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1Department of Biomedical Engineering, University of California, Irvine, California; 2Institute of Neurology, Medical Center of Fudan University, Shanghai, People's Republic of China; and 3Division of Endocrinology, Department of Medicine, University of California, Irvine, California
Submitted 11 October 2005 ; accepted in final form 16 November 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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nitrite; strain; stress; elastic modulus; ovariectomy
Epidemiological studies have shown that the incidence of cardiovascular disease is higher in men than in premenopausal women but increases in postmenopausal women (5). The increase appears to be related to the diminishing concentration of female hormone that characterizes this stage (35). In contrast, the Women's Health Initiative (WHI) and the Heart and Estrogen/Progestin Replacement Study (HERS) clinical trials showed that estrogen plus medroxyprogesterone does not prevent primary or secondary arteriosclerotic heart disease (11, 38). Although the effects observed in the WHI trial have been criticized because of the formulation and regimens (22), more definitive animal and clinical studies are required to underscore the significance of estrogen in modulation of vascular function.
Increasing evidence suggests that estrogen-induced augmentation of nitric oxide (NO) production by vascular endothelium may contribute to its vasculoprotective effects (4, 32). The NO plays a pivotal role in regulation of blood pressure and regional blood flow (31). It also inhibits platelet aggregation, thrombus formation, and leukocyte adhesion and relaxes the vascular smooth muscle cells (24). In the endothelium, estrogen enhances production and release of NO by increasing endothelial isoform of NO synthase (eNOS) activity or expression of eNOS gene or both (18). Inhibition of eNOS may lead to reduced basal vasodilation and hence results in hypertension (13). Huang et al. (12) reported that ovariectomy increased the basal tone of microvessels of female rats, and estrogen replacement restored the myogenic tone of arterioles to a level identical to that of normal female rats. Acs et al. (1) described that ovariectomy can increase total peripheral resistance by affecting the morphological caliber of resistance microvessels, which is an independent risk factor for several other cardiovascular diseases.
The present study was designed to determine the effects of short-term (4–6 wk) estrogen deficiency on the structural and biomechanical properties of the aorta and carotid and femoral artery by using an ovariectomized (ovx) mouse model. We hypothesized that estrogen modulates the mechanical homeostasis in the blood vessel wall through NO production. An additional hypothesis was that mechanical strain is the most sensitive measure of mechanical status of the blood vessel wall. The former hypothesis is based on a recent study that showed that the vasodilatory effect of estradiol (E2) is estrogen receptor-mediated stimulation of NO production (8). The latter hypothesis is based on our observation that strain is remarkably uniform throughout the vasculature (9). In conjunction with the changes in the mechanical properties, we assessed the biochemical changes, including nitrite and nitrate (NOx) in the vessel wall and the distribution of NOS in the endothelium. Our novel findings provide a direct relation between NO and the mechanical status of vessel wall that underscores the role of endogenous estrogen, NO, and eNOS on the mechanical homeostasis of elastic and muscular arteries.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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Sixty-three homozygous inbred mice (C57BL/6 strain) were used in this study. Thirty-two female mice with body weight of 23.7 ± 3.3 g (mean ± SD) and age 12.4 ± 2.5 wk underwent ovariectomy at age 6 or 7 wk by the vendor (Jackson Laboratory). The ovx mice were ovariectomized for 
1 mo. Nine female mice lacking the gene for eNOS (eNOS–/–) with body weight of 19.7 ± 1.3 g and age 11.3 ± 1.5 wk were also provided by Jackson Laboratory. The remaining 22 mice, including 12 intact female (20.6 ± 1.6 g body wt and 11.1 ± 1.6 wk old) and 10 males (27.7 ± 2.7 g body wt and 11.5 ± 1.8 wk old), were used for comparison.
The mice were anesthetized with intraperitoneal injections of ketamine (80 mg/kg) and xylazine (8 mg/kg). Arterial blood pressure was measured by inserting a catheter into the common carotid artery connected to a pressure transducer. Heparin (200 U/ml) was used to prevent blood clots in the vessels via a femoral artery catheter. All animal experiments were performed in accordance with national and local ethical guidelines, including the Institute of Laboratory Animal Research Guide, Public Health Service policy, Animal Welfare Act, and an approved University of California-Irvine protocol regarding the use of animals in research.
