Smooth Muscle Dysfunction in Resistance Arteries of the Staggerer Mouse, a Mutant of the Nuclear Receptor RORα
Retinoic acid receptor–related orphan receptor α (RORα) is a member of the nuclear receptor superfamily. The mouse mutant staggerer (sg/sg) carries a deletion within the RORα gene. RORα plays a major role in cellular differentiation during development and growth. In the present study, we found a lower mean arterial blood pressure in sg/sg than in +/+ mice (80.1±1.2 and 87.0±0.9 mm Hg, respectively; P<0.0002) and a smaller increase in blood pressure after in vivo injections of phenylephrine. To elucidate the mechanisms responsible for this phenotype, we investigated the vascular reactivity of large vessels (aorta and carotid arteries) and small resistance mesenteric arteries in response to mechanical forces or vasoactive agents. Arteries from sg/sg and +/+ mice were studied in vitro in arteriographs. Vascular responses of large vessels to all stimuli were similar in both groups. However, we found a markedly altered vascular function in mesenteric arteries from sg/sg mice. Flow-induced dilation, pressure-induced myogenic tone, responses to endothelium-dependent or -independent vasodilators, and responses to vasoconstrictors were significantly reduced in sg/sg compared with +/+ mice. We also determined by Western blot analysis the expression of smooth muscle (SM)-myosin, calponin, and heavy (h)-caldesmon, in large and small arteries of sg/sg and +/+ mice, and found a marked decrease in the expression of these contractile proteins in mesenteric arteries of sg/sg mice. Our findings provide the first evidence that functional RORα is required for normal contractile phenotype of smooth muscle cells (SMCs) in small resistance arteries and suggest that RORα might be involved in the differentiation of SMCs in mesenteric arteries.
Retinoic acid receptor–related orphan receptor α (RORα) is a member of the nuclear hormone-receptor superfamily.1 Its primary sequence associates it with the class of retinoic acid receptors.2 As other nuclear receptors, RORα is a transcriptional factor whose activity has to be modulated by a specific ligand, although so far, this ligand remains unknown. RORα exists in four alternatively spliced isoforms: RORα1 through 3 and RORα4 (also termed RZRα).2 The typical structure of a nuclear receptor includes a ligand binding domain (LBD) linked to a DNA domain (DBD) via a hinge region and a modulator binding site in the N-terminal region of the receptor. A spontaneous mutation consisting of a 122-bp deletion on the LBD of the RORα gene has been identified in mice.1 The homozygous staggerer mutant mouse (sg/sg), which carries this deletion, shows cerebellar ataxia and early neurodegeneration2,3⇓ and exhibits immune abnormalities.4 In addition, lipopolysaccharide-activated peripheral macrophages from sg/sg mice overexpress IL-1β.5 We recently reported that homozygous sg/sg mutant mice fed a high-fat diet develop increased atherosclerotic lesions compared with wild-type C57BL/6 mice6 and show hypoalphalipoproteinemia, associated with a decrease in apolipoprotein (apo)-AI gene transactivation.7 However, the function of RORα in vascular biology remains unknown for the most part. In the present work, we tested the hypothesis that RORα might be involved in the control of arterial blood pressure (BP) and found that BP and the in vivo response to a vasoconstrictor agonist, phenylephrine, were decreased in sg/sg compared with +/+ mice. To further investigate the mechanisms responsible for this phenotype, we studied the role of RORα in the control of the vasomotor tone in large arteries and in small mesenteric arteries that play a key role in the maintenance of arterial BP. We investigated the vascular reactivity of aorta, carotid artery, and mesenteric arteries in response to mechanical forces (pressure and flow) or vasoactive agents in sg/sg and wild-type littermate mice. We found altered responses to all stimuli, mechanical and pharmacological, specifically in small resistance arteries of sg/sg mice. Finally, we determined by Western blot analysis that the expression of smooth muscle cell (SMC) contractile protein (smooth muscle [SM]-myosin, calponin, and heavy [h]-caldesmon) was altered in mesenteric arteries from sg/sg mice.
Materials and Methods
The staggerer mutant mice used in this study were maintained on a C57BL/6J genetic background in our colony. Sg/sg mutant mice and their +/+ littermates were obtained by crossing known heterozygous (sg/+) mice. Homozygous sg/sg offspring were identified by their clinical ataxia, whereas +/+ and sg/+ mice were genotyped by PCR. All mice were maintained in a temperature-controlled room (25°C) with a 12-hour light-dark cycle. The animals received a standard diet (A04, Usine d’Alimentation Rationelle) and water ad libitum.
