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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1122-1129.)
© 1996 American Heart Association, Inc.

Articles

Chronic ₁-Adrenoreceptor Stimulation Increases DNA Synthesis in Rat Arterial Wall

Modulation of Responsiveness After Vascular Injury

D. deBlois; S.M. Schwartz; E.M. van Kleef; J.E. Su; K.A. Griffin; A.K. Bidani; M.J.A.P. Daemen; D.M. Lombardi

the Department of Pathology (D. deB., S.M.S., J.E.S., D.M.L.), University of Washington, Seattle, the Department of Pathology (E.M. van K., M.J.A.P.D.), University of Limburg, Maastricht, Netherlands, and the Department of Medicine (K.A.G., A.K.B.), Loyola Medical Center, Maywood, Ill.

Correspondence Dr D. deBlois, Centre de Recherche Hotel-Dieu de Montreal, 3840 St. Urbain St, Montreal, Quebec H2W 1T8, Canada. E-mail debloisd@ere.umontreal.ca.

	Abstract

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Despite indirect evidence from studies using adrenergic antagonists or sympathectomy, catecholamines have never been shown directly to stimulate vascular smooth muscle cell (SMC) DNA replication in vivo. We studied whether a chronic infusion of catecholamine stimulates SMC replication in vivo in both uninjured arteries and arteries with a neointima formed after vascular injury. Animals were killed after 2 weeks of continuous infusion of bromodeoxyuridine (to label replicating DNA) and either phenylephrine, norepinephrine, or vehicle solution, starting early (third week) or late (ninth week) after balloon injury to the left common carotid artery. In catecholamine-infused animals, the uninjured carotid artery or thoracic aorta showed a marked increase in cross-sectional area (>25%) and frequency of cells undergoing DNA synthesis among medial SMCs (4- to 10-fold) and endothelial cells (13-fold). With catecholamine infusion at 9 to 10 weeks after injury, the media or neointima of the injured carotid artery showed a smaller increase in SMC DNA replication (4-fold) than did the normal arterial media. In contrast, catecholamine infusion at 3 to 4 weeks did not cause significant SMC growth in the injured vessel. Catecholamine infusion caused labile elevations of systolic blood pressure. Taken together with our previous observation that ₁-blockers suppress arterial SMC replication without preventing severe hypertension in the rat, the present data strongly suggest that ₁-adrenoreceptors stimulate SMC DNA synthesis in vivo in arteries with or without intimal thickening, although not during the first weeks after balloon injury. The stimulation of DNA synthesis in vascular cells via the ₁-adrenoreceptor pathway may contribute to the vascular remodeling that occurs in hypertension and atherosclerosis.

Key Words: vascular smooth muscle • endothelium • DNA replication • catecholamine

	Introduction

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Several lines of evidence suggest that sympathetic mediators may act as stimulants of SM growth during vascular development and pathogenesis. For example, medial SMC DNA replication and vascular cross-sectional area are markedly reduced in the growing rabbit ear artery or the growing rat saphenous artery after local surgical sympathectomy.^{1 2 3} Increased sympathetic stimulation of the blood vessels may contribute to the arterial hypertrophy that precedes the development of hypertension in young spontaneously hypertensive rats.^{4 5 6 7 8} Chronic administration of catecholamines also accelerates the progression of atherosclerosis in several animal species, including nonhuman primates.^{9 10 11}

A principal sympathetic mediator is the catecholamine NE, which acts on SMCs via a heterogeneous population of adrenoreceptor subtypes.¹² In vitro studies have revealed a mitogenic effect of adrenoreceptors of the ₁ subtype in SMCs.^{13 14 15} Recent studies with receptor antagonists have implicated ₁-adrenoreceptors in two models of accelerated vascular growth in vivo. Chronic blockade of ₁-adrenoreceptors markedly suppresses the accumulation of SMCs in the intima after balloon injury to arteries in adult rats¹⁶ and rabbits.^{17 18 19} Blockade of ₁-adrenoreceptors also reduces Ang II–stimulated SMC DNA replication in the media of uninjured arteries in the rat without preventing the development of severe hypertension.²⁰ Despite all this indirect evidence, there has been no direct evidence for the stimulation of vascular SMC replication by ₁-adrenoreceptor agonists in vivo. We have reported that a bolus injection of the selective ₁-adrenoreceptor agonist PE rapidly induces gene expression for growth-related molecules in SMCs in the rat thoracic aorta, including ornithine decarboxylase, c-fos, c-myc, and the platelet-derived growth factor A subunit.²¹ These events, however, do not result in increased DNA replication or histone synthesis within 48 hours of treatment.²¹ Because a single pulse with PE in vivo may be too short to stimulate SMC growth, the present studies explored the possibility that chronic stimulation of ₁-adrenoreceptors might be required to increase DNA replication in the vascular wall.