Mechanical Testing: Pressure-Diameter-Length Relation
The left femoral artery was carefully exposed and cannulated with a heat-stretched catheter prepared from Micro-Renathane tubing. Water-resistant carbon particles were used to mark the femoral artery segment to measure axial changes. The cannulated femoral artery was filled with Krebs-Ringer-lactate (KRL) solution and was then ligated in the proximal (cephalad) position. The artery was preconditioned with five cyclic changes in pressure from 0 to 150 mmHg in a triangular form. The perfusion pressure was increased in 30-mmHg step increments from 60 to 150 mmHg in a staircase manner. We have verified that the vessel segments studied were leak-free such that there was no pressure drop, and hence the pressure applied was the pressure transmitted to the vessel wall.
The external geometry of the femoral artery, at the pressurized state, was photographed at x50 magnification (3.8-µm spatial resolution) to obtain the loaded outer diameter and the in vivo axial length. A similar mechanical protocol was later repeated in the left carotid artery. The mouse was then euthanized with an overdose of ketamine and xylazine via the jugular vein. The thoracic aorta was carefully exposed and cannulated by a 23-gauge needle at the middle position. The cannulated thoracic aorta in situ was filled with KRL solution and was then ligated at the distal position close to diaphragm. Cab-O-Sil (0.35% by wt), a colloidal silica, was mixed into the KRL solution to prevent flow through the microvessels and hence attain zero-flow condition. A similar pressure-diameter-length relation was recorded for the aorta.
No-load and Zero-Stress State
After the mechanical testing, the marked blood vessel segments of the aorta and carotid and femoral arteries were carefully dissected and placed into a Ca2+-free Krebs solution aerated with 95% O2-5% CO2. The vessel segment was then cut transversely into five or six rings with lengths of 0.15–0.2 mm. Each ring was photographed at x100 magnification (1.9-µm spatial resolution) in the no-load state (zero transmural pressure) and then cut radially by a scissor to reveal the zero-stress state. The ring opened into a sector and gradually approached a constant opening angle. The cross section of each sector was photographed 30 min after the radial cut. The morphological measurements of the in vitro axial length, inner and outer circumference, wall thickness (WT), and area in the no-load and zero-stress state were made from the images by using a morphometric analysis system (Sigma Scan).
Nitrite/Nitrate Measurements
The right femoral and carotid arteries were isolated immediately, and the corresponding left vessels were used for mechanical testing after the mouse was euthanized. The right arteries were coarsely grounded in methanol in ice bath. Homogenate (50 µl in total volume) was centrifuged at 800 g at 4°C for 10 min, and the supernatant was then assayed for nitrite (NO2–) and nitrate (NO3–) by using the Greiss reagent. The nitrite and nitrate concentrations in solution were measured by the combination of a diazo coupling method and high-performance liquid chromatography (ENO-20 NOx Analyzer; EiCom, Kyoto, Japan). The method for NOx analysis has been previously described in detail (20). Briefly, the peak of detected voltage for nitrite and nitrate was converted into nitrite and nitrate concentration by use of calibration solution. The signals were corrected for contamination by subtraction of the control nitrite concentration. The endogenous NO production was evaluated as the concentration of nitrite or nitrate per volume of arterial wall (nmol/mm3).
Western Blot Analysis
The thoracic and abdominal aorta, excluding the segment used for biomechanical testing, was removed and homogenized in a lysis buffer containing 50 mmol/l 
-glycerophosphate, 100 µmol/l sodium orthovanadate, 2 mmol/l magnesium chloride, 1 mmol/l EGTA, 0.5% Triton X-100, 1 mmol/l DL-dithiothreitol, 20 µmol/l pepstatin, 20 µmol/l leupeptin, 0.1 U/ml aprotinin, and 1 mmol/l phenylmethylsulfonyl fluoride and then incubated on ice for 1 h. The sample was centrifuged at 1,000 g for 15 min at 1°C, and the supernatant was drawn off. The total protein was measured by BCA kit (Bio-Rad). Equal amounts of protein (25 µg) were loaded and electrophoresed in 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane. After being blocked for 2 h in 8% dried milk in TBS-Tween buffer, the membrane was incubated overnight at 4°C with specific primary antibody (1:1,000 dilution in blocking buffer, BD Transduction Laboratory). The membrane was then rinsed and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h (1:3,000 dilution in blocking buffer, Bio-Rad). Specific eNOS protein was detected by enhanced chemiluminescence (ECL, Amersham) and evaluated by densitometry (Sigma Scan). All samples from each group were simultaneously probed with anti--actin, a mouse monoclonal antibody (primary antibody 1:1,000 dilution in blocking buffer, Santa Cruz Biotechnology) to correct for sample loading. Positive control protein was obtained from cultured human vascular endothelial cells provided by BD Transduction Laboratory.