Genomic DNA was extracted from tail biopsies and amplified in two sets of reaction, one for each allele. The staggerer allele primers were 5′-CGTTTGGCAAACTCCACC-3′ and 5′-GATTGAAAGC-TGACTCGTTCC-3′. The + allele primers were 5′-TCTCCCTTCT-CAGTCCTGACA-3′ and 5′-TATATTCCACCACACGGCAA-3′. The amplified fragments (+, 318 bp; sg, 450 bp) were detected by electrophoresis on agarose gel.
In Vivo Studies
Homozygous sg/sg and wild-type +/+ male mice (10 to 12 weeks old) were anesthetized with isoflurane for BP measurement through a catheter placed in the right carotid artery as previously described.8 After BP stabilization, a 100-μL bolus of phenylephrine (PE) (10 μmol/L) was injected in the femoral artery, and peak values of BP were recorded. After BP stabilization, 100 μL of acetylcholine (Ach) (10 μmol/L) was then injected to evaluate in vivo the endothelium-dependent relaxation.
Study of Pressure- and Flow-Dependent Tone in Isolated Arteries
Vessels were collected from 10- to 12-week-old homozygous sg/sg and wild-type +/+ male mice. The animals were anesthetized with isoflurane for BP measurement through a catheter in the right carotid artery as previously described.8 After BP measurement, mice were killed, and the mesentery, the left carotid artery, and the aorta were isolated. The procedure followed in the care and euthanasia of the study animals was in accordance with the European Community standards on the care and use of laboratory animals (Ministère de l’Agriculture, France, authorization No. 00577).
The left carotid artery and a segment of mesenteric artery (≈100 μm) were then cannulated at both ends in a video-monitored perfusion system9 (LSI) as previously described.8,10⇓ Briefly, arteries were bathed in a physiological salt solution (PSS) (pH 7.4; Po2 160 mm Hg, Pco2 37 mm Hg). Pressure was controlled by a servo-perfusion system and flow was generated by a peristaltic pump. Diameter changes were measured when intraluminal pressure was increased from 10 to 150 mm Hg. Pressure was then set at 75 mm Hg and flow increased by steps. At the end of each experiment, arteries were perfused and superfused with a Ca2+-free PSS containing EGTA (2 mmol/L) and sodium nitroprusside (SNP) (10 μmol/L), and pressure steps were repeated to determine the passive diameter of the artery.8,10⇓
Pharmacological Study in Isolated Arteries
Segments of mesenteric artery (≈160 to 180 μm in external diameter), carotid artery, and aorta were mounted in a myograph as previously described11 and bathed in PSS. Isometric tension was recorded and collected by a Biopac data acquisition system (Biopac MP 100). PE and serotonin (5-HT) cumulative concentration-response curves were obtained. Data were expressed as mN force per mm (carotid and mesenteric arteries) or as mN force per mg weight (aorta). Ach, isoprenaline (IP), and SNP concentration-response curves were obtained after preconstriction of the artery with PE (10 to 100 nmol/L, ≈70% of the maximal contractile response). Data were expressed as percent dilation of PE-induced preconstriction.
In a series of experiments, the aorta, the left carotid artery, and the mesenteric arteries were removed from sg/sg and +/+ mice, and quickly frozen in liquid nitrogen. Total proteins were extracted from frozen tissues as previously described.12 Polyclonal antibodies against the following proteins were used: calponin, 13 h-caldesmon,13 SM-myosin,13 and endothelial nitric oxide synthase (eNOS) (Santa Cruz). Specific protein was detected by chemiluminescence reaction (ECL+ kit, Amersham), and chemiluminescent signals were visualized using a computer-based imaging system (Fuji LAS 1000 plus, Fujifilm Medical Systems).
Results were expressed as mean±SEM. EC50 or IC50 (concentration of agonist required to induce half the maximum response) and Emax (maximal response) were calculated for each artery.8 Significance of the differences between groups was determined by analysis of variance (one- or two-factor ANOVA or ANOVA for consecutive measurements, when appropriate). Means were compared by paired t test or by Bonferroni test for multigroup comparisons. A value of P<0.05 was considered to be significant.
Body weight was lower in sg/sg (n=8) than in +/+ (n=10) mice (16.7±0.5 and 19.7±0.5 g, respectively; P<0.001). Mean BP was significantly decreased in sg/sg (n=11) compared with +/+ mice (n=12) (80.1±1.2 versus 87.0±0.9 mm Hg, n=12; P<0.0002).
