through a Grass system, and the data were collected by a digital data acquisition system (PharmLab; AstraZeneca). Paired vessel segments from animals fed the control or high-cholesterol diets were investigated in the same vessel chamber. After the mounting procedure, each preparation was stretched to 3 mN (resting tension) and equilibrated for 30 min. Subsequently, segments were stretched further to 10 mN and stabilized for 10 min.
Before the onset of the experiment, all vessel strips were pre-activated by the addition of KCl (100 mmol/l) and NE [noradrenaline (norepinephrine); 10−8 mol/l; Sigma Chemicals, St. Louis, MO, U.S.A.] in order to produce maximal contraction. After subsequent equilibration, the endothelium-dependent vasodilatory responses were studied by means of ACh (acetylcholine)-induced vasodilation (10−9–10−5 mol/l). SNP (sodium nitroprusside; 10−5–10−4 mol/l; Sigma) was used to evaluate the endothelium-independent relaxation. A similar protocol for ACh and SNP was repeated following incubation with the non-selective NO-inhibitor L-NNA (NG-nitro-L-arginine; 10−4 mol/l; Sigma).
En face quantification of regional plaque burden
The additional group of ApoE−/− mice used for plaque quantification was fixed with paraformaldehyde at 100 mmHg. The aortae were cut along the length of the vessel and mounted for microscopic inspection and quantification. Percentage plaque area was assessed in the proximal descending and mid-thoracic aorta respectively.
After fixation with 4% paraformaldehyde at 100 mmHg for 5–10 min, the aortic arch and heart were paraffin-embedded and serially sectioned. The sections were stained with Picro-Sirius Red for quantification of collagen, and Miller's elastin for IEL (internal elastica lamina) measurement. Computerized morphometry was performed by means of the Olympus Micro Image analysis software (version 4.0; Olympus Optical, Tokyo, Japan). The plaque area and the IEL length were measured and the vessel areas were calculated based on IEL length to avoid any folding artefacts. Lumen area was calculated as vessel area minus plaque area.
All data are expressed as means±S.E.M. For analysis of vascular reactivity, non-linear regression analysis (one-site competition) was used (Prism™ 3.0; GraphPad, San Diego, CA, U.S.A.) and ED50, maximum precontraction and dilation values were obtained. Mann–Whitney test was used to compare the cholesterol-fed and control groups with regard to flow velocities, cardiac functions, body weight, lumen area, MAP, collagen content, regional plaque burden, percentage plaque area and for vascular reactivity parameters mentioned above. Correlation between flow velocity and percentage plaque area in the aortic root was calculated using Spearman's non-parametric test. A value of P<0.05 was considered to be statistically significant.
MAP levels, recorded invasively via the femoral arteries, were similar in the cholesterol-fed and control groups (Table 1).
High-resolution Doppler ultrasound
Cholesterol-fed ApoE−/− mice had a significantly lower BW (body weight) compared with the control mice (15±0.5 compared with 22±0.3 g respectively; P<0.001). There were no significant differences in EDV, SV, EF or FS (Table 1).
Doppler measurement of the aortic root flow velocity
Treatment for 5 weeks with a high-cholesterol diet caused a significant increase in aortic root flow velocity in ApoE−/− mice compared with the control mice (1.25±0.07 compared with 1.01±0.07 m/s respectively; P<0.05), whereas flow velocities in the aortic arch were not affected (results not shown). In the cholesterol-fed group, 84% had a flow velocity >1.2 m/s, whereas 86% of animals in the control group had a flow velocity <1.2 m/s.
The cross-sectional area of the aortic root, calculated based on IEL, was similar in both the cholesterol-fed and control groups (0.99±0.06 compared with 1.00±0.05 mm2 respectively). Cholesterol-fed mice had significantly increased total plaque area compared with control mice (0.33±0.07 compared with 0.10±0.07 mm2 respectively; P<0.01; Figure 2), resulting in a significantly decreased lumen area (0.66±0.03 compared with 0.91±0.05 mm2 respectively; P<0.01).
Total collagen content normalized to plaque area did not differ between the cholesterol-fed and control ApoE−/− mice (results not shown).
