Donate Help Contact The AHA Sign In Home
American Heart Association
Hypertension
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Hypertension. 1997;29:340-344

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by deBlois, D.
Right arrow Articles by Hamet, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by deBlois, D.
Right arrow Articles by Hamet, P.
Right arrowPubmed/NCBI databases
*Substance via MeSH

(Hypertension. 1997;29:340.)
© 1997 American Heart Association, Inc.

Arthur C. Corcoran Memorial Lecture

Smooth Muscle Apoptosis During Vascular Regression in Spontaneously Hypertensive Rats

Denis deBlois; Bun-Seng Tea; Than-Vinh Dam; Johanne Tremblay; Pavel Hamet

From the Centre de Recherche de l'Hòtel-Dieu de Montréal, and the Departments of Pharmacology (D.deB., B.-S.T.) and Medicine (J.T., P.H.), Université de Montréal, Québec, Canada. E-mail debloisd{at}ere.umontreal.ca.

Correspondence to Dr Pavel Hamet, Centre de Recherche Hòtel-Dieu de Montréal, 3840 St Urbain St, Montréal, Québec H2W 1T8, Canada


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
We previously reported that apoptosis is increased in smooth muscle cells cultured from the aorta of spontaneously hypertensive rats versus normotensive controls. As an initial in vivo exploration, we now examined smooth muscle cell apoptosis regulation during the regression of vascular hypertrophy in the thoracic aorta media of spontaneously hypertensive rats receiving the antihypertensive drug enalapril (30 mg·kg-1·d-1), losartan (30 mg·kg-1·d-1), nifedipine (35 mg·kg-1·d-1), hydralazine (40 mg·kg-1·d-1), propranolol (50 mg·kg-1·d-1), or hydrochlo-rothiazide (75 mg·kg-1·d-1) for 1 to 4 weeks starting at 10 to 11 weeks of age. Three criteria were used to evaluate smooth muscle cell apoptosis: (1) oligonucleosomal fragmentation of the extracted aortic DNA, (2) reduction in aortic DNA content, and (3) depletion of smooth muscle cells in the arterial media. Arterial DNA synthesis was evaluated by [3H]thymidine incorporation in vivo. After 4 weeks of treatment, systolic blood pressure was reduced significantly by <42% with losartan, enalapril, and hydralazine, and by 23% with nifedipine, versus control values of 220±5 mm Hg. However, these agents affected vascular growth and apoptosis differently. Losartan, enalapril, and nifedipine stimulated smooth muscle cell apoptosis threefold to fivefold before there was a significant reduction in DNA synthesis (<25%), vascular mass (<19%), or vascular DNA content (<38%), and these treatments markedly reduced (by 38% to 50%) medial cell number as measured at 4 weeks by the three-dimensional disector method. Losartan and nifedipine stimulated smooth muscle cell apoptosis before reducing blood pressure. In contrast, hydralazine did not affect vascular mass, apoptosis, or DNA synthesis, although blood pressure was lowered. Propranolol or hydrochlorothiazide failed to affect hypertension or vascular growth. Thus, smooth muscle cell apoptosis represents a novel therapeutic target for the control of hypertensive vessel remodeling in response to therapeutic agents.


Key Words: apoptosis • smooth muscle cell • angiotensin II • calcium channel antagonist


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Studies in humans and in experimental models of hypertension have clearly demonstrated the importance of vascular structure in the regulation of blood pressure.1–6 Increased vascular mass is an important feature of hypertensive vessels. At the level of small muscular arteries, vascular hypertrophy accompanied by smooth muscle cell (SMC) hypertrophy or hyperplasia acts as an amplifier for elevated vascular resistance and blood pressure.7,8 At the level of the aorta and its main branches, increased mass and rigidity of the arterial wall contribute to systolic hypertension and represent an independent risk factor for left ventricular hypertrophy.9 The remodeling of vascular structure, including the regression of vascular hypertrophy, is now considered a key therapeutic target in the effort to reduce mortality and morbidity associated with high blood pressure.10–13

Increased vascular mass associated with the replication and accumulation of smooth muscle DNA, as in genetically determined or secondary hypertension,14–18 represents a mode of structural remodeling that is less readily reversible than vascular hypertrophy that is solely due to increased protein synthesis without de novo DNA synthesis. Thus, the DNA content of the vessel wall (due to SMC hyperplasia or polyploidy)16 may be considered as a record of past episodes of vascular growth, contributing to the persistence of the vascular disease. Apoptosis is a highly regulated form of programmed cell death that is involved in tissue morphogenesis and homeostasis as the essential counterpart of cell replication.19,20 In this context, apoptosis is potentially involved in the regulation of vascular remodeling, via the deletion of SMC in the vessel wall.20 We have previously reported that the heightened DNA replication of SMC cultured from the aorta of spontaneously hypertensive rats (SHR) occurs in parallel to an increase in apoptosis.21 These studies suggested that increased apoptosis may in part counterbalance the heightened cell growth in hypertensive vessels. In this view, the balance between apoptosis and cell (or DNA) replication would constitute a key determinant of vascular mass and a potential therapeutic target for achieving long-term regression of vascular hypertrophy.

The present studies were designed to investigate whether SMC apoptosis contributes to the regression of vascular hypertrophy during antihypertensive therapy in the SHR. Agents tested belong to several of the major classes of drugs used in the clinic to control elevated blood pressure. Our results suggest that SMC apoptosis is a novel therapeutic target for the pharmacological control of vascular structure in hypertension.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Drug Treatments
Male SHR weighing 250 to 275 g were purchased from Charles-River (St Constant, Que) and housed for at least 10 days before initiation of drug treatment at 10 to 11 weeks of age. Treated rats were given one of the following drugs: the calcium channel blocker nifedipine (35 mg·kg-1·d-1), the angiotensin-converting enzyme inhibitor enalapril (30 mg·kg-1·d-1), the AT1 receptor antagonist losartan (30 mg·kg-1·d-1), the smooth muscle relaxant hydralazine (40 mg·kg-1·d-1), the ß-adrenoceptor blocker propranolol (50 mg·kg-1·d-1), or the diuretic hydrochlorothiazide (HCT; 75 mg·kg-1·d-1). The doses of antihypertensive drug were chosen on the basis of published reports of others or our own preliminary data showing effectiveness in reducing hypertension or cardiovascular hypertrophy in the SHR.22–25 All drugs were dissolved in the drinking water except for nifedipine, which was mixed with powdered food. The subsets of control rats receiving powdered rat chow without nifedipine showed similar results as the control rats given regular chow in pellets, hence the data from these control groups were pooled for analysis. Food and water, with or without drug, were renewed three times weekly and administered ad libitum. The rats were weighed each week and the dosage of drug adjusted according to their body weight and the amount of water or food consumed. All drugs were from Sigma, except nifedipine, which was provided courtesy of Bayer Canada, Toronto, and losartan and enalapril, which were courtesy of Merck Frosst, Montreal, Canada. The ability of the antihypertensive drugs to reduce aortic hypertrophy was initially examined after 4 weeks of therapy. Drugs that proved effective at 4 weeks were selected for the studies at 2 weeks and 1 week of treatment. An age-matched control group was included in each experiment.

Blood Pressure Measurements
Systolic blood pressure was measured between 8 and 12 AM in each rat by use of the tailcuff plethysmograph method (IITC). Measurements were started 1 week before treatment and performed on conscious, restrained rats previously trained for the procedure. Pressure measurements were made at weekly intervals during the treatment period and at least 2 days before animal sacrifice for tissue isolation to avoid interfering with the vascular growth response.