The ability of catecholamines to stimulate growth in SMCs under in vitro culture conditions may be relevant to only certain phenotypic states of SMCs in vivo. Heterogeneity in SMC phenotype has been described in human atherosclerotic arteries^{22 23 24 25} as well as in rat arteries with a neointima after balloon injury.^{26 27 28 29} For instance, SMCs in the rat arterial neointima show a greater replicative response to Ang II in vivo, at least during the third and fourth weeks after vascular injury, than do SMCs in the media of the normal or injured artery.²⁷ Moreover, adrenergic pathways participate in several effects of Ang II in vivo,³⁰ notably in the stimulation of SM growth in the media of uninjured arteries.²⁰ Thus, the present studies considered the possibility that SMCs in the neointima might exhibit an altered replicative response to catecholamines. This possibility was explored by infusing a catecholamine for 2 weeks at either 3 to 4 or 9 to 10 weeks after balloon injury, ie, during either an earlier or later phase of the vascular response to injury. Our observations indicate that chronic activation of ₁-adrenoreceptors in the rat stimulates SMC DNA replication in the media of uninjured arteries as well as the media and neointima of arteries at 9 to 10 but not 3 to 4 weeks after balloon injury.

	Methods

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Vascular Injury
A 2F balloon embolectomy catheter (V. Mueller) was used to cause vascular injury to the left common carotid artery of 400- to 450-g male Sprague-Dawley rats (Zivic Miller). Briefly, rats were anesthetized with a single injection of ketamine (80 mg/kg IP) and xylazine (4 mg/kg IP). The external branch of the left carotid artery was exposed and nicked to insert the balloon catheter into the common carotid artery. The balloon was passed to the level of the aortic arch, inflated, and withdrawn to the carotid artery bifurcation with a twisting motion to deendothelialize and dilate the whole length of the common carotid artery. This procedure was performed a total of three times, after which the external branch of the carotid artery was ligated. The right carotid artery was not manipulated. All surgical procedures were conducted in accordance with institutional guidelines.

Drug Infusion
Two weeks after arterial injury, 52 Sprague-Dawley rats were randomly assigned to four groups of 13 animals and infused for the following 2 weeks with either PE (25 mg·kg^-1·d^-1 in 0.2% ascorbate; both from Sigma Chemical Co), NE (2.5 mg·kg^-1·d^-1 in 0.2% ascorbate; Research Biochemicals International), a catecholamine vehicle composed of 0.2% ascorbate in Ringer's lactate (for a resulting dosage of 0.05 mg·kg^-1·d^-1), or Ringer's lactate alone (24 µL·kg^-1·d^-1). This time course for drug infusion after vascular injury is similar to that in our studies with Ang II in vivo.²⁷ Ascorbate was used as a preservative against the possible oxidative degradation of the catecholamines in the pump; hence the Ringer's lactate vehicle was tested to control for possible effects of ascorbate. The above rate of NE infusion induces gene expression for c-fos and platelet-derived growth factor A chain within 6 hours in the rat aorta.²¹ The relative doses of NE and PE were chosen based on the relative potency of the two analogues to elicit a contractile response in isolated rat aortic rings, an assay in which NE is 10 times more potent than PE.³¹ To explore catecholamine effects at a later time after neointimal formation, two groups of 12 rats were infused for 2 weeks with PE (25 mg·kg^-1·d^-1) or the ascorbate vehicle starting 8 weeks after injury. Drugs were infused continuously via osmotic pumps (Alzet model 2002, Alza Corp) implanted subcutaneously in the back under ether anesthesia or general anesthesia as described above. All rats were also implanted with a second osmotic pump that delivered a continuous infusion of the nonradioactive thymidine analogue BrdU (0.8 mg·kg^-1·d^-1), which is incorporated into DNA during replication in vivo.³²

