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(Hypertension. 1997;29:1156-1164.)
© 1997 American Heart Association, Inc.

Articles

Effect of Mechanical Loading on Vascular _1D- and _1B-Adrenergic Receptor Expression

Mary L. Clements; Albert J. Banes; ; James E. Faber

From the Departments of Physiology (M.L.C., J.E.F.) and Vascular Surgery (A.J.B.), The University of North Carolina, Chapel Hill.

	Abstract

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Abstract Heterogeneous distribution and function of ₁-adrenergic receptor subtypes on arterial and venous vessels, together with evidence for altered -adrenergic receptor expression in hypertension, led us to examine whether mechanical load influences expression of _1B -and _1D-adrenergic receptors in rat aortic smooth muscle cells (SMCs). We used RNase protection and radioligand binding assays to measure mRNA and ₁-adrenergic receptor density. In the first model, SMCs were subjected to phasic loading using flexible culture plates. As a positive control for the load stimulus, postconfluent, quiescent passage 5 cells demonstrated the expected load-dependent morphological realignment. However, no changes were detected in expression of either _1D- or _1B-adrenergic receptor mRNAs or receptor density after 24 to 48 hours of loading. ß-Actin and SMC-specific -actin mRNA, as well as cell number and per-cell total RNA and protein, were also unaffected. In a second model, intact thoracic aortas, in either the presence or absence of endothelial cells, were cultured for 48 hours under tonic load. Like cultured cells, 48 hours of load did not affect SMC expression of ₁-adrenergic receptor mRNAs. We used suprarenal aortic coarctation to examine effects of increased pressure in vivo. As with the previous in vitro and in situ models, hypertension (30 days) had no effect on expression of _1B- and _1D-adrenergic receptor mRNAs in the suprarenal aorta compared with sham coarctation. To separate pressure per se from humoral influences, we also measured mRNAs in the subrenal, normotensive aorta. _1B mRNA levels decreased to 68±14% of sham-coarcted controls in subrenal aorta exposed to normal blood pressure but also to systemic humoral changes induced by coarctation. As a positive control for a load effect, SMC-specific -actin mRNA increased for loaded aorta in organ culture and in hypertensive aorta in vivo, whereas expression of ß-actin mRNA was unaffected. These results from cell culture, organ culture, and in vivo models suggest that pressure (load) alone has no effect on _1B- and _1D-adrenergic receptor expression. In coarctation hypertension, smooth muscle protected from the hypertension showed a decline in _1B mRNA that may be due to a humoral factor or factors.

Key Words: muscle, smooth, vascular • receptors, adrenergic, alpha • gene expression • growth

	Introduction

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The ₁-Adrenergic receptors (ARs) on smooth muscle cells (SMCs) mediate sympathetic regulation of peripheral resistance by small arteries/arterioles (resistance vessels) and venous return by venules/veins (capacitance vessels). We have previously shown in rat skeletal muscle that arteriole ₁-AR constriction, which appears to be mediated by the _1D subtype (see Graham et al¹ for a description of the nomenclature for ₁-ARs used in this article), is inhibited by reduced blood flow and tissue oxygen, whereas venule ₁ constriction, which is mediated by the _1B subtype, is unaffected.^{2 3} These segment-specific differences in subtype expression and local modulation, if generalizable, may underlie the well-known metabolic antagonism of adrenergic constriction of resistance but not capacitance vessels that serves to optimize autoregulation of tissue oxygen while at the same time preserving reflex control of venous return.⁴ Although to date few other vessels have been examined, a similar differential ₁-AR subtype distribution mediates constriction of rat thoracic aorta (_1D)^{5 6 7} versus vena cava (_1B).⁸ However, little is known concerning the factor or factors that might direct differential expression of these ₁-AR subtypes by vascular smooth muscle of arteries and veins.

A major physical factor in the vascular SMC microenvironment that could influence expression of _1D-ARs by arterial and _1B-ARs by venous SMCs is the artery-to-vein intravascular pressure gradient that imposes a different mechanical load on SMCs in the two regions. Also congruent with the hypothesis that mechanical load may modulate expression of ₁-ARs are reports that ₁-AR density is increased in some^{9 10} but not all^{11 12 13 14} models of hypertension. In addition, an increase in cardiac myocyte ₁-AR density has been observed in the pressure-overloaded failing ventricle in guinea pigs.¹⁵ However, no studies have identified whether pressure or mechanical loading of muscle cells per se can induce changes in -AR expression, owing to the difficulty in isolating pressure from other factors present in vivo.