Tissue Isolation and Vascular Mass Measurements
To evaluate DNA synthesis in vivo, rats were injected IP with [methyl-3H]thymidine (0.5 mCi/kg, New England Nuclear) at 17, 9, and 1 hour before death. Rats were anesthetized with a single IM injection of ketamine (80 mg/kg), xylazine (4 mg/kg), and acepromazine (2 mg/kg), and killed by perfusion of 200 mL isotonic saline via the abdominal aorta. The thoracic aorta was isolated from the diaphragm to above the first intercostal artery and it was cleaned of fat and adventitial connective tissue. The length and wet weight of the aorta were measured before the artery was cut longitudinally to allow removal of the endothelium by scrubbing the intimal surface with a cotton tip applicator. The aorta:body weight ratio was calculated and used as an index of vascular mass. The aortic media was snap frozen in liquid nitrogen and kept at -80°C until further processing. To further confirm changes in vascular mass, we also measured the cross-sectional area of aortae isolated after 4 weeks of effective antihypertensive therapy (a blindly selected subset of tissues was used for the control and enalapril groups). A 3-mm-long ring of aorta was cut between the third and fourth intercostal arteries after weighing the vessel. The aortic rings were fixed in 4% paraformaldehyde overnight and processed according to routine histological procedures for morphometric measurements in cross-sections of paraffin-embedded arteries. The medial cross-sectional area was evaluated in 5-µm-thick, hematoxylin-stained sections of aorta. Photomicrographs of the aorta sections were taken at 200x magnification, digitized, and analyzed using the NIH Image 1.6 image analysis freeware (NIH). Two measure of each tissue section were done to ensure the reproducibility of the image analysis.

DNA Extraction and Characterization
The whole aortic media was homogenized in liquid nitrogen using a mortar and pestle. An aliquot of the pulverized aortic media was weighed and total tissue DNA was extracted by the phenol and chloroform procedure, following tissue digestion steps with proteinase K and RNase A in the presence of EDTA, as described previously.26 DNA concentration was determined by spectrophotometry and total DNA content per unit of aortic length was calculated using the following equation: total aortic DNA content=(µg of DNA/mg of aorta)x(mg of aorta/cm of aorta).

Oligonucleosomal DNA fragmentation into 180- to 200-bp integer fragments is a hallmark of apoptosis.27 This specific pattern of DNA fragmentation appears as a "ladder" of DNA fragments after conventional agarose gel electrophoresis in various cell types undergoing apoptosis,27,28 including SMC.21,29–33 To quantify the degree of oligonucleosomal DNA fragmentation in the aortic media, 1 µg of extracted DNA was radiolabeled using terminal deoxynucleotidyl transferase and [32P]dCTP as described previously.26 Because DNA molecules from apoptotic nuclei have an increased number of free 3'OH ends as a result of enhanced endonuclease activity, these show greater incorporation of labeled dCTP. Increasing quantities of radiolabeled DNA (0.025, 0.05, 0.1, 0.2, and 0.4 µg) were loaded in adjacent lanes of 1.5% agarose gels. After electrophoresis, DNA was transferred onto nylon membrane (Hybond N; Amersham) and the radioactivity associated with 150 to 1500 bp DNA fragments was quantified using a PhosphorImager (Molecular Dynamics) (Fig 1A). The PhosphorImager data were used to construct a regression line for each sample and radioactivity per unit area (cpm per pixel) was plotted as a function of DNA loaded on the gel (µg DNA). We found this approach to be useful in increasing reproducibility of the measurements. Thus, the slope of the linear regression was defined as the "DNA fragmentation index" (Fig 1B). Commercially available DNA molecular size markers underwent radiolabeling, electrophoresis, and transfer at the same time as the extracted DNA to control for variability in the procedure. Cleavage of DNA into large fragments (eg, 20 to 50 kbp) at the site of attachment to scaffold proteins has also been described in cells undergoing apoptosis. Therefore, radiolabeling was also quantified for DNA fragments of 20 to 30 kbp, as described above.



View larger version (42K):
[in this window]
[in a new window]
  		FIG 1. A, DNA laddering obtained by gel electrophoresis of DNA extracted from the aorta of control SHR (CON) or SHR treated for 1 week with nifedipine (NIF; 35 mg·kg-1·d-1) or losartan (LOS; 30 mg·kg-1·d-1). Increasing amounts of DNA (0.025, 0.05, 0.1, 0.2, and 0.4 µg) radiolabeled with terminal deoxynucleotidyl transferase were fractionated on a 1.5% agarose gel. B, Quantification of radioactivity associated with the 150- to 1500-bp fragments as a function of the amount of DNA loaded, using a Phosphorlmager as described in "Methods." The slope of the linear regression was defined as the "DNA fragmentation index" (cpm per pixel per microgram DNA), indicative of apoptosis.

DNA synthesis in the aortic media was quantified by measuring the incorporation of [3H]thymidine into the extracted DNA. To rule out the possibility that drug-induced changes in DNA specific activity (cpm/100 µg DNA) result from changes in thymidine transport into the cells rather than changes in DNA synthesis activity per se, the specific activity of the whole aortic tissue (cpm/mg aorta) was also measured at 1 and 2 weeks of treatment.

Determination of Smooth Muscle Cell Number
SMC number in the aortic media was measured using the three-dimensional dissector method, as adapted from Mulvany et al.18 Briefly, three consecutive sections (5 µm in thickness) were obtained from aortic rings sectioned perpendicularly to the longitudinal axis of the vessel. The tissue sections were stained with hematoxylin, photographed, and printed at a final magnification of 1000x. In the top section, an area, ad, of vessel wall was delineated by two parallel lines approximately perpendicular to the wall edges. A disector was defined as the three-dimensional probe bounded by ad and the top surface of the "bottom" section. The disector has volume vd=adxhd, where ad is the area of the disector and hd is the height of the disector. The term hd is obtained by the following equation: hd=(s-1)xt, where s is the number of serial sections and t the average section thickness. Within ad, the number of nucleus profiles, nt, was determined and in the subsequent sections, each of the nt nuclei was followed and marked. In the final section, the number of nuclei still present was determined, nb. The number nd=(nt-nb) is then the number of "downward-pointing" nucleus ends within the disector when counting from top to bottom. Although binucleate SMC reportedly exist in SHR arteries, these cells account for less than 0.5% of the SMC in the thoracic aorta of 12-week-old SHR.34 Therefore, on the basis that each cell contains only one nucleus, an estimate of cell numerical density, Nv, is given by Nv=nd/vd. The number of cells per unit vessel length then was estimated from a1xNv, where a1 is the medial cross-sectional area determined with the image analyzer as described above. Nuclear length was measured using the following equation: nuclear length=(nt/nd)xhd.