Correlation between aortic root flow velocity and percentage plaque area
Percentage plaque area, representing the degree of lumen narrowing, was calculated as the ratio between plaque area and cross-sectional area in the aortic root. Percentage plaque area was 33±2 and 9±2% in the cholesterol-fed and control groups respectively (P<0.01). A significant correlation was found between maximum flow velocity and percentage plaque area in the aortic root in the whole study population (Spearman r=0.7180, P<0.01). This correlation was also significant when cholesterol-fed ApoE−/− mice only were studied (Spearman r=0.9276, P<0.05).
Regional plaque burden
Percentage plaque area was significantly greater in the proximal descending aorta compared with the mid-thoracic region of ApoE−/− mice (3.53±0.65 compared with 0.18±0.12% respectively; P<0.01).
Ex vivo vascular function
NE sensitivity was similar in both the cholesterol-fed and control groups (results not shown). The maximal ACh-induced vasodilatory response in the proximal descending aorta was significantly greater in the control mice than in the cholesterol-fed mice. Mean percentage maximal vasodilation was 37.7±5.6 and 57.3±5.7% in the cholesterol-fed and control groups respectively (P<0.05). SNP induced similar near-maximal vasodilation (100%) in both of the groups (92±5% in the cholesterol-fed group compared with 96±3% in the control group). Following incubation with L-NNA, the ACh-induced vasodilatory response was completely abolished in both of the groups (Figure 3a).
In the mid-thoracic aorta, ACh-induced vasodilation did not differ between diet groups (maximal percentage vasodilation, 50.5±5.6% in the cholesterol-fed group compared with 62.6±8.3% in the control group). SNP also produced similar complete vasodilation in both groups (100±0% in the cholesterol-fed group compared with 90±10% in the control group). Incubation with L-NNA abolished the ACh-mediated vasodilation in the mid-thoracic aorta in both groups (Figure 3b).
A similar NE sensitivity was found in the C57Bl/6 groups, regardless of vascular region and diets (results not shown). The ACh-induced vasodilation was similar between the proximal descending and the mid-thoracic aorta in both diet groups (maximal percentage vasodilation in proximal descending aorta, 48.8±7.2% in the cholesterol-fed group compared with 44.5±5.8% in the control group; mid-thoracic aorta, 54.8±5.3% in the cholesterol-fed group compared with 49.3±3.8% in the control group). SNP induced similar near-maximal vasodilation (100%) in the two studied vascular regions in both the cholesterol-fed and control groups (maximal percentage vasodilation in proximal descending aorta, 92±3 compared with 93±2% respectively; mid thoracic aorta, 91±3 compared with 93±2% respectively). Following incubation with L-NNA, the ACh-induced vasodilatory response was completely abolished in both vascular regions independently of dietary treatment.
In the present study, we have shown that consumption of a high-cholesterol diet caused impaired arterial expansive remodelling in ApoE−/− mice with significantly altered in vivo haemodynamics as a consequence. Cholesterol-fed ApoE−/− animals also showed decreased NO-dependent vasodilation in atherosclerosis-prone regions, whereas endothelial function was intact in regions less prone to atherosclerosis in the descending thoracic aorta.
Arterial outward remodelling has been suggested to be a defence mechanism developed to minimize lumen occlusion and thereby restore normal blood flow and delay the development of stenosis . Local haemodynamic parameters, such as shear stress, are believed to greatly influence remodelling . A local increase in shear stress, caused by an increased flow, is known to induce increased NO production, causing vasodilation and thereby reducing the shear stress to normal levels. If high shear conditions frequently occur, it is possible that this mechanism may result in structural arterial expansive remodelling . Release of vasodilators, such as NO, in response to increased shear works as a homoeostatic control mechanism preserving normal shear load on the vascular wall. However, this scenario is based on the assumption of an intact and functional endothelium. Failure of FMD (flow-mediated vasodilation) causes chronic high shear conditions and this may induce release of other vascular factors affecting structural components of the vascular wall, e.g. PDGF (platelet-derived growth factor) [10,20] and MMPs (matrix metalloproteinases) [10,21].