Nevertheless, characteristics of the mesenteric and carotid arteries (Table 1) did not differ between sg/sg and +/+ mice. Similarly, passive diameter curves (Figure 1) were unaffected in sg/sg mice compared with controls.
In Vivo Studies
The effects of PE and Ach on BP were tested in vivo. PE (10 μmol/L) induced a smaller increase in BP in sg/sg mice compared with +/+ mice (73.5±2.9% versus 84.2±2.9% of basal BP; P<0.05).
After BP stabilization, Ach (10 μmol/L) induced a significantly larger decrease in BP in +/+ compared with sg/sg mice (104.8±4.6% versus 86.3±3.3% of PE-induced BP increase; P<0.01).
Pressure- and Flow-Dependent Tone in Isolated Arteries
To evaluate whether the difference in in vivo vasoactive responses observed in sg/sg mice resulted from changes in arterial contractile properties, vasoreactivity of large and small arteries was tested in vitro. In isolated vessels, pressure induces the development of a myogenic tone. This was significantly decreased in mesenteric resistance arteries (Figure 1) but not in carotid arteries of sg/sg compared with +/+ mice (data not shown).
Increasing flow by steps induced a progressive dilation in arteries from both groups of mice. However, in mesenteric resistance arteries, flow (shear stress)-induced dilation was significantly lower in sg/sg than in +/+ mice. In contrast, carotid artery dilation did not differ between the two groups (Figure 2).
Pharmacological Study in Isolated Arteries
The contractile capacity of mesenteric arteries, carotid arteries, and aorta were tested in vitro. The maximal vascular contraction was obtained with KCl (125 mmol/L). Contraction to KCl was not affected by the mutation of the RORα gene, although it tended to decrease in mesenteric arteries (Table 2).
5-HT and PE induced a concentration-dependent contraction of all three types of arteries. Contractions to 5-HT and PE were significantly decreased in mesenteric arteries of sg/sg mice compared with +/+ mice but were unaffected in both the aorta and the carotid arteries (Table 2 and Figure 3).
Endothelium-dependent dilation to Ach was decreased in mesenteric and carotid arteries of sg/sg mice (Table 2 and Figure 4). Endothelium-independent dilation to SNP (Figure 4) or IP (Figure 5) was also significantly decreased in mesenteric arteries of sg/sg mice compared with +/+ mice but was not affected in either the carotid arteries or the aorta (Table 2). In a separate series of experiments, SNP-induced dilation was performed in deendothelialized mesenteric resistance arteries. Under these conditions, SNP-induced dilation was also significantly decreased in sg/sg mice compared with +/+ mice (Table 2).
To investigate the mechanisms involved in the alteration of vascular reactivity in sg/sg mice, we determined protein levels of SM-myosin, calponin, h-caldesmon, and eNOS by Western blot analysis. No difference in protein expression was observed in the aorta between sg/sg and +/+ mice (Figure 6A). In contrast, in mesenteric arteries, we found a markedly decreased expression of the contractile proteins in sg/sg compared with +/+ mice, whereas eNOS protein level was not different between the two groups (Figure 6B).
The main finding of the present study is the demonstration of the involvement of the nuclear receptor RORα in the regulation of vascular tone. Small resistance arteries play a key role in the maintenance of arterial BP by controlling peripheral resistances. In the present study, we have shown that the staggerer homozygous mutant mice have a significantly lower mean BP and develop decreased in vivo vasoactive responses to PE compared with their wild-type littermates. The in vivo findings were supported by in vitro pharmacological studies showing that the responses of mesenteric arteries to both vasodilator and vasoconstrictor agents are reduced in sg/sg mice.
The flow-induced dilation reflects the endothelium-dependent dilation of arteries, which relax their SMCs in response to an increase in flow.14 Although passive diameter values were unaffected in sg/sg mice, flow-induced dilation was significantly decreased in mesenteric arteries of the mutant mice. To investigate whether this modification was due to a lack of NO production by the endothelial cells or to decreased SMC sensitivity to NO, we tested the response of the mesenteric arteries to Ach, a potent endothelium-dependent dilator, and to SNP, an NO donor that directly stimulates the cGMP pathway in SMCs. We observed a diminished response to SNP in sg/sg mice, suggesting a muscular rather than an endothelial basis for the dysfunction in the mesenteric arteries. In agreement with this finding, SNP-induced dilation in the absence of endothelium was also diminished in sg/sg mice compared with +/+ mice, and eNOS protein expression was found similar in sg/sg and +/+ mice. In addition, IP-induced dilation, which involves the cAMP-dependent pathway, was similarly decreased in mesenteric arteries isolated from sg/sg mice. The SMC dysfunction was further corroborated by a decreased response to 5-HT and PE in mesenteric arteries of sg/sg mice.