Statistical Analysis
Data were analyzed using analysis of variance and unpaired Student's t test with Bonferroni correction for multiple comparisons where appropriate. The DNA fragmentation index was analyzed using the nonparametric tests Kruskal-Wallis followed by Mann-Whitney because of unequal variance between the groups and because we have no evidence that the index is a normally distributed variable. Values are presented as mean±SEM, and P>.05 was considered statistically significant.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
The ability of the antihypertensive drugs to reduce vascular hypertrophy and growth in the thoracic aorta was initially examined after 4 weeks of therapy in the SHR (Table 1). Drugs that proved effective in reducing vascular hypertrophy at 4 weeks were selected for the studies at 2 weeks and 1 week of treatment (Table 2). In addition, to facilitate intergroup comparisons, selected changes in vascular growth parameters are also presented in Fig 3, where the data are expressed as a percentage of control values (from paired animals without treatment) and plotted as a function of time after initiation of therapy with selected drugs.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Effect of Antihypertensive Therapy for 4 Weeks on SHR Body Weight, and Aortic Medical Hypertrophy, DNA Content, DNA Fragmentation, and DNA Specific Activity


View this table:
[in this window]
[in a new window]
  		TABLE 2. Effect of Antihypertensive Therapy for 1 or 2 Weeks on SHR Body Weight, and Aortic Medial Hypertrophy, DNA Content, DNA Fragmentation, DNA Specific Activity, and Tissue Specific Activity



View larger version (32K):
[in this window]
[in a new window]
  		FIG 3. Changes in growth-related parameters in the aortic media of SHR receiving losartan (30 mg·kg-1·d-1), enalapril (30 mg·kg-1·d-1), nifedipine (35 mg·kg-1·d-1), or hydralazine (40 mg·kg-1·d-1) for 1 to 4 weeks. A, Vascular mass expressed as the aorta:body weight ratio. B, Aortic DNA content. C, Aortic DNA fragmentation index, indicative of apoptosis. D, Aortic DNA specific activity, indicative of DNA synthesis in vivo. The data were taken from Tables 1 and 2, expressed as a percentage of the corresponding control values and plotted as a function of time after initiation of drug therapy.

Systemic Changes During Drug Administration
Rats used in this study were hypertensive before initiation of therapy, with an average pretreatment value of systolic blood pressure of 176±2 mm Hg (n=110; no difference between control and experimental groups). During the experimental period, systolic pressure increased further in untreated animals, up to 203±5 mm Hg at 2 weeks (n=39) and 220±5 mm Hg at 4 weeks (n=25). Fig 2A shows the evolution of systolic blood pressure in control and treated rats over the 4-week experimental period. Systolic blood pressure was not significantly affected in rats receiving propranolol or HCT for 4 weeks (Fig 2B) but it was significantly reduced in rats receiving either hydralazine (47% reduction in final pressure), losartan (42% reduction), enalapril (44% reduction), or nifedipine (23% reduction). Significant reduction in blood pressure was achieved after 1 week with enalapril or hydralazine, after 2 weeks with losartan, and after 3 weeks with nifedipine (Fig 2A). Thus, at the high doses used in this study, the drugs showed the following order of potency for blood pressure reduction: enalapril >=hydralazine <losartan <nifedipine <<HCT >=propranolol.



View larger version (24K):
[in this window]
[in a new window]
  		FIG 2. A, Progressive increase in systolic blood pressure in untreated 10- to 11-week-old SHR and the reversal of hypertension in SHR treated for 4 weeks with losartan (30 mg·kg-1·d-1), enalapril (30 mg·kg-1·d-1), nifedipine (35 mg·kg-1·d-1), or hydralazine (40 mg·kg-1·d-1). B, Systolic blood pressure at the beginning and end of a 4-week treatment with propranolol (50 mg·kg-1·d-1) or hydrochlorothiazide (HCT; 75 mg·kg-1·d-1) in SHR. Systolic pressure was measured by tailcuff plethysmography as described in "Methods." Day 0 refers to the beginning of drug administration. Values are mean±SEM (n=5 to 14 rats). *First occurrence of a significant difference between treated and control groups (P>.05).

Pretreatment values of body weight were 260±2 g (n=110). All rats gained weight during the experimental period but final values of body weight were similar between corresponding control and experimental groups at 1, 2, and 4 weeks (not shown). Daily water consumption was increased in rats treated with losartan (by 20%) or enalapril (by 23%). Powdered food consumption was not different between the nifedipine and the corresponding control group (not shown).

Effect of Drugs on Vascular Hypertrophy and DNA Content
At 4 weeks the aorta:body weight ratio was not affected by hydralazine, propranolol, or HCT (Table 1). In contrast, there was a significant reduction in aortic mass at 4 weeks in rats given losartan (19% reduction), enalapril (22% reduction), or nifedipine (26% reduction). Further studies at earlier times revealed a significant reduction in aortic mass as early as 2 weeks with losartan or nifedipine but not enalapril (Table 2 and Fig 3A). The aorta of untreated animals showed no increase in vascular mass between tissues isolated at 1, 2, and 4 weeks, suggesting that the drug-induced reductions in vascular mass represented a regression rather than a prevention of vascular hypertrophy. As a further confirmation that vascular mass was decreased, the aortic cross-sectional area was significantly decreased by losartan, enalapril, or nifedipine but not hydralazine for 4 weeks (Table 3).


View this table:
[in this window]
[in a new window]
  		TABLE 3. Effect of 4-Week Antihypertensive Therapy on Medial Cross-Sectional Area, SMC Nuclear Length, SMC Numerical Density, and SMC Number in the Aortic Media of SHR

To further analyze vascular growth, aortic DNA content was evaluated per length of vessel. The length of the freshly isolated thoracic aorta was 30±1 mm (n=26) in untreated animals at 4 weeks, and this value was not affected by any of the treatments studied (not shown). Among all the drugs studied, only losartan and enalapril reduced aortic DNA content significantly, by 63% and 66%, respectively, in the aorta after 4 weeks of treatment (Table 1). In contrast, DNA content at 2 weeks showed a significant 60% to 65% reduction with nifedipine and losartan, and no evidence of reduction with enalapril (Table 2 and Fig 3B). Administration of losartan or nifedipine for a shorter period of time (1 week) did not affect total DNA content in the aorta. Because the enalapril-induced reduction in DNA content required more than 2 weeks of treatment, this drug was not included in the 1-week studies designed to examine apoptosis.

Effect of Drugs on SMC Apoptosis
The therapy-induced reduction in DNA content in the thoracic aorta suggested a stimulation of apoptosis and a corresponding reduction in SMC number. To test this hypothesis, the DNA fragmentation index was evaluated as described in "Methods." Losartan caused a threefold to fivefold increase in internucleosomal DNA fragmentation. This effect of losartan was highest at 1 week and sustained as late as 4 weeks after beginning of treatment (Tables 1 and 2, and Fig 3C). Enalapril also stimulated internucleosomal DNA fragmentation by threefold to fivefold, although the highest effect was observed at 4 weeks. In nifedipine-treated rats, internucleosomal DNA fragmentation was increased at 1 week only. The DNA fragmentation index for the large 20- to 30-kbp DNA fragments showed no significant change with treatment (not shown), compared with control values of 1.8±0.1 cpm per pixel per microgram DNA (n=40).