Immunohistochemistry
The ascending aorta was cut and fixed with 10% formalin in phosphate buffer for at least 6 h. The vessel was then embedded in paraffin and cross-sectioned at 4 µm. Sections were treated with 0.3% hydrogen peroxide-methanol solution for 10 min to inhibit endogeneous peroxidase activity and blocked for 30 min with buffer containing 1.5% normal goat serum. Sections were then incubated with an eNOS-specific primary antibody (1:200 dilution in buffer, BD Transduction Laboratory) for 1 h at 37°C. After being incubated for 30 min at room temperature with biotinylated anti-mouse antibody (1:200 dilution in buffer), the sections were visualized with VecStain ABC kit (Vector Laboratories) by using diaminobenzidine tetrahydrochloride. Sections were then counterstained with hemotoxylin, dehydrated, cleared, coverslipped with Permount, and examined by light microscopy. Controls were obtained by using arterial sections incubated without the corresponding primary antibody.
Statistical Analysis
All values for mechanical analysis and Western blot quantitative analysis were expressed as means ± SD. Significance of the differences between the various groups was evaluated by two-way ANOVA or t-test. The results were considered statistically significant when P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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The outer diameter of aorta and carotid and femoral artery was obtained by direct measurements in the loaded state and was found to increase with pressure (P < 0.01). The inner diameter increased (P < 0.01) while the WT (intima-media) decreased (P < 0.01) with increase in pressure for all arteries as computed from Eq. 1B (see APPENDIX). Figure 1, A and B, shows the WT and the WT-to-radius (WTTR) ratio for all arteries for male, female, ovx, and eNOS–/– mice at physiological pressure (120 mmHg), respectively. The WT of the carotid artery was significantly increased in ovx and eNOS–/– mice compared with those in controls (P < 0.01). In the femoral artery, the WT was significantly increased in ovx and eNOS–/– mice compared with female control (P < 0.001) but was not significantly different from male control. The WTTR ratio of the carotid artery was greater in ovx and eNOS–/– mice than in controls (P < 0.01) as shown in Fig. 1B. In the femoral artery, the WTTR ratio was significantly increased in eNOS–/– mice compared with female and male control mice (P < 0.05), which were not significantly different from ovx mice. In the aorta, there was no significant difference for both WT and the WTTR ratio among the four groups. Hence, the changes in WT and WTTR were only observed in the smaller vessels (carotid and femoral) but not the aorta.
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The reproducibility of the NOx measurements was initially assessed by obtaining measurements in quadruplicate. We found the variation to be <5%. Subsequently, all measurements were made in duplicate, and the mean was reported. The NO concentration (measured as nitrite and nitrate, the stable degradation by-products of NO) of the carotid and femoral artery for male, female, ovx, and eNOS–/– mice is shown in Fig. 4. A significant decrease in nitrite (P < 0.001) and nitrate (P < 0.05) concentration was found in ovx and eNOS–/– mice compared with controls for both femoral and carotid artery, which indicates that the NO production decreased in ovx and eNOS–/– mice.