BP and Body Parameters
Systolic BP was measured by using the tail-cuff plethysmograph method (Narco Biosystems) in selected rats infused with NE (n=5) or ascorbate (n=6). Measurements were started 2 weeks before treatment and performed on conscious, restrained rats that had been trained for the procedure. Because tail-cuff plethysmography was not a reliable method for BP measurements in PE-infused rats (see "Results"), a radiotelemetric method was used³³ to record systolic BP at 10-minute intervals in individually housed rats infused with PE (n=6). Each telemonitoring system (Data Sciences International) was implanted intraperitoneally under pentobarbital anesthesia and included a BP sensor (model TA11PA-C40) connected to a radiofrequency transmitter that was fixed to the peritoneum and hooked to a catheter inserted into the aorta below the level of the renal arteries. The signals transmitted from the pressure sensors were received by a Dataquest IV acquisition system (Data Sciences International).

Rats were weighed immediately before osmotic pump implantation and again at the time of death. Hearts were excised and dried at 60°C for 48 hours before being weighed.

Immunohistochemistry
To select the denuded segments of the injured arteries, Evans Blue was given intravenously 10 minutes before the animal was killed by an overdose of pentobarbital (25 mg/kg IV). Both common carotid arteries, and in certain experimental groups the thoracic aorta, were excised following vessel fixation in situ by intra-arterial perfusion of 4% paraformaldehyde at 100 mm Hg. With rats infused 3 to 4 weeks after injury, vessels were fixed by immersion in 4% paraformaldehyde. Nuclear incorporation of BrdU was used as a marker for de novo synthesis of DNA molecules during the 2-week infusion period. A segment of each paraformaldehyde-fixed vessel was processed according to routine histological procedures and embedded in paraffin. Tissue sections (5 µm) were obtained for immunodetection of incorporated BrdU in the nucleus of SMCs by using an indirect peroxidase-labeled antibody technique.²⁰ Briefly, deparaffinized tissue sections were treated with H₂O₂ (0.3% in methanol) for 20 minutes to block endogenous peroxidase, washed in 0.05 mol/L Tris-HCl (pH 7.6), digested with 0.2 mg/mL pepsin (Boehringer Mannheim Biochemicals) in 0.1N HCl for 30 minutes at 37°C to expose the DNA, incubated in 1.5N HCl for 30 minutes at 37°C to denature the DNA, and washed in 0.1 mol/L sodium tetraborate (pH 8.5) to fix the DNA. Incubation of sections for 1 hour at 37°C with a monoclonal mouse anti-BrdU IgG (Euro-Diagnostic BV) was followed by incubation with biotinylated horse anti-mouse IgG (rat adsorbed; Boehringer Mannheim Biochemicals) for 45 minutes at 22°C. An avidin-biotin complex (ABC Elite, Vector) conjugated to horseradish peroxidase was applied for 30 minutes at 22°C. The chromogen 3,3'-diaminobenzidine, which precipitates as a dark crystal within 10 minutes in the presence of peroxidase activity, was used to stain the immunoreactive nuclei. Sections were counterstained with hematoxylin before being permanently mounted under coverslips. The number of SMCs in the media and intima per cross section was evaluated by counting under light microscopy. Results are expressed separately for intima and media as the cumulative labeling fraction, ie, the percentage of nuclei having synthesized DNA at any given time during the 2-week period of subcutaneous drug administration.

Measurement of Vascular Cross-sectional Area
Cross sections (5 µm) of arteries were stained with the elastin fiber–specific stain orcein, and cross-sectional areas were measured in two nonconsecutive sections per vessel by using light videomicroscopy with a computerized morphometry system (BioScan) that was standardized with a microruler at the beginning of each day. Medial area was defined as the area enclosed between the external and internal elastic laminae, and intimal area was defined as the area between the internal elastic lamina and the lumen perimeter. Lumen area was calculated in vessels fixed in situ by using the formula C²/4, where C is the length of the fixed internal elastic lamina.³⁴

Statistical Analysis
ANOVA followed by Fisher's protected least-significant difference test was used to analyze the data. Student's t test was used when comparisons were made between two groups only. All groups were compared with the group receiving AA (0.2%). A probability value of less than .05 was considered significant.