The three-dimensional disector method was used to determine whether the reduced DNA content corresponded to a reduced SMC number in the arterial wall (Table 3). In the aorta of untreated rats, we found 116±6 SMC per micrometer of vessel length. This SMC number is comparable to what Owens35 reported in the aorta of untreated 3-month-old SHR, using a different method of evaluation. We found that the SMC number per length of aorta was significantly reduced by 50% with losartan, by 47% with enalapril, and by 38% with nifedipine. In contrast, the potent antihypertensive effect of hydralazine did not result in reduced SMC number in the arterial wall. Thus, there was a highly significant correlation between the aortic DNA content and the number of SMC in the arterial wall (P>.001; r=.6). Although losartan, enalapril, and nifedipine significantly reduced the cross-sectional area and therefore the total mass of the aorta, the reduction in SMC number was large enough so that the SMC numerical density was also significantly decreased in the arterial wall (Table 3). None of the treatments under study with the disector method affected significantly the average nuclear length of arterial SMC, which was 15±1 µm in the untreated aorta, a value comparable with the results obtained by Mulvany et al18 in SHR small mesenteric arteries using the same method.

Effect of Drugs on DNA Synthesis
As shown in Table 2, several treatments caused a significant reduction in arterial DNA specific activity without affecting radioactivity levels in the whole vascular media (at least at 2 weeks), thus suggesting an inhibition of SMC DNA synthesis. Within 4 weeks, the specific activity of aortic DNA was decreased by losartan, enalapril, and nifedipine but not by hydralazine, propranolol, or HCT (Tables 1 and 2Up). The kinetic but not the magnitude of the inhibitory effect varied between losartan, enalapril, and nifedipine (Fig 3D). Thus, DNA specific activity was significantly reduced by losartan at 2 and 4 weeks (25% and 31% reduction, respectively), by nifedipine at 2 weeks only (42% reduction), and by enalapril at 4 weeks only (32% reduction).

Effects of a Lower Dose of Losartan
The regulation of aortic growth was examined in rats treated with a lower dose of losartan for 4 weeks. As shown in Table 4, administration of losartan (10 mg·kg-1·d-1) to SHR was unable to reverse the preexisting hypertension but prevented the further development of high blood pressure. Interestingly, this treatment did not result in any significant change in aortic mass, DNA fragmentation, DNA specific activity, or DNA content at 4 weeks.


View this table:
[in this window]
[in a new window]
  		TABLE 4. Effect of 4-Week Antihypertensive Therapy With a Low Dose of Losartan (10 mg·kg-1·d-1) on SHR Systolic Blood Pressure, Body Weight, and Aortic Medial Hypertrophy, DNA Content, DNA Fragmentation, and DNA Specific Activity


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Increased vascular mass is an important feature of hypertensive vessels. In the adult SHR, the thoracic aorta shows medial hypertrophy and a higher SMC DNA content and synthesis rate as compared to normotensive animals.3,7,17,34–38 In the absence of high blood pressure, SHR SMC cultured in vitro maintain an increased growth behavior, including hyperresponsiveness to growth factors, accelerated entry into S phase of the cell cycle, reduced cell-contact inhibition, and high incidence of polyploidy, suggesting a pressure-independent dysregulation of growth.39–44 We have reported that SMC cultured from SHR aorta also exhibit a heightened propensity to undergo apoptosis, eg, in response to growth factor withdrawal.21 We recently reviewed45 the possible role of apoptosis in regulating vascular mass and DNA content, a hypothesis that has never been examined in hypertensive vessels. The balance between SMC DNA synthesis and degradation determines the total DNA content of the arterial media, which in turn is a primary determinant of vascular mass. Thus, the aim of the present studies was to investigate the regulation of SMC apoptosis during the regression of aortic medial hypertrophy induced by antihypertensive therapy in the SHR.

Time Window of Apoptosis
Within 4 weeks of effective antihypertensive therapy, we observed a reduction in aortic mass in rats treated with losartan, enalapril, or nifedipine but not hydralazine. Several lines of evidence suggest that the regression of vascular hypertrophy was associated with increased SMC apoptosis. First, there was a marked increase in internucleosomal fragmentation of the DNA in the aortic media. This specific pattern of DNA is a hallmark feature of apoptosis observed in several types of cells27 including SMC in culture.21,30–33 Second, there was a significant reduction in aortic DNA content in the weeks following the initial burst of internucleosomal DNA fragmentation. Finally, the number of SMC in the aortic wall was reduced after 4 weeks of losartan, enalapril, or nifedipine but not hydralazine, in correlation with the effects of these drugs on aortic DNA content. The present data on the kinetics of arterial SMC apoptosis during drug therapy are reminiscent of our recent observations in the heart adapting to pressure overload caused by aortic coarctation.26 In this model, the development of cardiac hypertrophy is preceded by a transient increase in apoptosis, notably in cardiomyocytes. We suggested that the design of therapeutic approaches aimed at controlling cardiovascular remodeling should take into account the presence of time windows of apoptosis during pathogenesis.46 The present studies provide evidence that windows of rapid change in apoptosis also occur in response to drug therapy.

SMC Apoptosis and Blood Pressure
The stimulation of SMC apoptosis could be dissociated from the antihypertensive effects of the drugs tested. Although hydralazine was very effective at reducing hypertension, it did not affect DNA fragmentation, DNA content, SMC number, or vascular mass at 4 weeks. Furthermore, the stimulation of SMC apoptosis was more rapid with losartan or nifedipine (1 week) than with enal- april (2 weeks), whereas blood pressure reduction was more rapid and pronounced with enalapril (1 week) than with losartan (2 weeks) or nifedipine (<2 weeks). Taken together, these data strongly suggest that drug-induced SMC apoptosis was not a secondary response to blood pressure reduction. Consistent with this, calcium channel blockers stimulate apoptosis independently of hemodynamic changes in cultured SMC.47

Several groups reported a dissociation between blood pressure reduction and aortic mass regression in the SHR on the basis of comparative studies between hydralazine and losartan,48 a calcium channel blocker,49 or an ACE inhibitor.50 The present results extend these findings by showing that nifedipine induced aortic mass regression before systolic pressure reduction (ie, at 2 and 3 weeks, respectively). Increased SMC apoptosis may account for this early structural effect of the calcium channel blocker. SMC apoptosis may also contribute, along with vasodilatation51 and decreased extracellular matrix accumulation,52 to increase the compliance of large conduit arteries in response to ACE inhibitors, AT1 antagonists or calcium channel blockers, a major beneficial effect of these drugs against left ventricular hypertrophy and systolic hypertension.22,53

The available evidence from previous studies suggests that in SHR the antihypertensive and antihypertrophic effects of ß-blockers and diuretics are modest, slow to develop, and may be more pronounced when the drugs are administered before hypertension is established.24,25,54–56 Thus, although propranolol and HCT did not significantly affect blood pressure or vascular structure at this established phase of SHR hypertension, we cannot rule out that an earlier or more prolonged schedule of administration may affect arterial SMC apoptosis.

SMC Apoptosis and DNA Synthesis
Heightened DNA replication rates are often associated with and even counterbalanced by an increase in SMC apoptosis. Cho et al29 reported that high rates of DNA synthesis in specific arteries of the neonatal lamb do not translate to a corresponding increase in vascular DNA content. In these arteries which show regression after birth the high rates of SMC DNA synthesis are counterbalanced by heightened apoptosis. SMC apoptosis is also markedly increased in the developing neointimal lesion after balloon catheter injury in rats.57,58 Apoptosis and DNA replication are both found mainly among SMC located near the luminal surface of the neointima, suggesting that apoptosis may regulate the cellularity of the lesion in the face of chronic cell proliferation.59 Bennett et al30 reported that SMC overexpressing the proto-oncogene and growth-related transcription factor c-myc showed an increased rate of DNA replication that was resistant to inhibition by serum withdrawal or interferon-{gamma}. In an apparent paradox, however, serum withdrawal or interferon-{gamma} indeed abolished the accumulation of c-myc cells. This discrepancy between the high rates of DNA synthesis and the low rates of cell accumulation could be explained by the markedly increased frequency of apoptosis in the c-myc cells in response to serum withdrawal or interferon-{gamma} treatment. Thus, c-myc overexpression stimulates both cell proliferation and death by apoptosis, and the balance between proliferation and death is controlled by survival and cytostatic signals from the environment. Overall, the data on SMC apoptosis regulation suggest that the need for survival signals may be increased in cells stimulated to undergo replication. It is intriguing to speculate that the early increase in aortic SMC apoptosis, preceding the inhibition of DNA synthesis, represented a failure of SMC to adapt rapidly to reduced levels of survival factors such as angiotensin II or extracellular calcium influx.