Unlike many cells, vascular SMCs are exposed to phasic and tonic changes in pressure-dependent mechanical loading of the vascular wall. Moreover, increases in pressure-induced stretch of SMCs induces myogenic contraction, which is a key physiological regulator of vascular smooth muscle tone. The cellular signaling mechanisms underlying myogenic tone include increases in intracellular calcium, activation of protein kinase C, and phosphorylation of contractile and regulatory proteins. Past studies have demonstrated that SMCs respond to cyclic mechanical loading in vitro with increases in DNA synthesis and expression of platelet-derived growth factor (PDGF) isoform genes.¹⁶ Also, cardiac myocytes subjected to cyclic mechanical loading in vitro increase expression of c-fos¹⁷ as well as increase protein synthesis¹⁸ and myelin basic protein activity.¹⁹ Recent studies have proposed the presence of shear stress mechanosensitive elements in the promoters of several genes. For example, bovine aortic endothelial cells and epithelial-like HeLa cells exhibit shear stress–induced gene expression of monocyte chemotactic protein-1, which is dependent on the presence of a region in the promoter.²⁰ Moreover, a specific 6-bp putative shear stress response element has been localized to several endothelial cell genes, including the PDGF B-chain promoter.²¹ Although the importance of such elements in SMCs is unclear, SMCs are optimized to physiologically detect and respond to alterations in mechanical load with changes in contractile activity and may also possess load-sensitive mechanisms to alter expression of adrenergic receptors.

In the present study, we examined the hypothesis that mechanical load directly modulates expression of _1B- and/or _1D-ARs using measurements of cellular mRNA content and ₁-AR density. Because load may also influence SMC growth¹⁶ and affect interpretations of these measurements, we also determined cell proliferation, -actin, ß-actin, cellular RNA, and protein as controls for an effect of load on growth and non–₁-AR gene expression. Indeed, to our knowledge only one study²² has examined the effect of SMC load per se on SMC growth. We used three models to isolate mechanical loading of SMCs from other factors. Load applied over 48 hours was examined in rat aortic SMCs cultured in vitro on flexible bottom culture plates and also maintained in situ in intact aortic organ culture. In a third model, we used prolonged hypertension produced for 30 days by suprarenal abdominal aortic coarctation to study the effect of increased mechanical load in vivo.

	Methods

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Cell Culture
Each primary culture of SMCs was obtained from pooled thoracic aortas from eight 200-g male rats (Sprague-Dawley, Sasko, Omaha, Neb) as previously described.^{23 24} Cells were maintained at 37°C in a humidified atmosphere of 95% air/5% CO₂ in medium 199 (M199) supplemented with 10% fetal bovine serum, 200 mg/mL L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin; 2.5 mg/L Fungizone was also present when cells were plated on Flex plates (Flexcell). Medium was changed every 2 days. Cells were grown from primary isolation on plastic culture dishes, subcultured at 90% to 95% confluence every 5 to 7 days with 0.05% trypsin–EDTA, seeded at a density of 5000 cells/cm², and studied at passage 5.

Application of Load to Cultured Cells
SMCs were seeded onto collagen-coated flexible membrane bottom plates (Flex). To favor the differentiated phenotype and quiescence, we studied passage 5 cells 4 days after they had reached confluence.^{24 25 26} A separate cell line was used in each experimental replicate. Aortic SMCs were subjected to 24 or 48 hours of cyclic stretch (1 Hz) and compared with time-matched control cells not exposed to stretch. Stretch was achieved by a computer-driven, vacuum-operated Flexercell system (Flexcell) that has been described in detail.²⁷ This system was set to apply a defined vacuum level (15 kPa) to provide a strain gradient across the culture plate membrane bottom and attached cells; the gradient extends from zero in the center to a maximum of 27% elongation at the periphery to achieve an 18% average elongation. Release of the vacuum allows the membrane to return to its original conformation, resulting in one complete cycle per minute. Viable cell number was determined by hemocytometry and trypan blue exclusion. Cell morphology was photographed with an Olympus BHTU microscope equipped with a PM-10ADS photomicrographic system.

Organ Culture
To examine the effects of load on SMCs maintained in situ, we used an organ culture system described in detail elsewhere²⁴ to study thoracic aorta with intact intimal endothelial cells, medial SMCs, and adventitial fibroblasts. Briefly, descending thoracic aortas were isolated under sterile conditions from 200-g rats, and loose connective and fatty tissue were carefully removed in M199 at 4°C with a stereomicroscope. For each experiment, 25-mm lengths of aorta (one per animal) were suspended horizontally from stainless steel wires in tissue culture medium. Circumferential wall tension was provided by a second wire connected to an adjustable weight (0.7 g/mm vessel length [submerged tension]). This load was chosen to provide a preload 1.7- to 1.8-fold in excess of that found to be optimal in contractile studies of isolated rat aortic rings.²⁴ Vessels in the control group were suspended in culture medium in the absence of load. In some experiments, we used a proline loop technique, which avoids mechanical damage and proliferative stimulation of the underlying smooth muscle media, to remove endothelial cells from vessels before placement in organ culture.^{24 28} Serum-free medium (50% Dulbecco's modified Eagle's medium and 50% F-12 containing 2.85 mg/mL insulin, 5 mg/L transferrin, 35.2 mg/L ascorbic acid, 6 µg/mL selenium, and 2.5 mg/L penicillin/streptomycin/Fungizone [GIBCO-BRL]) was used in all organ culture experiments because in preliminary experiments, load produced no differences in cultured SMCs in the presence of serum and in serum-free medium (P. Ping and J.E.F., unpublished observations, 1993). The absence of serum also enabled us to examine the effect of load without potentially confounding serum growth factors. A single experimental time point consisted of eight vessels (animals) each for load and no-load (control) groups to assure adequate population sampling and provide sufficient tissue for analysis. Consistent with other evidence, including catecholamine contraction, that these vessels remain viable in the organ culture environment for at least 48 hours,^{24 29} we found in preliminary studies of aorta immediately after isolation ("fresh") and after 48 hours in culture that total RNA (88±12% of fresh, n=5) and protein (84±17% of fresh, n=5) content per millimeter of vessel length and ß-actin levels (113±13% of fresh, n=3) remained constant.