Side Study in Wistar-Kyoto Rats
This report also includes data from a separate set of experiments that was conducted independently from the main studies described above in 12 male Wistar-Kyoto rats (250 to 300 g; Central Animal Facilities, University of Limburg, Netherlands). All experimental procedures were as described above, with the following differences: (1) pentobarbital anesthesia (5.4 mg/kg IP) was used before vascular injury or subcutaneous pump implantation; (2) animals were infused with a low dose of PE (10 mg·kg^-1·d^-1) with saline (0.9%) as the vehicle 3 to 4 weeks after vascular injury only; (3) only carotid arteries were isolated and examined after perfusion-fixation in situ with 4% paraformaldehyde; and (4) data from the PE group were compared with the saline group by using Student's t test. Although these experiments were conducted in a different strain of rat and with a lower dose of PE without ascorbate, similar conclusions were reached as in the main studies with the Sprague-Dawley rats (see "Results"). Thus, the data with the Wistar-Kyoto rats have been included in this report as a side study.

	Results

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Chronic infusion of NE (2.5 mg·kg^-1·d^-1) caused a mild and inconsistent increase in systolic BP that was significant on days 9 and 14 of infusion but not on days 5 and 13 as measured by tail-cuff plethysmography (Fig 1). Because we were unable to obtain reproducible pulse pressure in rats infused with PE (25 mg·kg^-1·d^-1), possibly due to vasospasm of the tail artery, a radiotelemetric method was used to monitor intraaortic pressure at 10-minute intervals before and during chronic infusion of PE in a subset of six rats. Chronic infusion of PE induced labile increases in systolic BP in free-moving rats (see Fig 2 for a typical tracing of a systolic BP recording). To analyze these data, the pressure recordings obtained at 10-minute intervals were averaged for individual rats over a 24-hour period before (day 0), halfway through (day 7), and prior to termination of (day 14) treatment. Compared with the pretreatment systolic BP values (128±4 mm Hg on day 0, mean±SEM; n=6 rats), PE caused a slowly developing and modest increase in the daily average pressure (136±4 mm Hg on day 7; NS; and 143±4 mm Hg on day 14; P<.05). (A study detailing the regulation of BP during chronic catecholamine infusion is in preparation.) Although BP was measured telemetrically in PE-infused rats and by tail-cuff plethysmography in NE-infused rats, both sets of data suggest that chronic catecholamine infusion did not cause a stable increase in systolic BP above 155 mm Hg; control values ranged between 125 mm Hg and 135 mm Hg.

View larger version (18K): [in this window] [in a new window]   Figure 1. Line graph shows borderline elevation of systolic BP in rats chronically infused with NE (2.5 mg·kg-1·d-1) or the ascorbate vehicle (AA; 0.05 mg·kg-1·d-1). Systolic pressure was measured by using tail-cuff plethysmography. Values are mean±SEM (n=5 or 6 rats). *P<.05 vs AA vehicle.

View larger version (42K): [in this window] [in a new window]   Figure 2. Representative tracing of labile changes in systolic BP in rats chronically infused with PE (25 mg·kg-1·d-1). Pressure was recorded radiotelemetrically every 10 minutes starting 10 days before drug infusion (days 0 through 14). Pressure data, averaged over 24-hour periods, showed a slowly developing and modest increase in daily pressure from 128±4 mm Hg (day 0, mean±SEM; n=6 rats) to 136±4 mm Hg (day 7; NS vs day 0) and 143±4 mm Hg (day 14; P<.05 vs day 0). The lapse in recording on day 5 is due to temporary power interruption in the telemetry system.