Using the high-cholesterol diet protocol, a lumen obstructive lesion was observed in the aortic root of ApoE−/− mice. Hypercholesterolaemia has been shown to give rise to outward remodelling both in humans  and, after moderate long-term hypercholesterolaemia, in the aortic root of ApoE−/− mice . However, in the 5 week protocol in the present study, no sign of outward remodelling was observed. Although possible direct effects of the diet on factors involved in the remodelling process cannot be excluded based on the present study, data from regional vascular function studies oppose a general toxic effect of the diet, since endothelial function is preserved in the non-plaque burdened vascular region of ApoE−/− mice. Thus it is conceivable that the lack of shear-stress-mediated functional outward arterial remodelling was one of the crucial pathophysiological mechanisms leading to the lumen obstructive lesions.
Endothelial function was assessed in vascular segments with significantly different atherosclerotic burden. To avoid any potential destruction of the plaque morphology in the ex vivo setting, we measured regional plaque burden in an additional group of animals. However, thanks to the genetic homogeneity of the animals, variation of plaque burden in ApoE−/− mice is relatively small between the individual animals. In the present study, despite the exaggerated hypercholesterolaemia, the ACh-mediated vasodilatory responses in vessel segments from mid-thoracic aorta were essentially preserved. However, in vessel segments from high turbulent flow regions, known to be prone to atherosclerosis , the NO-dependent vasodilation was impaired. These findings suggest that cholesterol-induced endothelial dysfunction is associated more with regional atherogenic processes rather than systemic effects. We have also shown that the maximum ACh-mediated vasodilatory response, rather than ACh sensitivity, was reduced in aorta from cholesterol-fed compared with control animals, and the difference was abolished following NO-blockade, suggesting decreased NO-releasing capacity in the cholesterol-treated animals. In vivo, impaired NO-dependent vasodilation is associated with decreased FMD . Thus it is likely that the NO-dependent vasodilation is impaired in the aortic root of cholesterol-fed ApoE−/− mice, with even more profound lesions, which may cause reduced FMD. Taken together, impaired NO-dependent vasodilation could be one possible physiological mechanism accelerating the lumen occlusive disease process.
To explore a potential cause and consequence relationship between plaque formation and impaired endothelial function further, ex vivo vascular function was assessed in a group of wild-type C57Bl/6 mice. No differences in endothelial function were observed in either the proximal descending or the mid-thoracic aortic regions between the normo- and hyper-cholesterolaemic C57Bl/6 mice. This observation may support further the hypothesis that impairment of endothelial function was related to the presence of atherosclerotic lesions rather than cholesterol in this experimental model. Furthermore, the similar vasodilatory capacity observed between the two vascular regions in the hypercholesterolaemic C57Bl/6 mice might contradict confounding effects of site-specific vascular structure on the impaired endothelial function in the cholesterol-fed ApoE−/− mice.
In man, numerous studies have shown both acute  and chronic  effects of cholesterol on endothelial function. For example, Celermajer and co-workers  have shown that impaired FMD was related to lipoprotein(a) levels in familial hypercholesterolaemic children. Indeed, acute incubation with oxLDL (oxidized low-density lipoprotein) has been shown to inhibit endothelium-dependent vasodilation in isolated porcine coronary arteries . In the present study, we aimed to investigate the intrinsic vasodilatory capacity of the vessels without potential influences from circulating systemic factors, such as cholesterol, lipoprotein(a) and oxLDL etc, which are present in the in vivo human setting. This might explain the discrepancy between the in vivo and our ex vivo observations, which demonstrated intact endothelial function in non-atherosclerotic vascular regions. Thus, in the absence of systemic metabolic factors, hypercholesterolaemia-induced endothelial dysfunction seems to be localized in atherosclerotic vascular regions in ApoE−/− mice. However, in vivo, in the presence of severe hypercholesterolaemia, it is highly conceivable that endothelium might be dysfunctional in these animals. To address this issue, further methodological development aimed at assessing in vivo mouse endothelial function, in line with the FMD approach, could be valuable. Interestingly, Woo et al.  have shown recently that endothelial dysfunction could be reversed to a large extent by exercise and dietary treatment in hypercholesterolaemic obese children, which may suggest that endothelial dysfunction is reversible in subjects without established atherosclerosis.