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-actin antibody reacted with a 42-kDa protein corresponding to the size of -actin. The final value for eNOS densitometry was computed as the ratio of eNOS to -actin. The eNOS protein expression significantly decreased in ovx mice compared with the controls (P < 0.001). As expected, there was no eNOS expression in eNOS–/– mice, which confirmed that the eNOS–/– mice lack detectable eNOS protein. Figure 6 shows immunohistochemical localization of eNOS protein in the aorta in female and male control, ovx, and eNOS–/– mice. As expected, immunoreactivity for eNOS was restricted to the endothelium. In contrast to the relatively heavy positive staining in female and male control mice, the immunoreactivity of eNOS appeared to be sparsely located in the endothelium in ovx mice (Fig. 6C). As expected, no eNOS staining was visible in the eNOS–/– mice (Fig. 6D).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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The systemic blood pressure is regulated through integration of cardiac, neuronal, humoral, and vascular mechanisms. Recently, Milsted et al. (23) have reported an elevation in blood pressure in normotensive rats after 7 wk of ovariectomy. Sasaki et al. (33) found that the systolic blood pressure of ovx rats was significantly higher than that of sham-operated rats, and this increase in systolic blood pressure was suppressed by estrogen supplementation. Contrary to these findings, Nickenig et al. (27) reported no significant differences in blood pressure between ovx or sham-operated rats. In the present study, we found no significant increase in arterial blood pressure in the 4–6 wk ovx mice compared with the female and male control mice. These differences may be explained by the duration of ovariectomy. Our results suggest that the short-term (<6 wk) absence of circulatory estrogen may not significantly affect arterial blood pressure in mice. Hence, the observed remodeling in ovx mice is not due to changes in pressure. We did find, however, that mice deficient in eNOS have increased blood pressure relative to female and male control mice. This result is consistent with an earlier report by Huang et al. (13), who demonstrated the development of hypertension for independently generated eNOS knockout mice. Moreover, Steudel et al. (36) reported that eNOS is a key enzyme responsible for providing basal pulmonary NO release and that congenital eNOS deficiency produces mild pulmonary hypertension in mice. Our data suggest that eNOS is important for maintenance of normal blood pressure. The eNOS–/– mice may lack the vasorelaxing pathway mediated by endothelium-derived NO on vascular smooth muscle cells.
Structural Properties
Because functional estrogen receptors exist in vascular endothelial cells and vascular smooth muscular cells, it is thought that estrogen may act directly on the arterial wall to inhibit intimal thickening (15). Leukocyte infiltration, endothelial injury, and synthesis of extracellular matrix are important steps in the development of intimal thickening. Beldekas et al. (2) have reported that estrogen may inhibit adhesion of leukocytes to endothelial cells in vitro. Moreover, estrogen replacement improves endothelium-dependent vasomotor responses, suggesting that estrogen could protect endothelial cells from injury (7). It is well known that estrogen enhances production of NO by increasing eNOS activity (4, 32, 12). Extensive evidence suggests that NO may inhibit vascular smooth muscle proliferation in vitro and in vivo (24). The long-term block of eNOS causes coronary microvascular remodeling and fibrosis in rats in vivo (28). In the present study, we have found an increase in WT (intima-media) of the femoral and carotid artery in ovx and eNOS–/– mice as shown in Fig. 1. Interestingly, we found no changes in WT of the larger vessel (aorta). The WTTR ratio mirrored the changes in WT for the elastic vessels (carotid artery and aorta). For the muscular vessel (femoral artery), however, the WTTR was significantly higher in the eNOS–/– but not the WT group. The increase in WTTR ratio is consistent with the increase in pressure in the eNOS–/– animals, which attempts to normalize the circumferential stress (16). The differences in WT between the small muscular and elastic vessels may be due to the presence of tone, which is likely absent during the in vitro experiments. Hence, the in vitro carotid artery is more similar to the in vivo conditions than is the femoral artery.
Mechanical Properties
Arterial mechanical properties can be influenced by several factors, such as heart rate, atherosclerotic plaque, blood pressure, and age. The effects of estrogen on the biomechanical properties of the mouse arterial wall have not been investigated. The novel results of this study show that ovariectomy significantly increases the circumferential elastic modulus in all arteries, suggesting an increase in arterial stiffness (Fig. 3). For the mice lacking the eNOS gene, we found that the circumferential modulus significantly increased only in the aorta but not in the carotid or femoral artery compared with controls. This is an interesting observation, which suggests that there may be a time lag in aortic wall remodeling that is responsible for the increase in stiffness. There are data to support the difference in time course of remodeling in different-sized vessels. For example, atherosclerotic lesions develop spontaneously in a time-dependent manner in apoE knockout mice (25, 29). Nakashima et al. (25) reported that lesion begins at the aortic root at 10 wk of age, and then the proximal coronary and the principal branches of the aorta, including pulmonary and carotid arteries, become involved in lesions later in time. In our study, we only chose age 10–12 wk eNOS–/– mice as an experimental group. It is possible that the development of vascular remodeling in eNOS–/– mice is also age dependent.