Rats infused with the vehicle solutions (Ringer's lactate or ascorbate) but not those infused with a catecholamine showed significant gains in body weight by the end of the 2-week treatment (Table 1). Dry heart weight was reduced with PE. To confirm that the treatments did not induce significant cardiac hypertrophy, we calculated for each animal the ratio of dry heart weight to body weight and found no difference among the four groups (Table 1).

View this table: [in this window] [in a new window]   Table 1. Body Parameters of Rats After Chronic Infusion of PE or NE or the Vehicle Solutions AA or Ringer's Lactate 3-4 Weeks After Vascular Injury

Catecholamines and the Normal Arterial Wall
Infusion of PE or NE significantly increased SMC DNA replication in the media of uninjured carotid arteries by four- to sevenfold (Figs 3 and 4). In the thoracic aorta media, there was a significant stimulation of SMC DNA synthesis with PE (BrdU-labeling fraction, 14.1±3.1%) versus ascorbate (1.4±0.2%; P<.001; n=12 rats/group). BrdU-positive nuclei in the arterial media were randomly distributed, although clusters of positive nuclei were often seen in vessels from catecholamine-infused rats (Fig 5B). As described in "Methods," we included two control groups receiving either Ringer's lactate alone or Ringer's plus ascorbate, the vehicle used to deliver the catecholamines. Addition of ascorbate to the infusate did not affect arterial SMC DNA synthesis compared with Ringer's lactate alone (Fig 3), suggesting that catecholamines are responsible for the trophic effects reported here. The number of nuclei per cross section of carotid artery media tended to be increased with infusion of PE (383±28) or NE (385±21) versus the ascorbate vehicle (357±14) or Ringer's lactate alone (359±15) (all NS).

View larger version (18K): [in this window] [in a new window]   Figure 3. Bar graphs show increased SMC DNA synthesis in normal but not balloon-injured carotid arteries of rats receiving a chronic infusion of catecholamine during weeks 3 and 4 after vascular injury. Rats received PE (25 mg·kg-1·d-1), NE (2.5 mg·kg-1·d-1), the vehicle solution composed of 0.2% AA in Ringer's lactate, or Ringer's lactate alone (R). Values are mean±SEM (n=13 rats/group) of the cumulative labeling fraction. *P<.05 vs AA.

View larger version (563K): [in this window] [in a new window]   Figure 5. Photomicrographs. Nuclear incorporation of BrdU in ECs (small arrow), SMCs (large arrows), and adventitial cells in carotid arteries of rats infused for 2 weeks with either PE (25 mg·kg-1·d-1; B and D) or the ascorbate vehicle (A and C) in combination with BrdU (0.8 mg·kg-1·d-1). A and B show uninjured carotid artery; C and D, injured carotid artery from rats infused at 9 to 10 weeks after balloon injury. Arrowheads indicate external elastic lamina (hematoxylin counterstain, magnification x400, bar=20 µm).

As a further indication of vascular growth stimulation by catecholamines, the medial cross-sectional area of the carotid artery showed a 31% increase with NE and up to a 42% increase with PE (Table 2). Further morphometric analysis of normal carotid arteries isolated after perfusion fixation at physiological pressure in situ (experiment at 9 to 10 weeks; Table 2) revealed a 9% reduction in lumen area with PE (0.68±0.03 mm²) versus the ascorbate vehicle (0.75±0.02 mm²; P<.03). Thus, there was a 58% increase in the ratio of wall area to lumen area with PE (0.28±0.01) versus vehicle (0.18±0.01; P<.001) in the uninjured carotid artery, predominantly as a result of an increase in wall mass. This was also the case in the thoracic aorta, where the wall-to-lumen ratio was increased by 68% with PE (0.31±0.01) versus vehicle (0.19±0.01; P<.001) as a result of an increase in medial cross-sectional area (PE, 1.01±0.03 mm² versus vehicle, 0.64±0.01 mm²; P<.001); no significant reduction in lumen area was noted (not shown).