Aortic Coarctation
We used hypertension produced by coarctation of the rat suprarenal abdominal aorta as an in vivo model to test the effect of load on SMC ₁-AR expression. This approach also allowed us to examine the effect of increased aortic pressure sustained over an extended 30-day interval. At this time, the hypertension is associated with increased angiotensin- and catecholamine-dependent vascular tone.^{30 31 32 33} Coarctation of the aorta was produced in 180- to 200-g male Sprague-Dawley rats using a modification of the techniques of Stanek et al.³² The aorta proximal to the renal arteries was exposed via a sterile abdominal approach. A sleeve of green plastic wrap was sewn in place with a 7-0 silk suture around the aorta between the celiac and superior mesenteric arteries to prevent growth of collateral vessels. Size 0 silk suture was then tied between the vessels around the aorta and a piece of 20-gauge needle tubing to completely occlude blood flow. The tubing was then removed within several seconds. Sham animals consisted of age-matched male littermates in which the abdominal aorta was exposed without subsequent coarctation. Animals received intraperitoneal penicillin and streptomycin and intramuscular cefpiramide sodium (Sepatren, Samitomo Pharmaceutical Co Ltd) and were housed three to a cage. Thirty days after surgery, blood pressures were determined from the femoral and carotid arteries with rats under ketamine (91 mg/mL) and acepromazine (0.91 mg/mL) anesthesia. Animals were then deeply anesthetized and transcardially perfused with 4°C saline. The heart and vessel segments were taken between the aortic arch and several millimeters above the site of constriction as well as from 1 cm below coarctation to the bifurcation of the aorta. Identical vessel segments were taken from sham animals. Left and right ventricles and 5-mm segments of aortas 1 cm above and below the stenosis/sham stenosis were reserved for determination of tissue wet and dry (60°C, 4 hours) weights. This protocol was approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill.

Protein and RNA Determinations
Total soluble protein concentration was determined by BCA assay (Pierce Chemical). Total cellular RNA was extracted from cultured cells using a modified acid guanidinium isothiocyanate/phenol/chloroform method.²⁴ In all organ culture experiments after a 48-hour protocol (see "Results"), thoracic aortas were incubated in an enzyme solution (Hanks' buffered salt solution [GIBCO-BRL], 100 µ/mL penicillin, 100 µg/mL streptomycin, 16 mmol/L sodium bicarbonate, 1 mmol/L calcium chloride dihydrate, pH 7.2, 2 mg/mL collagenase, 2 mg/mL soybean trypsin inhibitor, and 13.5 U/mL elastase [enzymes from Worthington Biochemical Corp]) for 23 minutes at 37°C in 95% air and 5% CO₂. The medial layer (100% SMCs) was then gently separated from the adventitia (>95% fibroblasts^{23 24} ) with fine forceps using a dissection microscope and 4°C tissue bath containing M199. Endothelial cells were then removed or sham removed in the already denuded group by gentle rubbing with a cotton-tipped applicator. Medial segments were washed several times, frozen in liquid nitrogen, and stored at -80°C. RNA was extracted within 2 weeks. Vessel segments from stenosed and sham-stenosed animals were removed, frozen in liquid nitrogen, and stored at -80°C. Vessels were ground to powder at -80°C and homogenized in guanidinium isothiocyanate solution, and RNA was extracted with acid/phenol/chloroform as described above. RNA integrity and concentration were assessed by gel electrophoresis and spectrophotometry.