It is interesting to note that there was no significant difference for the circumferential and axial elastic modulus between male and female mice (Fig. 3). Moreover, Western blot, immunostaining, and NOx analysis have confirmed that there was no difference for eNOS protein expression and NO concentration between female and male control mice. This implies that all three arteries have similar elasticity in female and male mice despite the reduced endogenous estrogen in male mice. It may be that there is a compensatory mechanism for NO that maintains a lower stiffness in the male mice.
A Novel Relation Between NO and Mechanical Properties
Some in vitro and in vivo studies have shown that estrogen enhances vasorelaxant activity by elevating NO release and NOS activity (4, 12, 32). NO is released by the endothelium, which is a potent vasodilator that inhibits extracellular matrix turnover and could thus modify the mechanical properties of the arterial wall (37). Recently, we have found that acute arterial wall vasodilation occurs in response to E2 in ovx mice, mediated through a rapid cross-talk from estrogen receptors to endothelial NO synthase (8). Hayashi et al. (10) reported basal release of NO from aortic rings of ovx rats and rabbits were lower than their respective female control animals. In contrast, pregnancy (a highly estrogenic state) results in vascular remodeling with an increased distensibility of mesenteric arteries in rats (21). Pregnancy increases uterine arterial NO-mediated vasodilatation via increased basal NO production in humans (26).
The present results demonstrate that ovariectomy increases the aortic, carotid, and femoral arterial stiffness, decreases eNOS protein expression, and reduces endogenous NO. The disruption of the eNOS gene results in a reduction in arterial elasticity in the aorta and in NO production in the femoral and carotid artery. In eNOS–/– mice, the nitrite (NO2–) level was decreased by 3–5-fold, and nitrate (NO3–) level was decreased by 2- to 3-fold compared with controls. Hence, the expression of the eNOS gene correlates with the endogenous NO concentration. Furthermore, a significant relation between NOx and the structural and mechanical properties is observed, as shown in Fig. 7. The stress and strain show a significant linear relation while the WT and modulus reveal an inverse relation. To our knowledge, this is the first direct connection between NO and structural and mechanical properties, which suggests causation between the NO bioavailability and the biomechanical status of the vessel wall.
It is interesting to note that the relations between NOx and stress, strain, or WT have higher correlation coefficients than that of elastic modulus. Clinically, the change in modulus is often used as a predictor of vascular dysfunction and atherosclerosis. The present data show that stress, strain, or WT has less scatter in their relation to NOx and should be considered as viable predictors of vascular disease.
Homeostasis of Strain
There exists a biomechanical homeostasis in the cardiovascular system that has important implications for mechanotransduction and for vascular growth and remodeling. We recently found that the circumferential strain (computed in reference to the zero-stress state) responds faster and recovers more quickly than the circumferential stress (19). We also found that although stress varies by a factor of 15, strain varies within a factor of 5 throughout the aorta and the coronary arterial tree down to 10-µm vessels (more than 1,500 difference in vessel diameter) (9). In the present study, our data show that the absence of estrogen and disruption of eNOS gene can significantly decrease the circumferential strain in all arteries. The strain was decreased by 12–18% in the aorta, 31–40% in the carotid artery, and 16–30% in the femoral artery, respectively. For the circumferential stress, however, significant change was observed only in the carotid artery for both ovx and eNOS–/– mice. In the aorta and femoral artery, the stress decreased in ovx and eNOS–/– mice, but it failed to be significant.
In summary, strain is the only mechanical parameter that shows significant changes in all arteries in ovx and eNOS–/– mice compared with males and females. These findings suggest that the strain is the most sensitive mechanical parameter compared with stress or elastic modulus. This is reasonable as strain is related to deformation or conformational changes at the molecular level and is a measurable quantity, unlike stress and modulus, which have to be computed.