View this table: [in this window] [in a new window]   Table 2. Cross-sectional Area (mm2) of Normal and Balloon-Injured Common Carotid Arteries of Rats After Chronic Infusions of PE or NE or the Vehicle Solutions AA or Ringer's Lactate 3-4 or 9-10 Weeks After Vascular Injury

Perfusion fixation of carotid arteries in situ preserves endothelium integrity during vascular tissue isolation, allowing the quantification of endothelial DNA replication. The BrdU-labeling fraction of ECs was markedly increased by PE (48.9±3.6%) versus the ascorbate vehicle (3.6±1.2%; P<.001) (Fig 5A and 5B). These changes were accompanied by a 2.8-fold increase in the number of EC nuclei per cross section (PE, 122±6 versus vehicle, 44±13; P<.001). BrdU labeling was also increased by PE in adventitial cells (Fig 5A and 5B).

Catecholamines and the Injured Media
In control rats infused with vehicle solution 3 to 4 weeks after injury the rates of DNA synthesis in the media were similar between the uninjured carotid artery and the contralateral, injured carotid artery (Fig 3), indicating that the injury-induced wave of medial SMC replication was over by the third week after injury.³⁵ In contrast to the unmanipulated carotid artery, however, SMC DNA replication was not significantly increased in the media of the injured arteries with catecholamine infusion at 3 to 4 weeks (Fig 3). Infusion of PE at 9 to 10 weeks caused a significant increase in DNA replication in the injured carotid artery media (Fig 4), where BrdU-positive nuclei were randomly distributed. This replicative response was weaker than that in the normal media and did not result in a detectable change in the total number of SM nuclei per cross section in the injured artery media (PE, 470±34 versus vehicle, 481±22).

View larger version (17K): [in this window] [in a new window]   Figure 4. Bar graphs show increased SMC DNA synthesis in normal and balloon-injured carotid arteries of rats receiving a chronic infusion of catecholamine during weeks 9 and 10 after vascular injury. Rats received PE (25 mg·kg-1·d-1) or the vehicle solution composed of 0.2% AA in Ringer's lactate. Values are mean±SEM (n=12 rats/group) of the cumulative labeling fraction. *P<.05 vs AA.

In contrast to the hypertrophic effect of catecholamines in the unmanipulated carotid artery, catecholamines failed to cause a significant increase in medial cross-sectional area in injured carotid arteries, even 9 to 10 weeks after injury (Table 2). The lack of hypertrophic effect of catecholamines at 3 to 4 weeks was not due to a preexisting hypertrophy of the injured artery media, since the injured and uninjured arteries showed similar values of medial cross-sectional area in the control groups (Table 2).

Catecholamines and the Neointima
In control rats, a significant proportion of SMCs in the carotid artery neointima were still undergoing DNA replication during the third and fourth weeks after injury (Fig 3). During this early period, none of the experimental drug treatments affected neointimal DNA replication (Fig 3), cross-sectional area (Table 2), or number of nuclei per cross section (not shown). At 9 to 10 weeks, however, DNA replication was no longer prominent in the control neointima (Fig 4).³⁵ Infusion of PE during this period caused a significant increase in neointimal DNA replication (Fig 4), although there was no significant increase in the total number of nuclei per cross section (PE, 1565±106 versus ascorbate, 1538±90) or in the cross-sectional area of the neointima (Table 2). The BrdU-positive nuclei in the neointima were seen at the lumenal surface as well as in the deeper layers of the lesion (Fig 5D).

Side Study in Wistar-Kyoto Rats
As described in "Methods," a separate set of studies in Wistar-Kyoto rats infused with a lower dose of PE (10 mg·kg^-1·d^-1) 3 to 4 weeks after injury was conducted independently from the main studies in Sprague-Dawley rats. Despite the differences in rat strain and drug regimen, results of arterial DNA replication were comparable in the two studies, and the data obtained in Wistar-Kyoto rats have been included in this report as a side study. In the Wistar-Kyoto rats, a 2-week infusion of PE significantly increased SMC DNA synthesis in the media of the uninjured carotid artery by sixfold (vehicle, 0.2±0.1% versus PE, 1.3±0.2%; mean±SEM of the BrdU-labeling fraction; n=6). In the injured carotid artery of the same animals, however, PE infusion 3 to 4 weeks after denudation failed to affect DNA synthesis in the media (vehicle, 0.5±0.1% versus PE, 0.2±0.1%) or the neointima (vehicle, 17.5±0.1% versus PE, 17.3±0.1%). In these animals, PE (10 mg·kg^-1·d^-1) caused no change in the cross-sectional area of the uninjured or injured carotid artery.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Experiments with adrenergic antagonists have implicated ₁-adrenoreceptors in the accelerated growth of SMCs seen in both the balloon-injured arterial wall^{16 17 18 19} and the uninjured arterial media during Ang II–induced hypertension.^{20 27} Similarly, ₁-adrenoreceptor stimulation increases SMC proliferation in vitro.^{13 14 15} The present study extends those observations by showing for the first time in vivo that chronic stimulation of ₁-adrenoreceptors induces significant DNA replication in SMCs in both the normal and injured arterial wall. Our data also suggest that ₁-adrenoreceptors or the trophic response to these receptors may be downregulated in the SMCs during the time course of the response to injury.