mRNA Measurement
We used RNase protection assays (RPAs) to measure mRNA levels. Assay details, specificity of our RPAs, and [³²P]CTP-labeled probe construction have been previously described.^{24 34} Briefly, a 709-bp fragment was cleaved from the _1D cDNA and ligated into pGEM 4Z (Promega). This fragment contains part of the coding region of the mRNA corresponding to the putative third intracellular loop; DNA-dependent RNA polymerase T7 produces a cRNA probe of 159 bases that protects 117 bp when hybridized to _1D mRNA. A 306-bp fragment of the _1B-AR cDNA between the putative second and third intracellular loops ligated into pGEM 3Z yields a T7 transcribed cRNA probe of 342 bases and a 306-bp protected hybrid. Plasmids containing the 3' untranslated region of the - and ß-actin mRNAs were provided by G.K. Owens (University of Virginia), and RPAs were developed for these transcripts to serve as controls.²⁴ T7 produces a 303-base cRNA probe that protects 191 bp of the -actin mRNA. A plasmid containing 526 bases of the 3' untranslated mRNA sequence was subcloned into pGEM-4, and the DNA-dependent RNA polymerase SP6 was used to produce the ß-actin riboprobe. A plasmid containing the partial rat cyclophilin cDNA (Ambion) was used in an RPA as internal control for the _1D- and _1B-AR RPAs in the coarctation studies. The cytosolic protein cyclophilin (cyclosporin A binding protein) is ubiquitous in its tissue and phylogenetic distribution, and the SMC content of its low-abundance mRNA is refractory to many stimuli (including mechanical loading, see "Results"), making it a useful internal control gene for low-abundance target ₁-AR mRNAs. Total cellular RNA was incubated with riboprobes in solution, specific hybrids isolated with RNase (either RNase ONE [-actin RPA] or RNase A and T1 [Boehringer Mannheim]), and subjected to electrophoresis. Gels were dried and exposed to autoradiographic film (X-OMAT, Eastman Kodak). mRNA levels were measured according to film densitometry (laser scanner [UMAX, UC630] and the Image program (National Institutes of Health]). Exposure times were adjusted to maintain film signal densities within a linear range.

Determination of Receptor Density
Receptor number was determined by radioligand binding with methods described in detail previously.³⁴ Briefly, aortic SMCs were washed twice with ice-cold phosphate-buffered saline, scraped in 5 mmol/L Tris (pH 7.5) and 5 mmol/L EDTA buffer, and homogenized and centrifuged at 18 000 rpm for 10 minutes at 4°C. The resulting pellets were frozen at -80°C. Within 2 weeks, pellets were thawed on ice, rehomogenized in 5 mmol/L Tris (pH 7.5) and 5 mmol/L EDTA buffer, and centrifuged at 18 000 rpm for 10 minutes at 4°C. The crude membrane was resuspended in incubation buffer (5 mmol/L Tris [pH 7.5] and 5 mmol/L MgCl₂) and protein concentration determined by BCA assay. Membranes were diluted to 1 mg/mL, and saturation binding was determined with [³H]prazosin (New England Nuclear). Nonspecific binding was determined in the presence of 10 µmol/L phentolamine.

Statistics
Data are expressed as mean±SEM; "n" sizes represent separate experiments, each from a separate cell line in cell culture studies and pooled aortic segments from four to eight aortas (one per animal) per time point in organ culture and in vivo studies. Values were compared by ANOVA and/or Student's t test for unpaired observations where appropriate (see figure legends). B_max and K_d values for radioligand binding assays were determined by nonlinear regression analysis (Graphpad). Differences were considered significant at a value of P<.05.

	Results

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Effect of Mechanical Load on _1D- and _1B-AR and Actin Isoform mRNA Expression and Cultured Aortic SMC Growth
Cyclic deformation of postconfluent aortic SMCs for 24 or 48 hours did not significantly alter _1D- or _1B-AR mRNA levels (Fig 1). RPAs were performed on a constant amount of RNA (50 µg) since cell number and cell size (RNA and protein per cell) were unaffected by load (see below). _1D and _1B probes were hybridized with total cellular RNA in the same reaction to examine the specificity of load effects for either ₁ subtype mRNA. Actin isoform mRNA levels were also determined as controls for the specificity of any response to cyclic cell deformation. ß-Actin mRNA content was similar in SMCs subjected to mechanical load for 24 or 48 hours compared with nonloaded time-matched control cells (Fig 1). Expression of smooth muscle–specific -actin mRNA was also unaffected (Fig 1). -Actin and ß-actin were determined in separate RPAs.

View larger version (39K): [in this window] [in a new window]   Figure 1. Effect of cyclic cell deformation on expression of 1D- and 1B-adrenergic receptor (AR) mRNAs in cultured smooth muscle cells. Top, Total RNA (50 µg) from aortic smooth muscle cells subjected to 24 or 48 hours of cyclic load (L, 15 kPa, 60 cycles per minute, 18% average cell elongation) was assayed for 1D- and 1B-AR mRNA using ribonuclease protection assay. Lane 1, probes alone (P); lane 2, probes plus RNases A and T1 (PX); (C); lane 3, 24-hour control; lane 4, 24-hour cyclic load; lane 5, 48-hour control; and lane 6, 48-hour cyclic load. Bottom, Expressions of 1D-AR, 1B-AR, -actin, and ß-actin mRNAs were not significantly altered (ANOVA) in loaded cells compared with nonloaded time-matched control cells for 24 or 48 hours (n=3; separate experiments each from a different cell line).

Since 48 hours of cyclic deformation induces DNA synthesis in vascular smooth muscle,¹⁶ we determined cell number and total cell protein and RNA contents in all experiments as measures of proliferation and hypertrophy, respectively. Mechanical loading did not alter cell number compared with nonloaded time-matched control cells (Fig 2). Cell viability remained at 98% to 100% in all experiments. Total RNA and protein levels per cell were also unaffected (Fig 2). Cell alignment induced by cyclic deformation was examined as a positive control for the effects of deformation on SMCs. As noted by other researchers,^{35 36} after 24 hours of cyclic deformation, SMCs had realigned their long axes perpendicular to the axis of strain (ie, in an annular fashion).