Pressure-induced tone (myogenic tone) is a characteristic of small resistance arteries and of some veins.15–17⇓⇓ It is opposed by flow-induced dilation in vitro, as well as in vivo.18,19⇓ These two mechanical stimuli determine the basal vascular tone in resistance arteries and allow for a rapid adaptation to changes in blood flow and pressure. Whereas myogenic tone is mainly independent of endothelial17 and humoral20 factors, flow (shear stress) triggers an endothelium-dependent dilation.21 In the present study, we have shown that myogenic tone was strongly attenuated in mesenteric arteries of sg/sg mice. Interestingly, the reactivity of large conductance arteries was not affected by the mutation of the RORα gene. Indeed, response of the carotid artery and the aorta to Ach or SNP, as well as the contraction to PE or 5-HT, was not different in sg/sg compared with wild-type mice.
Response to mechanical factors is different between large and small resistance arteries. Large arteries, in response to increased pressure, dilate in a passive way involving an endothelium-independent mechanism. In resistance arteries, the increase in BP leads to the contraction of SMCs (myogenic tone) to maintain constant blood supply to tissues. This difference between small resistance arteries and large arteries is best explained by the disparity in the structure and function of the two types of vessels.15 Contrasting dependencies on RORα gene expression between large and small arteries possibly reflect phenotypical differences in SMCs between the two types of arteries.
Previous studies have shown RORα as a regulator in various developing systems. In differentiating adipocytes, RORα was upregulated during late adipogenesis.22 More importantly, RORα is involved in the differentiation of myoblasts23 and osteoblasts.24 The exogenous expression of a dominant-negative RORα vector represses the expression of the muscle-specific helix-loop-helix transcription factor MyoD and the general transcriptional coactivator p300.23 RORα is also highly expressed in the Purkinje cells of the cerebellum during the postnatal development of brain and plays a major role in differentiation and survival of Purkinje cells.25 Taken together, these previous studies suggest that RORα is involved in the cascade of events leading to the cell differentiation during development. Our present findings also indicate that RORα might be involved in the differentiation of small-artery SMCs. We found that the mutation of RORα leads to a reduced expression of differentiation markers, including calponin and h-caldesmon, in mesenteric arteries, but not in aorta. Furthermore, the expression of the contractile protein SM-myosin is also altered in mesenteric arteries from sg/sg mice. This provides a mechanistic explanation for the observed decrease in contractile functions of small arteries in sg/sg mice. Decreased expression of SMC marker proteins, calponin and h-caldesmon, and of contractile protein SM-myosin, is a hallmark of dedifferentiation of SMCs in culture,26 which lose their contractile properties and adopt a synthetic phenotype.26–28⇓⇓ However, this is unlikely to account for the alteration in the relaxing response in mesenteric arteries of sg/sg. Further studies are required to elucidate the mechanisms responsible for this phenotype.
In conclusion, the present study demonstrates a physiological role for RORα in the control of vasomotor tone in small resistance arteries. We conclude that functional RORα is required for a normal contractile and dilatory phenotype of SMCs in small resistance arteries.
Sandrine Besnard is a recipient of a grant from Société Française d’Athérosclérose.
Original received March 13, 2001; resubmission received October 2, 2001; revised resubmission received February 21, 2002; accepted February 21, 2002.
- ↵Trenkner E, Hoffmann MK. Defective development of the thymus and immunological abnormalities in the neurological mouse mutation staggerer. J Neurosci. 1986; 6: 1733–1737.
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- ↵Henrion D, Iglarz M, Levy B. Chronic endothelin-1 improves NO-dependent flow-induced dilation in resistance arteries from normotensive and hypertensive rats. Arterioscler Thromb Vasc Biol. 1999; 19: 2148–2153.
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- ↵Lau P, Bailey P, Dowhan DH, Muscat GE. Exogenous expression of a dominant negative RORα1 vector in muscle cells impairs differentiation: RORα1 directly interacts with p300 and myoD. Nucleic Acids Res. 1999; 27: 411–420.
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