Chronic catecholamine infusion did not cause a stable increase in systolic BP above 155 mm Hg. Moreover, the oscillations in intraaortic systolic pressure induced by PE infusion (Fig 2) are similar to the hemodynamic changes observed during a 2-day infusion of NE to rats.³⁶ Although the possibility exists that transient increases in pressure may have partially contributed to the stimulation of vascular growth, there is strong evidence suggesting that catecholamines may act directly to stimulate DNA synthesis in the arterial wall independently of alterations in BP. First, arterial SMC DNA synthesis is suppressed by ₁-adrenoreceptor blockers in Ang II–infused rats, even though severe hypertension (systolic pressure >190 mm Hg) is not prevented.^{27 37} Sympathetic activity is important for vascular SMC growth even during hypertension. For instance, rat cerebral arteries denervated by unilateral ganglionectomy show less medial hypertrophy during the development of essential hypertension than do the contralateral innervated arteries.³⁸ Second, in the present study, catecholamines failed to alter the ratio of heart weight to body weight, suggesting that cardiac afterload and BP were not increased enough to cause cardiac hypertrophy. These data also tend to rule out the possibility that vascular growth occurred as a consequence of a significant cardiostimulatory action of NE via ß-receptors in the heart. Finally, both PE and NE are mitogenic for SMCs in culture.^{11 13 14 15}

Previous studies with NE and selective adrenergic antagonists suggest that a balance between - and ß-adrenergic stimulation may be an important feature of SMC growth control. In SMCs cultured from rat uninjured arteries, stimulation of ß-adrenoreceptors partially blunts the mitogenic action of ₁-adrenoreceptors.¹⁴ Also, in vivo stimulation of ß-receptors inhibits ₁-receptor stimulation of ornithine decarboxylase activity in the cockerel aorta media.³⁹ The present data with NE indicate that a significant increase in SM DNA synthesis occurs in the uninjured arterial wall as a net result of - and ß-receptor stimulation, as it occurs with the more selective ₁-receptor agonist PE.

The present data do not establish whether the stimulation of DNA replication caused an increase in SMC ploidy (DNA content per cell) or number in the arterial wall. Mulvany et al⁴⁰ report that a 40% increase in SMC number was not detectable by counting nuclei in arterial sections in resistance vessels of hypertensive rats, mainly because of the systematic error associated with the sampling of particles of different size, shape, and orientation in cross sections of a volume.⁴¹ Of interest, polyploid SMCs are more frequent in the thoracic aorta of rats following a 2-week infusion of NE⁴² or in response to cell stimulation with the catecholamine in vitro.⁴³ Also, the possibility exists that SMC apoptosis (programmed cell death) occurred more often in arteries with catecholamine infusion, as seen in remodeling arteries early after balloon injury,⁴⁴ with changes in blood flow,⁴⁵ or in atherosclerosis.^{46 47} However, a possible increase in SMC number cannot be ruled out in the present study. In fact, clusters of BrdU-positive SMC nuclei were often seen in arteries following catecholamine but not vehicle infusion (Fig 5), suggesting that SMC mitosis was indeed stimulated by catecholamines in vivo, as shown in vitro.^{13 14}