View larger version (23K): [in this window] [in a new window]   Figure 2. Effect of cyclic cell stretch on growth of cultured smooth muscle cells. Cell number, total cellular protein, and total cellular RNA were unaffected (ANOVA) by 24 or 48 hours of cyclic cell deformation compared with time-matched nonloaded control cells (n=3).

Effect of Load on Receptor Number
Membrane receptor density for total ₁-ARs was determined in cultured aortic SMCs subjected to cyclic loading for 48 hours. Total ₁-AR binding to prazosin (B_max) was similar for control SMCs and loaded cells (Table 1). The antagonist dissociation constant (K_d) was also similar for loaded versus nonloaded control cells. At the K_d value, nonspecific binding was 12±0.4% for control and 11±0.7% for loaded cells.

View this table: [in this window] [in a new window]   Table 1. Effect of Mechanical Load on Antagonist Dissociation Constant (Kd) for Prazosin and 1-Adrenergic Receptor Density (Bmax) of Cultured Aortic Smooth Muscle Cells

Effect of Load on SMCs Maintained in Organ Culture
Expression of smooth muscle–specific -actin mRNA was increased 1.7-fold in the aortic medial layer after it was separated from intact aorta that had been subjected to 48 hours of tonic load in organ culture (Fig 3). This effect was specific for the expression of -actin because, in contrast, load did not alter _1D, _1B, and ß-actin mRNA levels. -Actin mRNA was similarly increased in loaded aorta from which endothelial cells had been removed before placement in organ culture compared with nonloaded media also denuded of endothelial cells (2.1±0.2-fold increase, n=3 experiments, P<.05). Removal of endothelial cells before organ culture did not significantly change mRNA levels for ß-actin, _1D, or _1B in loaded media compared with nonloaded time-matched controls. In agreement with previous studies,²⁴ SMC -actin and _1B-AR mRNA were not detected in adventitial fibroblast RNA (from 10 and 50 µg total RNA assayed, respectively). Load did not alter levels of _1D or ß-actin mRNA in adventitia (data not shown). No changes in total protein, total RNA, or dry weight of the medial layer (determined on a per millimeter of vessel media length basis) were evidenced by aorta subjected to 48 hours of tonic load in organ culture (Fig 3).

View larger version (46K): [in this window] [in a new window]   Figure 3. Effect of 48 hours of tonic load on mRNA expression and growth in aortic medial smooth muscle. Top, Representative autoradiograms of ribonuclease protection assays in organ culture experiments for effect of tonic load on -actin and ß-actin mRNAs. Lane 1, -Actin probe plus RNase ONE (Px); lanes 2 and 3, 10 µg total RNA from medial layer isolated from nonloaded control (C) and loaded (L) aorta after 48 hours in organ culture and hybridized with -actin probe (191-bp protected fragment); and lanes 4 and 5, 5 µg total RNA from control and loaded aortic media hybridized with ß-actin probe. Bottom, 48 hours of tonic load did not affect expression of 1D-AR, 1B-AR, or ß-actin mRNAs in cultured intact thoracic aorta. However, unlike cell culture, -actin mRNA was 1.7±0.23-fold increased in loaded vessels. Total protein and RNA per millimeter vessel length were unaltered after 48 hours of tonic load. Numbers at the base of bars represent separate experiments each consisting of eight pooled vessels for control or load group. *P<.05 vs control by unpaired t test.

Effects of Increased Load via Aortic Coarctation on SMCs In Vivo
Thirty days after abdominal aortic coarctation, all measurements (body weight, blood pressure, tissue wet weight) and the thoracoabdominal aorta above the point of stenosis were obtained from stenosed (hypertensive) and sham-stenosed (normotensive) rats. The abdominal aorta was also taken beginning 1 cm below the site of ligation (normotensive in both stenosed and sham-operated animals). Carotid artery pressure was 61% higher in stenosed rats, and femoral artery pressure was identical in both stenosed and sham-operated rats, as expected^{30 31 32 33} (Table 2). Whereas right ventricular wet weight was not affected by aortic stenosis, left ventricular wet weight was significantly increased by 48%. To document any vascular hypertrophic effect of the hypertension, we determined aortic mass as a percentage of total body weight (Fig 4). Wet and dry weights from the suprarenal aorta of stenosed rats were significantly higher than those of sham-stenosed (control) animals (Fig 4). Interestingly, aortic wet and dry weights of the subrenal aorta below the constriction were also significantly greater in stenosed rats than control rats. Total protein and RNA per millimeter of vessel from stenosed suprarenal aorta were increased 1.3- and 1.5-fold, respectively, over sham vessels, whereas subrenal aorta had comparable total protein and RNA content in both stenosed and sham animals (Fig 4). These results indicate that aorta exposed to chronic hypertension hypertrophied in association with an increase in cell size and/or cell number. In contrast, hypertrophy seen in the normotensive subrenal aorta of stenosed animals (Fig 4) may reflect nonprotein increases in carbohydrate and/or lipid. This subrenal hypertrophy suggests that a humoral factor or factors, to which aorta both above and below the site of constriction was exposed, can increase vascular wall mass in the absence of increased wall load.