Several groups reported that the aorta of SHR has an increased percentage of polyploid SMC, a feature which correlates in part with the severity of hypertension and which is significantly reduced by ACE inhibitors or losartan but not hydralazine or diuretics.7,16,38,43,60 Assuming equal frequencies of apoptotic cell death among diploid and tetraploid SMC, the decrease in aortic DNA content would overestimate SMC mortality by one third. Indeed, in the present study treatment with losartan caused a greater reduction in DNA content than in SMC number in the aorta after 4 weeks (63% reduction in DNA versus 47% reduction in cellularity, respectively). A similar pattern was observed with enalapril (66% versus 50% reduction, respectively). In contrast, nifedipine caused a smaller reduction in DNA content than in SMC number (25% versus 38% reduction, respectively). It is intriguing to speculate that these differences may reflect differential rates of apoptosis in polyploid versus diploid SMC. Clearly, further studies are needed to investigate apoptosis regulation and arterial SMC polyploidy.

Detailed studies by Owens35 did not detect a change in vascular DNA content or SMC number in the aorta of SHR treated with the ACE inhibitor captopril. Differences in experimental design may explain the discrepancies between the latter and present studies. First, captopril was administered to SHR for 3 months beginning at 2 months of age, ie, before the onset of hypertension. In contrast, rats used in the present study were already hypertensive when drug therapy was initiated and their aorta was examined at earlier times thereafter. Second, we observed a marked reduction of the preexisting hypertension (at least with inhibitors of the angiotensin II pathway), whereas the treatment with captopril35 mainly prevented the development of high blood pressure over time. It is intriguing to speculate that stimulation of SMC apoptosis in the arterial media may require aggressive antihypertensive therapy causing abrupt yet specific changes in the homeostatic environment of the cells in the arterial wall. In support of this view, we showed that a lower dose of losartan that prevented, but did not reverse, the progression of hypertension in SHR failed to affect aortic mass, DNA fragmentation index, or DNA content at 4 weeks. Previous studies support the view that rates of blood pressure change over time may be critical in determining SMC growth behavior. For instance, the gradual increase in blood pressure in SHR, Goldblatt rats, and DOCA-salt and norepinephrine-induced models of hypertension results in increased aortic SMC polyploidy.15,16 In contrast, the abrupt increase in intra-aortic blood pressure that follows suprarenal aortic coarctation is associated with the rapid and significant increase in aortic SMC hyperplasia.61 The regulation of SMC apoptosis in normotensive rats, which show less prominent hemodynamic responses to antihypertensive drugs than SHR, remains to be determined. An additional though nonexclusive possibility is that the depletion of SMC in the arterial wall is transient and reversible within the first months of drug therapy. Indeed, our data with nifedipine at 2 and 4 weeks (Fig 3) are consistent with such a complex regulation of SMC growth in the arterial wall. Ultimately, it is conceivable that vascular remodeling without a net change in vascular mass or cell number (eutrophic vascular remodeling)62 may result from the reversible depletion of SMC via a balanced process of SMC apoptosis and replication.

Molecular Mechanisms Regulating SMC Apoptosis
It is important to note that the triggering of apoptosis is dependent on a balance of environmental cues that are far from being specific to apoptosis. In vitro studies have identified several factors that can modulate, in parallel or in opposition, SMC DNA replication and apoptosis. For instance, DNA replication is inhibited and apoptosis is increased via the cAMP pathway, the nitric oxide pathway, interferon-{gamma}, or calcium channel blockers.21,32,33,47,47,63 In contrast, platelet-derived growth factor or insulin-like growth factor-1 inhibits apoptosis and promotes DNA replication.31,64,65 In addition, overexpression of the transcription factor c-myc or its adenoviral functional homologue E1A increases both DNA replication and apoptosis in SMC.66,67 Finally, basic fibroblast growth factor and epidermal growth factor stimulate DNA replication without affecting SMC apoptosis.31,68 We have recently reported further evidence for the independent regulation of DNA synthesis and cell death by apoptosis in cultured SMC.69 To our knowledge, the present study is the first to describe the regulation by pharmacological agents of arterial SMC apoptosis in vivo.

Angiotensin II acting via AT1 receptors may act as a survival factor for SMC. Both the ACE inhibitor enalapril and the angiotensin II AT1 receptor antagonist losartan potently increased SMC apoptosis in the aorta. Angiotensin II binding to AT1 receptors may inhibit SMC apoptosis either directly or indirectly by stimulating the production of autocrine survival factors, including platelet-derived growth factor and insulin-like growth factor-1,31,64,65 or by stimulating the production of specific extracellular matrix molecules, such as osteopontin,70 in the arterial wall. Disruption of binding to the {alpha}Vß3 integrin receptor for osteopontin induces apoptosis in angiogenic blood vessels.71 In fact, a growing body of evidence suggests that extracellular matrix proteins regulate apoptosis by interacting with cellular integrin receptors and modulating intracellular protein tyrosine phosphatase activity.72 Stimulation of the AT2 receptor subtype for angiotensin II is a potential pathway for the rapid stimulation of SMC apoptosis in losartan-treated animals, a condition where plasma levels of angiotensin II are markedly increased.73 Recently, the angiotensin AT2 receptor subtype has been implicated in the reduction of high blood pressure,74,75 the inhibition of arterial SMC DNA replication,76 and the induction of apoptosis in fibroblasts, ovarian cells and neuronal cells via the activation of protein tyrosine phosphatase pathways.77,78

Enalapril effects on SMC apoptosis, DNA synthesis, and vascular mass were comparable in magnitude though delayed compared with losartan, which was the most potent inducer of apoptosis or inhibitor of DNA synthesis. The faster onset for blood pressure reduction with enalapril may involve the reduced breakdown of endogenous vasodilatory kinins.79 Effects of kinins on apoptosis are unknown, but it is possible that kinin-induced secretion of NO by endothelial cells may contribute to stimulate SMC apoptosis in vivo.63

Nifedipine had a transient effect on arterial growth and apoptosis. Recent retrospective epidemiological studies suggested that treatment of hypertensive patients with rapidly acting formulations of nifedipine (as in the present study) might increase the risk of coronary mortality80 or cancer.81 In the latter study, inhibition of apoptotic cell death by nifedipine was proposed as a putative mechanism for the promotion of neoplastic growth. The present study does not provide evidence supporting this hypothesis. Clearly, these clinical issues remain to be clarified within the framework of prospective studies. Notably, it remains unclear whether the putative risk increases might represent a class effect of calcium channel blockers, a secondary effect related to sympathetic activation with short acting formulations of nifedipine82 (in the case of coronary mortality), or simply a medication-independent association between hypertension and cancer.83 The present data suggest that antihypertensive medications have complex effects on growth and apoptosis which need to be explored further in the long term.