In the present study, we validated a non-invasive technique for measurement of flow velocity in the mouse aortic root. This was achieved by using 2D high-resolution echocardiography to guide a defined pulsed-Doppler gate used for flow velocity measurements. In a previous study, Doppler ultrasound measurements in aged ApoE−/− mice revealed a 49% increase in ascending aortic root velocity compared with age-matched C57Bl/6J mice . However, compared with the present study, the Doppler technique used was blinded and a large Doppler gate was used making it difficult to pinpoint the exact region of the vascular tree from which the signals were derived. The technique employed in the present study makes it possible to positively distinguish between Doppler signals from the aortic root, aortic arch and descending aorta. When measuring flow velocities in the aortic arch, there were no differences between the groups, indicating that the increase in flow velocity detected in the aortic root in the cholesterol-fed group is a locally derived incident. Histological analysis confirmed that cholesterol-fed animals had significantly reduced lumen areas, which suggests that the increased flow velocity in cholesterol-fed mice is a local haemodynamic phenomenon secondary to morphological alterations, whereas the velocities were normalized in the aortic arch. Finally, the significant correlation between the peak flow velocity and the percentage plaque area in the aortic root provides direct evidence of the validity of this non-invasive imaging method to assess atherosclerosis burden in mice.
MAP was collected following 20–30 min of stabilization of the arterial pressure trace after which data were averaged over a period of 2 min. Although only the anaesthetized MAP was assessed, we still consider it relevant as a control for potential systemic haemodynamic influences during Doppler echocardiography, which was indeed performed during anaesthesia. By using the current blood pressure acquisition equipment, only MAP values could be collected in a reliable way, and the possible impact of pulse pressure on the flow velocity profile could be of interest for further investigation. Nevertheless, the correlation between the peak flow velocity and the degree of aortic root narrowing still suggests that morphological change is the major factor accounting for the altered haemodynamic conditions in the aortic root of cholesterol-fed ApoE−/− mice.
It has been suggested that systolic cardiac function and peripheral vascular resistance may influence maximum flow velocity in the aortic root . In the present study, anaesthetized MAP, as discussed above, and CO did not differ significantly between the groups; these results suggest that peripheral vascular resistance was also similar. Furthermore, based on our echocardiographic data, no significant differences were detected regarding the systolic cardiac function, e.g. EF and FS, between the groups. Thus it appears likely that the increased flow velocities in the aortic root are closely linked to the atherosclerotic process.
To test the validity of our technique further, we applied the protocol to a group of male ApoE−/− mice undergoing a similar treatment protocol in a parallel study in our laboratory (M. E. Johansson, A. Wickman, S. M. Fitzgerald, L. Gan and G. Bergström, unpublished work). In these mice, a significant increase in aortic root velocity was also observed in the cholesterol-fed group, closely matching the histologically evaluated degree of lumen obstruction, suggesting that the non-invasive imaging technique is feasible and reproducible.
In summary, cholesterol feeding caused a failure of arterial expansive remodelling and induced lumen obstructive plaque formation in ApoE−/− mice with significant haemodynamic consequences. This phenomenon is associated with impaired NO-dependent vasodilation in regions prone to developing atherosclerosis.
This study was supported by the Swedish Medical Research Council (LG14601, 14602 and 15168, and GB12580), the Swedish Heart Lung Foundation, the Lundberg Foundation, King Gustaf V and Queen Victoria Foundation (to A.W.), the Åke Wiberg Foundation, the Memorial Foundation of Lars Hierta, and the Magnus Bergvall Foundation. SWEGENE supported the postdoctoral position of A.W. We thank Mrs Jia Jing for her excellent technical assistance. This study was performed with the assistance of SWEGENE Centre for Mouse Physiology.
Abbreviations: 2D, two-dimensional; ACh, acetylcholine; ApoE−/−, apolipoprotein E deficient; BW, body weight; CO, cardiac output; EDV, end-diastolic volume; EF, ejection fraction; ESV, end-systolic volume; FMD, flow-mediated vasodilation; FS, fractional shortening; HR, heart rate; IEL, internal elastica lamina; L-NNA, NG-nitro-L-arginine; LV, left ventricular; MAP, mean arterial pressure; NE, noradrenaline; NO, nitric oxide; SNP, sodium nitroprusside; SV, stroke volume
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