View this table: [in this window] [in a new window]   Table 2. Baseline Data 30 Days After Suprarenal Aortic Coarctation or Sham (Control) Coarctation of Rats

View larger version (29K): [in this window] [in a new window]   Figure 4. Top, Effect of aortic coarctation hypertension on aortic wall composition. Suprarenal aorta was stenosed or sham-stenosed (control). Thirty days later, the thoracoabdominal aorta above the stenosis (suprarenal) and extending 1 cm below the stenosis to the iliac bifurcation (subrenal) were removed. Bottom, Total protein and RNA obtained from 5-mm segments of aorta above stenosis and the segment from 10 to 15 mm below stenosis, normalized to comparable segments from sham-stenosed (control) animals. Numbers at the base of bars represent separate aortic segments taken from separate animals (except for RNA determinations, in which n represents four pooled segments from four animals). *P<.05, **P<.005 by unpaired t test.

To differentiate the effects of pressure per se from that of humoral factors on mRNA expression, we performed RPAs using RNA from subrenal aorta exposed to normal pressure as well as suprarenal aorta exposed to high pressure in stenosed rats and compared them with results from sham-stenosed (control) vessels. Cyclophilin mRNA was not significantly different in all four vessel segment groups and was therefore used as an internal assay control to normalize mRNAs in each ₁-AR and -actin RPA. Besides load, we have also found that cyclophilin mRNA is unaffected by exposure of cultured aortic SMCs to 4 to 48 hours of norepinephrine (1 µmol/L), angiotensin II (1 nmol/L to 1 µmol/L), hypoxia (21 versus 150 mm Hg PO₂), estrogen (10 nmol/L), dexamethasone (10 nmol/L), cGMP (25 nmol/L), insulin (50 mmol/L), KCl (40 mmol/L), epidermal growth factor (100 ng/mL), retinoic acid (10 nmol/L), endothelin-1 (10 nmol/L), histamine (100 nmol/L), prostaglandin F₂ (100 nmol/L), serotonin (10 µmol/L), and thrombin (10 nmol/L) (unpublished results, 1996). There was no difference in expression of _1D-AR mRNA in stenosed versus sham-stenosed (control) aorta, both from suprarenal as well as subrenal aorta (Fig 5). _1B-AR mRNA was not altered in suprarenal aorta, but levels in subrenal stenosed aorta were decreased to 68±14% of those in sham-stenosed aorta. This suggests the presence of a humoral factor or factors in the stenosed animals that favors reduction of _1B expression in aorta protected from hypertension.

View larger version (43K): [in this window] [in a new window]   Figure 5. Effect of aortic coarctation hypertension on aortic mRNA levels. Top, Representative autoradiograms of ribonuclease protection assay done on aortic total RNA from sham-stenosed (C) or stenosed (S) rats for Fig 4 experiment. Lane 1, 1B-AR and cyclophilin probes (P); lane 2, probes plus 80 µg tRNA and digested with RNases A and T1 (PX); lanes 3 and 5, suprarenal aorta from sham-stenosed rats; lanes 4 and 6, suprarenal aorta from stenosed rats; lane 7, subrenal aorta from sham-stenosed rats; and lane 8, subrenal aorta from stenosed rats. Lanes 3 through 8 were each loaded with total RNA from vessel segments together equaling 80 mm of length. Bottom, mRNA levels in hypertensive suprarenal aorta and normotensive subrenal aorta compared with levels from the same segments in sham coarctation normotensive animals (control). mRNA levels were determined by densitometry, and values shown were normalized to the lower band of cyclophilin protected fragment to correct for RNA loading and within-assay variability. Cyclophilin expression did not differ among the four vessel segment groups. Numbers at the base of bars represent mRNA from four aortic segments pooled from a given vessel segment group. *P<.05 by unpaired t test.