It has been suggested that long-term benefits from antihypertensive therapies may depend on the remodeling of vascular structure with the normalization of wall-to-lumen ratio.10–12 Recent studies in hypertensive humans indicated that blood pressure reduction is accompanied with significant remodeling of small muscular arteries in patients treated with ACE inhibitors or a calcium channel antagonist but not ß-blockers.84–86 The present studies suggest that SMC apoptosis is one of the earliest events in the sequence of changes taking place during drug-induced regression of vascular hypertrophy. It remains unclear what is the long-term significance of apoptosis on SMC population dynamics in the arterial wall. Another critical question is whether the regulation of apoptosis is vessel specific, as it has been suggested for SMC growth behavior.70,87

In summary, nifedipine and inhibitors of the angiotensin pathway stimulated SMC apoptosis early during regression of vascular hypertrophy in the thoracic aorta, before SMC DNA replication was inhibited, and independently of blood pressure reduction. Thus, a major goal of research should be to better characterize mechanisms regulating apoptotic SMC death in vivo and its putative role in hypertensive vessel remodeling in disease and in response to therapeutic agents.


*    Acknowledgments
 
This work was supported by grants from the Medical Research Council of Canada (MT-10803 and MT-12563), the Heart and Stroke Foundation of Canada, and Bayer Canada. B.-S. Tea received a studentship from the Medical Research Council of Canada and T.-V. Dam received a fellowship from Merck Frosst Canada. Special thanks are extended to Dr Pierre Moreau and Dr Stephen M. Schwartz for fruitful discussion and to Suzanne Carioto, Manon Léveillé, Zachary Perras, and Nathalie Rousseau for assistance in animal care and surgery.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Folkow B. Physiological aspects of primary hypertension. Physiol Rev. 1982; 62 : 347 –504.[Free Full Text]
  2. Mulvany MJ. Are vascular abnormalities a primary cause or secondary consequence of hypertension? Hypertension. 1991; 18 (suppl I): I-52 –I-57.[Medline] [Order article via Infotrieve]
  3. Lee RMKW, Smeda JS. Primary versus secondary structural changes of the blood vessels in hypertension. Can J Physiol Pharmacol. 1985; 63 : 392 –401.[Medline] [Order article via Infotrieve]
  4. Schiffrin EL. Reactivity of small blood vessels in hypertension: Relation with structural changes. Hypertension. 1992; 19 (suppl II): II-1 –II-9.
  5. Heagerty AM, Aalkjaer C, Bund SJ, Korsgaard N, Mulvany MJ. Small artery structure in hypertension: dual processes of remodeling and growth. Hypertension. 1993; 21 : 391 –397.[Free Full Text]
  6. Heistad DD, Armstrong ML, Baumbach GL, Faraci FM. Sick vessel syndrome: recovery of atherosclerotic and hypertensive vessels. Hypertension. 1995; 26 : 509 –513.[Abstract/Free Full Text]
  7. Lee RM, Owens GK, Scott-Burden T, Head RJ, Mulvany MJ, Schiffrin EL. Pathophysiology of smooth muscle in hypertension. Can J Physiol Pharmacol. 1995; 73 : 574 –584. Review.[Medline] [Order article via Infotrieve]
  8. Folkow B. "Structural factor" in primary and secondary hypertension. Hypertension. 1990; 16 : 89 –101.[Medline] [Order article via Infotrieve]
  9. Safar ME, Laurent S. Behaviour of conduit arteries in hypertension. Clin Exp Hypertens. 1993; 15 : 1033 –1045. Review.[Medline] [Order article via Infotrieve]
  10. Boudier HAJS, van Bortel LMAB, De Mey JGR. Remodeling of the vascular tree in hypertension: drug effects. Trends Pharmacol Sci. 1990; 11 : 240 –245.[Medline] [Order article via Infotrieve]
  11. Mulvany MJ. Determinants of vascular structure. J Cardiovasc Pharmacol. 1992; 19 (suppl 5): S1 –S6.
  12. Mulvany MJ. The development and regression of vascular hypertrophy. J Cardiovasc Pharmacol. 1992; 19 (suppl 2): S22 –S27.[Medline] [Order article via Infotrieve]
  13. Safar ME, Levy BI, London GM. Arterial structure in hypertension and the effects of angiotensin converting enzyme inhibition. J Hypertens Suppl. 1992; 10 : S51 –S57. Review.[Medline] [Order article via Infotrieve]
  14. Owens GK, Schwartz SM. Vascular smooth muscle cell hypertrophy and hyperploidy in the Goldblatt hypertensive rat. Circ Res. 1983; 53 : 491 –501.[Abstract/Free Full Text]
  15. Yamori Y, Mano M, Nara Y, Horie R. Catecholamine-induced polyploidization in vascular smooth muscle cells. Circulation. 1987; 75 (suppl I): I-92 –I-95.[Medline] [Order article via Infotrieve]
  16. Owens GK. Control of hypertrophic versus hyperplastic growth of vascular smooth muscle cells. Am J Physiol. 1989; 257 : H1755 –H1765.[Medline] [Order article via Infotrieve]
  17. Walker SV, Hamet P. Enhanced DNA synthesis in heart and kidney of newborn spontaneously hypertensive rats. Hypertension. 1986; 8 : 520 –525.[Abstract/Free Full Text]
  18. Mulvany MJ, Baandrup U, Gundersen HLG. Evidence for hyperplasia in mesenteric resistance vessels of spontaneously hypertensive rats using a three-dimensional dissector. Circ Res. 1985; 57 : 794 –800.[Abstract/Free Full Text]
  19. Kerr JFR, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972; 26 : 239 –257.[Medline] [Order article via Infotrieve]
  20. Thompson GB. Apoptosis in the pathogenesis and treatment of disease. Science. 1995; 267 : 1456 –1462.[Abstract/Free Full Text]
  21. Hamet P, Richard L, Dam T-V, Teiger E, Orlov SN, Gaboury L, Gossard F, Tremblay J. Apoptosis in target organs of hypertension. Hypertension. 1995; 26 : 642 –648.[Abstract/Free Full Text]
  22. Frohlich ED, Sasaki O, Chien Y, Arita M. Changes in cardiovascular mass, left ventricular pumping ability and aortic distensibility after calcium antagonists in Wistar-Kyoto and spontaneously hypertensive rats. J Hypertens. 1992; 10 : 1369 –1378.[Medline] [Order article via Infotrieve]
  23. Oddie CJ, Dilley RJ, Bobik A. Long-term angiotensin II antagonism in spontaneously hypertensive rats: effects on blood pressure and cardiovascular amplifiers. Clin Exp Pharmacol Physiol Suppl. 1992; 19 : 392 –395.
  24. Igawa T, Ikeno K, Watanabe K, Kimura Y. Dose-related inhibitory effects of the beta-adrenoceptor blocking drug carteolol and propranolol ion cardiac hypertrophy in spontaneously hypertensive rats. Eur J Pharmacol. 1984; 104 : 93 –99.[Medline] [Order article via Infotrieve]