Infusion with catecholamines revealed important differences in the control of medial SM growth between normal and injured vessels. The ability of SMCs to replicate DNA in response to exogenous PE was suppressed as early as 3 to 4 weeks after injury and was only partially restored by 9 to 10 weeks. Four nonexclusive explanations may be considered. First, the neointima at the earlier time after vascular injury may already be replicating at a maximal rate. Against this hypothesis, however, DNA replication in the neointima can be stimulated further by Ang II infusion at 3 to 4 weeks.²⁷ Second, expression of ₁-adrenoreceptors may be suppressed as part of the SM response to vascular injury, as a consequence, eg, of increased sympathetic output with downregulation of adrenoreceptors at the site of vascular injury. Consistent with this, vascular repair after injury to the rabbit iliac artery results in the local facilitation of adrenergic neurotransmission, with decreased contractile responses to exogenous catecholamines.⁴⁸ To date, four subtypes of ₁-adrenoreceptor have been cloned,^{12 49} but no data are available on receptor expression in injured arteries or in growing versus quiescent SMCs. Third, the intrinsic replicative properties of SMCs might be changed after balloon injury.⁵⁰ Intimal SMCs have been reported to exhibit many properties that are different from SMCs found in the media of the normal vessel wall.^{26 50 51 52 53 54} Decreased arterial responsiveness to catecholamines may also occur as a consequence of increased production of nitric oxide by SM after balloon injury.^{55 56 57} Finally, it is also possible that the injured wall has altered catabolism of catecholamines.

Taken together with our previous data showing that ₁-blockers suppress arterial DNA replication during Ang II–induced hypertension,^{20 37} the present data suggest that a significant proportion of the in vivo mitogenic effects of Ang II on the normal wall may be mediated indirectly via the adrenergic pathway. Consistent with this view, Ang II increases sympathetic neurotransmission^{58 59} as well as vascular SM sensitivity to ₁-agonists.⁶⁰ The failure to stimulate DNA replication in the neointima with catecholamines 3 to 4 weeks after injury is consistent with our earlier data showing that prazosin does not suppress Ang II–stimulated replication in the neointima during the same period early after injury.³⁷ Thus, a role for the adrenergic pathway in the mitogenic effect of Ang II in the neointima is unlikely at 3 to 4 weeks but it cannot be ruled out at later times (eg, 9 to 10 weeks) after balloon injury.⁶¹

Endothelial dysfunction is often associated with the pathogenesis of hypertension and atherosclerosis.^{62 63} The present study provides the first evidence that catecholamines stimulate growth in ECs. In PE-infused rats, the dramatic increase in both the BrdU-labeling fraction and the total number of EC nuclei per arterial cross section suggests an increase in EC mitosis or a decrease in apoptosis. In cultured ECs, stimuli for proliferation include pulsatile strain⁶⁴ and vasoactive mediators such as Ang II (via angiotensin receptor subtype 1).⁶⁵ Thus, the increased growth of ECs in PE-infused rats could represent a secondary response to increased BP lability and/or a direct response to ₁-adrenergic stimulation.

In summary, chronic stimulation of ₁-adrenoreceptors in vivo stimulated growth in ECs and SMCs in arteries with or without intimal thickening, though not during the first weeks after balloon injury. The stimulation of DNA synthesis in vascular cells via the ₁-adrenoreceptor pathway may contribute to the vascular remodeling that occurs in hypertension and atherosclerosis.

	Selected Abbreviations and Acronyms

 

AA = ascorbic acid Ang II = angiotensin II BP = blood pressure BrdU = 5-bromo-2'-deoxyuridine EC = endothelial cell NE = norepinephrine PE = phenylephrine SM = smooth muscle SMC = smooth muscle cell

	Acknowledgments

 
This work was supported in part by a fellowship grant from the Medical Research Council of Canada to Dr deBlois at the Department of Pathology, University of Washington, and by grants from the National Institutes of Health (HL 26405) and the Netherlands Heart Foundation (93165). We are grateful to Patti Polinski, Ben Rampp, Hillel Schwartz, and Foon Tsui for their excellent technical assistance in surgery or histology, and to Marie-France Ross and Andrea Tancredi for their expertise in image analysis.

Received August 9, 1995; revision received March 20, 1996;

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