-Actin mRNA expression in aorta exposed to hypertension was increased twofold, whereas -actin mRNA in subrenal aorta was not different from that in sham-stenosed aorta (Fig 5). Thus, in agreement with the organ culture model, pressure alone appears capable of increasing -actin expression. ß-Actin mRNA was unaffected in all groups.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Different -AR subtypes may mediate SMC constriction of arteries, arterioles, venules, and veins.^{2 5 6 7 8} The declining pressure gradient, and thus SMC load, experienced across these vascular segments, together with evidence that increased load may alter ₁-AR expression,^{9 10 15} suggests that load may influence ₁-AR expression by SMCs. We used three models—cell culture, organ culture, and in vivo aortic stenosis—to examine the effects of load on _1D- and _1B-AR expressions. Mechanical loading of early passage cultured aortic SMCs over 48 hours did not alter _1D- or _1B-AR mRNA expression or ₁ receptor density. The absence of effect was not due to the cell culture environment because 48 hours of load also failed to affect expression of these transcript levels in intact aortic media maintained in organ culture. In vivo suprarenal aortic coarctation, which we used to elevate pressure in the aorta above the point of stenosis for a more prolonged period (30 days), also did not alter _1D- or _1B-AR mRNA expression in the hypertensive aorta. The lack of changes in expression was not due to failure of the SMCs to experience load in these in vitro, in situ, and in vivo studies. Load increased -actin expression in the in situ and in vivo (and tended to in the cell culture) models and caused the expected load-induced realignment of cultured cells.^{35 36} The absence of effect also cannot be attributed to complications associated with proliferation or hypertrophy of SMCs. No changes in proliferation were induced in cultured cells, and no cell hypertrophy (that is, no concomitant increase in both RNA and protein levels per cell or millimeter of vessel length²⁴ ) or changes in ß-actin expression were detected in cultured cells or organ cultured aortic wall media (where dry weight was also unaffected). In prolonged coarctation hypertension, the hypertrophy evidenced by both the hypertensive and normotensive aorta, which were similar in magnitude, was not correlated with a change in _1B or _1D expression. Although these studies were confined to aortic SMCs, in two other experiments, each conducted on separate cell lines of passage 5 cultured rat vena caval SMCs, cyclic load over 48 hours also had no effect on mRNA levels for _1B, _1D, -actin, or ß-actin nor on cell number, per-cell protein, or RNA (data not shown). Thus, these results indicate that the mechanical load imposed on SMCs, at least over 2 to 30 days, is not a direct determinant of _1B- and _1D-AR expressions.

A limitation of our radioligand binding results is that only total ₁-AR number was measured and only in the cultured cell model. This is because of inadequate tissue quantity for binding studies obtained from the approximately 100 rats used for each of the organ culture and in vivo studies as well as the absence of suitable antagonists or antibodies available for clear separation of _1B and _1D subtypes in binding or immunodetection studies of cell types such as SMCs with a characteristic very low total ₁-AR density.³⁴ It should also be noted that we did not examine _1A-AR expression in the present study because we are unable to detect _1A mRNA in aortic or vena caval SMC cultures in early passage (passages 2 through 5) using a sensitive _1A RPA against even 100 µg RNA (J.E.F. et al, unpublished results, 1996). Whether SMCs in the intact aorta express _1A-ARs remains unclear because the few previous investigations have not distinguished between SMC, endothelial cell, and adventitial fibroblast sources of RNA extracted from whole vessels. It remains possible, even though mRNA levels did not change, that opposing changes in _1B- and _1D-AR densities may have occurred without a change in overall ₁-AR number as reflected by [³H]prazosin binding. Also noteworthy are differences in the mechanical loads and duration of presentation in the three models we used. Loading was phasic in cell culture and tonic in organ culture, and both phasic and tonic increases over control were produced by coarctation hypertension. In the latter study, pulse pressures from paired carotid and femoral determinations in the same animals were 95±10 mm Hg in the suprarenal and 13±2 mm Hg in the subrenal aorta of coarctation animals (n=12), and 28±4 mm Hg in the suprarenal and 49±3 mm Hg in the subrenal aorta of sham-stenosed animals (n=6). However, despite these differences in the three models, increased load had no effect on _1B or _1D expression.

In the subrenal normotensive aorta of coarcted animals, no change in _1D-AR mRNA expression was detected, but _1B mRNA levels were decreased. Pulse pressure in the subrenal aorta was 74% lower and in the suprarenal aorta 339% higher compared with that in sham-coarcted control segments. These differences may suggest that reduced phasic load in the subrenal aorta below the stenosis decreases _1B-AR expression. However, the lack of an increase in _1B mRNA in the suprarenal aorta, together with no effect of load in cell or organ culture experiments, argues against a role for SMC load in _1B- or _1D-AR expression.

A perhaps more likely mechanism for the decrease in _1B mRNA in the subrenal aorta may involve an interaction of load with a humoral factor present in in vivo hypertension. Plasma renin activity increases within minutes after a reduction in renal perfusion pressure³⁷ and after aortic coarctation.³⁸ While renin levels return to normal or near-normal levels in 1 to 2 weeks, hypertension persists above the point of stenosis in association with increased angiotensin and sympathetic dependence.^{33 38} Since mean pressure was unaffected below the stenosis, the decrease in _1B-AR expression evidenced therein may be due to the presence of a circulating factor or factors such as angiotensin. Although speculative, the increase in pressure above the stenosis may oppose such a humoral-dependent reduction in _1B mRNA levels. The decrease in _1B mRNA expression was specific since no changes in cyclophilin mRNA were detected in the same assay and no effects on _1D mRNA expression levels were observed. Besides a possible role for angiotensin II to induce selective downregulation of _1B expression, differences in the nature of the hypertrophy above versus below the coarctation (see below) could also be involved in the decrease in _1B-AR mRNA below the coarctation.