Critique of Method
It is very well known that blood vessels possess viscoelastic material properties that include creep, relaxation, and hysteresis. In the present study, we only considered the elastic response of the vessels. The imposed pressure had a staircase profile with a 30-s interval for each staircase, i.e., each pressure value was maintained for this duration. This duration was necessary to ensure the captured image was in focus. In initial experiments, we acquired video images to continuously record the change in diameter of the vessel after pressure change (creep). We noted that the change in diameter reached an asymptotic value within 20 s, and the magnitude of creep was small compared with the initial elastic response (2–10%, depending on the vessel). Hence, digital photographs were taken at similar times for all animal groups to decrease analysis time. In summary, the present report relates to the elastic properties, and future studies are needed to address the changes in the viscoelastic properties that may affect circulatory dynamics and function.
Although eNOS expression was evaluated in the aorta while NO metabolites were evaluated in the carotid and femoral arteries, a definitive conclusion can be made that eNOS expression in the vessel wall is related to nitrate or nitrite. In several specimens, we sampled eNOS expression in the carotid and femoral arteries and NOx in aortic tissue. The results were consistent with our conclusions.
Summary and Conclusions
The present study shows that estrogen deficiency increases the aortic, carotid, and femoral artery stiffness as quantified by the elastic modulus in ovx mice. The observations in ovx and eNOS–/– mice indicate that estrogen and NO derived from eNOS play an important role in the development and maintenance of the structural and mechanical properties of elastic and muscular arteries. Furthermore, we show that the structural and mechanical properties are significantly correlated with the NOx status of the blood vessel wall. Finally, we found that strain is the most sensitive mechanical parameter, which may underscore its role in mechanotransduction and remodeling. These findings provide evidence for the significant role of NO-mediated estrogen on the maintenance of vascular mechanical homeostasis and hence function.
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APPENDIX |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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Incompressibility condition.
The incompressibility condition for a cylindrical vessel can be expressed as
 | (1A) |
z= l/lo is the stretch ratio in the axial direction, where l and lo are the vessel length in the loaded and zero-stress state, respectively; and Ao is the wall area in the no-load state. The WT at the loaded state was computed as the difference between the outer and inner radius of the vessel at various pressures as
 | (1B) |
z were measured quantities.
Strain and stress.
The circumferential deformation of the artery may be described by Green strain (
) as
 | (2A) |
is the midwall circumferential stretch ratio ( = Cm/C
), Cm refers to the midwall circumference (average of inner and outer circumference) of the vessel in the loaded or no-load state, and C refers to the midwall circumference in the zero-stress state as described in the previous section. The axial Green strain is given by
 | (2B) |
z is defined above. At equilibrium, the average circumferential Cauchy stress in a cylinder can be computed as
 | (3A) |
 | (3B) |
is the circumferential stretch ratio. The axial second Piola-Kirchhoff stress, under similar assumptions, is given by
 | (3C) |
Elastic moduli.
In analogy to an isotropic tube, Dobrin and Doyle (6) expressed the relationship between the circumferential and axial strains for an anisotropic vessel as
 | (4A) |
 | (4B) |
signifies a change in a quantity and 
is the Poisson's ratio ( = –/z). Equations 4A and 4B can be solved for the respective incremental moduli in the axial and circumferential directions as
 | (5A) |
 | (5B) |
tends to 
/, which is the definition of the tangent modulus for a uniaxial experiment.
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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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TOPABSTRACTMETHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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-estradiol on the biosynthesis of collagen in cultured bovine aortic smooth muscle cells. Biochemistry 20: 2162–2167, 1981.[CrossRef][Medline]
-, but not -estrogen receptor. Circ Res 90: 413–419, 2002.This article has been cited by other articles:
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  E. S. Buys, P. Sips, P. Vermeersch, M. J. Raher, E. Rogge, F. Ichinose, M. Dewerchin, K. D. Bloch, S. Janssens, and P. Brouckaert Gender-specific hypertension and responsiveness to nitric oxide in sGC{alpha}1 knockout mice Cardiovasc Res, July 1, 2008; 79(1): 179 - 186. [Abstract] [Full Text] [PDF]
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