  25. Kobayashi H, Sano T, Tarazi RC, Fouad-Tarazi FM. Effects of antihypertensive drugs on heart and resistance vessels. Cardiovasc Res. 1990; 24 : 137 –143.[Abstract/Free Full Text]
  26. Teiger E, Dam TV, Richard L, Wisnewsky C, Tea BS, Gaboury L, Tremblay J, Schwartz K, Hamet P. Apoptosis in pressure overload-induced heart hypertrophy in the rat. J Clin Invest. 1996; 97 : 2891 –2897.[Medline] [Order article via Infotrieve]
  27. Bortner CD, Oldenburg NBE, Cidlowski JA. The role of DNA fragmentation in apoptosis. Trends Cell Biol. 1995; 5 : 21 –26.[Medline] [Order article via Infotrieve]
  28. Oberhammer F, Wilson JW, Dive C, Morris ID, Kichman JA, Wakeling AE, Walker PR, Sikorska M. Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 1993; 12 (9) : 3679 –3684.
  29. Cho A, Courtman DW, Langille BL. Apoptosis (programmed cell death) in arteries of the neonatal lamb. Circ Res. 1995; 76 : 168 –175.[Abstract/Free Full Text]
  30. Bennett MR, Evan GI, Newby AC. Deregulated expression of the c-myconcogene abolishes inhibition of proliferation of rat vascular smooth muscle cells by serum reduction, interferon-gamma, heparin, and cyclic nucleotide analogues and induces apoptosis. Circ Res. 1994;74 : 525 –536.
  31. Bennett MR, Evan GI, Schwartz SM. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest. 1995; 95 : 2266 –2274.[Medline] [Order article via Infotrieve]
  32. Miura M, Shu H, Rotello R, Hartwieg EA, Yuan J. Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C elegans cell death gene ced-3. Cell. 1993; 75 : 653 –660.[Medline] [Order article via Infotrieve]
  33. Orlov SN, Dam TV, Tremblay J, Hamet P. Apoptosis in vascular smooth muscle cells: role of cell shrinkage. Biochem Biophys Res Commun. 1996; 221 : 708 –715.[Medline] [Order article via Infotrieve]
  34. Owens GK. Differential effects of antihypertensive drug therapy on vascular smooth muscle cell hypertrophy, hyperploidy, and hyperplasia in the spontaneously hypertensive rat. Circ Res. 1985; 56 : 525 –536.[Abstract/Free Full Text]
  35. Owens GK. Influence of blood pressure on development of aortic medial smooth muscle hypertrophy in spontaneously hypertensive rats. Hypertension. 1987; 9 : 178 –187.[Abstract/Free Full Text]
  36. Owens GK, Schwartz SM. Alterations in vascular smooth muscle mass in the spontaneously hypertensive rat: role in cellular hypertrophy, hyperploidy, and hyperplasia. Circ Res. 1982; 51 : 280 –289.[Abstract/Free Full Text]
  37. Loeb AL, Bean BL. Antihypertensive drugs inhibit hypertension-associated aortic DNA synthesis in the rat. Hypertension. 1986; 8 : 1135 –1142.[Abstract/Free Full Text]
  38. Dominiczak AF, Devlin AM, Lee WK, Anderson NH, Bohr DF, Reid JL. Vascular smooth muscle polyploidy and cardiac hypertrophy in genetic hypertension. Hypertension. 1996; 27 : 752 –759.[Abstract/Free Full Text]
  39. Scott-Burden T, Resink TJ, Baur U, Burgin M, Buhler FR. Epidermal growth factor responsiveness in smooth muscle cells from hypertensive and normotensive rats. Hypertension. 1989; 13 : 295 –304.[Abstract/Free Full Text]
  40. Hamet P, Hadrava V, Kruppa U, Tremblay J. Transforming growth factor ß1 expression and effect in aortic smooth muscle cells from spontaneously hypertensive rats. Hypertension. 1991; 17 : 896 –901.[Abstract/Free Full Text]
  41. Hamet P, Hadrava V, Kruppa U, Tremblay J. Vascular smooth muscle cell hyper-responsiveness to growth factors in hypertension. J Hypertens Suppl. 1988; 6 : S36 –S39.[Medline] [Order article via Infotrieve]
  42. Hadrava V, Tremblay J, Sekaly RP, Hamet P. Accelerated entry of aortic smooth muscle cells from spontaneously hypertensive rats into the S phase of the cell cycle. Biochem Cell Biol. 1992; 70 : 599 –604.[Medline] [Order article via Infotrieve]
  43. Conyers RB, Kwan CY, Lee RM. Alterations in beta-adrenoceptors and polyploidy in cultured aortic smooth muscle cells from different age groups of spontaneously hypertensive rats and Wistar-Kyoto rats. J Hypertens. 1995; 13 : 507 –515.[Medline] [Order article via Infotrieve]
  44. Lee RM, Conyers RB, Kwan CY. Incidence of multinucleated and polyploid aortic smooth muscle cells cultured from different age groups of spontaneously hypertensive rats. Can J Physiol Pharmacol. 1992; 70 : 1496 –1501.[Medline] [Order article via Infotrieve]
  45. Hamet P, deBlois D, Dam T-V, Richard L, Teiger E, Tea B-S, Orlov SN, Tremblay J. Apoptosis and vascular wall remodeling in hypertension. Can J Physiol Pharmacol. 1996; 74 : 850 –861.[Medline] [Order article via Infotrieve]
  46. Hamet P, Moreau P, Dam T-V, Orlov SN, Tea B-S, deBlois D, Tremblay J. The time window of apoptosis: a new component in the therapeutic strategy for cardiovascular remodeling. J Hypertens. 1996; 14 (suppl 5): S65 –S70.
  47. Leszczynski D, Shao Y, Luokkamaki M, Foegh ML. Apoptosis of vascular smooth muscle cells: protein kinase C and oncoprotein Bcl-2 are involved in regulation of apoptosis in non-transformed rat vascular smooth muscle cells. Am J Pathol. 1994; 145 : 1265 –1270.[Abstract]
  48. Soltis EE. Alterations in vascular structure and function after short-term losartan treatment in spontaneously hypertensive rats. J Pharmacol Exp Ther. 1993; 266 : 642 –646.[Abstract/Free Full Text]
  49. Ferrante F, Abbate F, Ciriaco E, Laura R, Amenta F. Influence of isradipine treatment on the morphology of the aorta in spontaneously hypertensive rats. J Hypertens. 1994; 12 : 523 –531.[Medline] [Order article via Infotrieve]
  50. Benetos A, Albaladejo P, Levy BI, Safar ME. Acute and long-term effects of angiotensin converting enzyme inhibition on larger arteries and cardiac hypertrophy: mechanical and structural parameters. J Hypertens Suppl. 1994; 12 : S21 –S29.
  51. Safar ME, Levy BI. Mechanical function and histological structure of the arterial wall. The response to antihypertensive treatment. Drugs. 1993; 46 (suppl 2): 68 –74. Review.[Medline] [Order article via Infotrieve]
  52. Albaladejo P, Bouaziz H, Duriez M, Gohlke P, Levy BI, Safar ME, Benetos A. Angiotensin-converting enzyme inhibition prevents the increase in aortic collagen in rats. Hypertension. 1994; 23 : 74 –82.[Abstract/Free Full Text]