Although not the main focus of this study, we obtained new findings regarding whether load directly influences growth of SMCs in the intact vascular wall. Although substantial correlative evidence links the degree of blood pressure elevation with the amount of vascular hypertrophy,³⁹ there is little evidence that mechanical load directly causes medial SMC hypertrophy. Application of load over 48 hours did not result in changes in growth of postconfluent, quiescent cultured SMCs or intact aortic media in organ culture. Holycross et al²⁹ also did not find an increase in protein or DNA synthesis in rat aortic rings subjected to 0.6 g/mm tension versus no load over 16 hours in organ culture, in which contractile responses to angiotensin II and phenylephrine were maintained. However, Bardy et al²² used an elegant organ culture model and found that total protein synthesis in the medial layer of the rabbit aorta increased sixfold, when pressurized to 150 mm Hg for 3 days, over basal protein synthesis levels observed at 0 and 80 mm Hg but only when the vessels were maintained in the presence of serum. In our studies, the absence of serum or a shorter duration of loading (2 days) may have prevented detection of an increase in vessel wall protein, RNA, or dry weight, although SMC -actin was increased. We did observe that a prolonged increase in load by aortic coarctation increased wet and dry weights and total protein and RNA for a given amount of vessel length above the point of stenosis. This may reflect the increase in cell size and absence of cell proliferation noted by other researchers,⁴⁰ although hyperplasia was detected by Owens and Reidy⁴¹ in a rat aortic coarctation model in which hypertension was, however, not sustained. Wet and dry weights of the subrenal aorta in stenosed animals also increased but without changes in RNA or protein, indicating wall hypertrophy, possibly from increased carbohydrate and/or lipid in the extracellular matrix. This presence of wall hypertrophy below the coarctation is supported by similar reports of structural remodeling of the vasculature and an increase in the minimal resistance below the stenosis.^{42 43} Interestingly, fibronectin mRNA and protein were increased in the aorta both above and below coarctation when examined 14 days after surgery, whereas laminin was increased and chondroitin sulfate decreased only above the stenosis.⁴⁴ Other studies with prolonged angiotensin infusion confirm a selective increase in fibronectin expression and vascular hypertrophy that are not solely dependent on an increase in pressure.^{45 46 47} Future studies concerning alterations in wall composition are needed that consider both humoral and pulse-pressure differences⁴⁸ as potential influences in these changes above versus below stenosis.

Vascular hypertrophy associated with hypertension has been postulated to result from direct load-induced alterations in the expression of certain growth factors. Wilson et al¹⁶ detected increased PDGF-A expression, thymidine incorporation, and cell number in passages 16 through 29 SMCs of the R22D cell line exposed to 48 hours of phasic loading. However, increased PDGF levels have been associated with reduced -actin mRNA levels in SMCs.²⁶ Moreover, in the present study, -actin mRNA increased in organ cultured SMCs exposed to tonic load and in aorta exposed to hypertension in vivo. This is the first demonstration of a direct effect of load to increase -actin expression in SMCs both in vitro and in vivo. Interestingly, smooth muscle -actin mRNA is reexpressed by adult rat myocardium 24 hours after aortic coarctation–induced load.⁴⁹ Furthermore, the -actin increase we observed does not require concomitant growth since it occurred in organ culture and coarctation models to the same degree in the absence versus presence of vascular wall hypertrophy. Although these increases in -actin mRNA require confirmation at the protein level, SMC expression of collagen^{50 51} and expression of cytoskeletal proteins in skeletal muscle⁵² are also induced by a maintained increase in mechanical load. These effects have been postulated to oppose the increased stress placed on the cells, and our observed increase in -actin mRNA may underlie a similar adaptive response.

In summary, no evidence was obtained suggesting that load is a direct determinant of _1B- or _1D-AR expression by SMCs. We have also not located the presence of the putative shear-sensitive response element²¹ in the known DNA sequence flanking the coding region of the rat _1B and _1D genes. Load does appear to increase -actin gene expression and wall hypertrophy, but in coarctation hypertension, the normotensive vasculature appears to also undergo hypertrophy that is, however, different in composition. Whether these differences or the observed decrease in _1B-AR mRNA in this region and absence of its downregulation in the hypertensive aorta are involved in the increase in catecholamine sensitivity reported in both the suprarenal and subrenal coarcted aorta^{53 54} remain to be determined. Interestingly, unlike the increased norepinephrine sensitivity of the subrenal aorta, which was abolished after endothelial cell removal, in the suprarenal aorta, increased sensitivity was specific for the SMCs and unaffected by endothelial cell inactivation.⁵⁴

	Acknowledgments

 
This study was supported by The National Heart, Lung, and Blood Institute (HL-52610 to J.E.F.) and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR-38121 to A.J.B.); partial predoctoral support from Glaxo, Research Triangle Park, NC; and a predoctoral fellowship award from the American Heart Association, North Carolina Affiliate.

	Footnotes

 
Reprint requests to James E. Faber, Department of Physiology, 474 Medical Sciences Research Building, University of North Carolina, Chapel Hill, NC 27599-7545.

Received August 20, 1996; first decision October 15, 1996; accepted November 25, 1996.

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