  53. Safar ME, Frohlich ED. The arterial system in hypertension: a prospective view. Hypertension. 1995; 26 : 10 –14.[Abstract/Free Full Text]
  54. Yamori Y, Tarazi RC, Ooshima A. Effect of beta-receptor-blocking agents on cardiovascular structural changes in spontaneous and noradrenaline-induced hypertension in rats. Clin Sci. 1980; 59 : 457s –460s.[Medline] [Order article via Infotrieve]
  55. Benetos A, Poitevin P, Prost PL, Safar ME, Levy BI. Life survival and cardiovascular structures following selective beta-blockade in spontaneously hypertensive rats. Am J Hypertens. 1994; 7 : 186 –192.[Medline] [Order article via Infotrieve]
  56. Contard F, Glukhova M, Marotte F, Narcisse G, Schatz C, Swynghedauw B, Guez D, Samuel JL, Rappaport L. Diuretic effects on cardiac hypertrophy in the stroke prone spontaneously hypertensive rat. Cardiovasc Res. 1993; 27 : 429 –434.[Abstract/Free Full Text]
  57. Bochaton-Piallat ML, Gabbiani F, Redard M, Desmoulière A, Gabbiani G. Apoptosis participates in cellularity regulation during rat aortic intimal thickening. Am J Pathol. 1995; 146 : 1059 –1064.[Abstract]
  58. Han DK, Haudenschild CC, Hong MK, Tinkle BT, Leon MB, Liau G. Evidence for apoptosis in human atherogenesis and in a rat vascular injury model. Am J Pathol. 1995; 147 : 267 –277.[Abstract]
  59. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury: smooth muscle growth in the absence of endothelium. Lab Invest. 1983; 49 : 327 –333.[Medline] [Order article via Infotrieve]
  60. Devlin AM, Gordon JF, Davidson AO, Clark JS, Hamilton CA, Morton JJ, Campbell AM, Reid JL, Dominiczak AF. The effects of perindopril on vascular smooth muscle polyploidy in stroke-prone spontaneously hypertensive rats. J Hypertens. 1995; 13 : 211 –218.[Medline] [Order article via Infotrieve]
  61. Owens GK, Reidy MA. Hyperplastic growth response of vascular smooth muscle cells following induction of acute hypertension in rats by aortic coarctation. Circ Res. 1985; 57 : 695 –705.[Abstract/Free Full Text]
  62. Mulvany MJ, Baumbach GL, Aalkjaer C, Heagerty AM, Korsgaard N, Schiffrin EL, Heistad DD. Vascular remodeling. Hypertension. 1996; 28 : 505 –506.[Medline] [Order article via Infotrieve]
  63. Fukuo K, Hata S, Suhara T, Nakahashi T, Shinto Y, Tsujimoto Y, Morimoto S, Ogihara T. Nitric oxide induces upregulation of Fas antigen and apoptosis in vascular smooth muscle cells. Hypertension. 1996; 27 : 823 –826.[Abstract/Free Full Text]
  64. Itoh H, Mukoyama M, Pratt RE, Gibbons GH, Dzau VJ. Multiple autocrine growth factors modulate vascular smooth muscle cell growth response to angiotensin II. J Clin Invest. 1993; 91 : 2268 –2274.[Medline] [Order article via Infotrieve]
  65. Naftilan AJ, Pratt RE, Dzau VJ. Induction of platelet-derived growth factor A-chain and c-myc gene expressions by angiotensin II in cultured rat vascular smooth muscle cells. J Clin Invest. 1989; 83 : 1419 –1424.[Medline] [Order article via Infotrieve]
  66. Bennett MR, Evan GI, Newby AC. Deregulated expression of the c-myc oncogene abolishes inhibition of proliferation of rat vascular smooth muscle cells by serum reduction, interferon-{gamma}, heparin, and cyclic nucleotide analogues and induces apoptosis. Circ Res. 1994; 74 : 525 –536.[Abstract/Free Full Text]
  67. Bennett MR, Evan GI, Schwartz SM. Apoptosis of rat vascular smooth muscle cells is regulated by p53 dependent and independent pathways. Circ Res. 1995; 77 : 266 –273.[Abstract/Free Full Text]
  68. Jackson CL, Schwartz SM. Pharmacology of smooth muscle cell replication. Hypertension. 1992; 20 : 713 –736.[Abstract/Free Full Text]
  69. Hamet P, Orlov SN, Tremblay J. Dissociation of DNA synthesis and apoptosis in vascular smooth muscle cells. Hypertension. 1996; 28 : 529 . Abstract.
  70. deBlois D, Lombardi DM, Su JE, Clowes AW, Schwartz SM, Giachelli CM. Angiotensin II induction of osteopontin expression and DNA synthesis in rat arteries. Hypertension. 1996; 28 : 1055 –1063.[Abstract/Free Full Text]
  71. Brooks PC, Montgomery AMP, Rosenfeld M, Reisfeld RA, Hu T, Klier G, Cheresh DA. Integrin {alpha}Vß3 antagonists promote tumor regression by inducing apoptosis in angiogenic blood vessels. Cell. 1994; 79 : 1157 –1164.[Medline] [Order article via Infotrieve]
  72. Meredith J, Fazeli B, Schwartz MA. The extracellular matrix as a cell survival factor. Mol Biol Cell. 1993; 4 : 953 –961.[Abstract]
  73. Timmermans PBMWM, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JAM, Smith RD. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev. 1993; 45 : 205 –251.[Medline] [Order article via Infotrieve]
  74. Ichiki T, Labosky PA, Shiota C, Okuyama S, Imagawa Y, Fogo A, Niimura F, Ichikawa I, Hogan BL, Inagami T. Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor. Nature. 1995; 377 : 748 –750.[Medline] [Order article via Infotrieve]
  75. Hein L, Barsh GS, Pratt RE, Dzau VJ, Kobilka BK. Behavioural and cardiovascular effects of disrupting the angiotensin II type-2 receptor in mice. Nature. 1995; 377 : 744 –747.[Medline] [Order article via Infotrieve]
  76. Nakajima M, Hutchinson HG, Fujinaga M, Hayashida W, Morishita R, Zhang L, Horiuchi M, Pratt RE, Dzau VJ. The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer. Proc Natl Acad Sci U S A. 1995; 92 : 10663 –10667.[Abstract/Free Full Text]
  77. Yamada T, Horiuchi M, Dzau VJ. Angiotensin II type 2 receptor mediates programmed cell death. Proc Natl Acad Sci U S A. 1996; 93 : 156 –160.[Abstract/Free Full Text]
  78. Tanaka M, Ohnishi J, Ozawa Y, Sugimoto M, Usuki S, Naruse M, Murakami K, Miyazaki H. Characterization of angiotensin II receptor type 2 during differentiation and apoptosis of rat ovarian cultured granulosa cells. Biochem Biophys Res Commun. 1995; 207 : 593 –598.[Medline] [Order article via Infotrieve]
  79. Bao G, Gohlke P, Qadri F, Unger T. Chronic kinin receptor blockade attenuates the antihypertensive effect of ramipril. Hypertension. 1992; 20 : 74 –79.[Abstract/Free Full Text]
  80. Furberg CD, Patsy BM, Meyer JV. Nifedipine: dose-related increase in mortality in patients with coronary heart disease. Circulation. 1995; 92 : 1326 –1331.[Abstract/Free Full Text]
  81. Pahor M, Guralnik JM, Salive ME, Corti MC, Carbonin P, Havlik RJ. Do calcium channel blockers increase the risk of cancer? Am J Hypertens. 1996; 9 : 695 –699.[Medline] [Order article via Infotrieve]