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Cardiovascular Research 2002 55(1):178-189; doi:10.1016/S0008-6363(02)00400-5
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Copyright © 2002, European Society of Cardiology

Aortic smooth muscle cell phenotypic modulation and fibrillar collagen deposition in angiotensin II-dependent hypertension

Gian Paolo Rossia,*, Martina Cavallina, Anna S Bellonib, Giuseppina Mazzocchib, Gastone G Nussdorferb, Achille C Pessinaa and Saverio Sartorec

aDepartment of Clinical and Experimental Medicine, Clinica Medica 4, University of Padua, via Giustiniani 2, Padua 35126 Italy
bDepartment of Human Anatomy and Physiology (section of Anatomy), University of Padua, Padua, Italy
cDepartment of Biomedical Sciences—CNR Unit for Muscle Biology and Pathophysiology, University of Padua, Padua, Italy

* Corresponding author. Tel.: +39-49-821-3304 or 2301; fax: +39-49-880-2252 gianpaolo.rossi{at}unipd.it

Received 27 November 2001; accepted 5 March 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Background: We investigated the effect of nifedipine, AT-1 and ET-1 receptor blockade on arterial smooth muscle cell phenotypes and collagen deposition in TGRen2 transgenic rat (TGR). Methods: Four-week-old TGR were blood pressure (BP)-matched and allocated to receive a placebo (n = 8), the calcium antagonist nifedipine (n = 6), the AT-1 specific receptor antagonist irbesartan (n = 6), the ETA/ETB antagonist bosentan (n = 6) or the ETA-selective antagonist BMS-182874 (n = 5). Sprague–Dawley normotensive rats served as controls (n = 6). After 4 weeks of treatment animals were euthanized and the left ventricle (LV) and the structural changes in intracardiac arterioles and aorta were assessed histomorphometrically. Smooth muscle cell phenotypes and fibrillar collagen content of the aortic wall were evaluated by immunostaining, using differentiation markers—specific antibodies and Syrius red staining, respectively. The changes in ETA and ETB receptor density were also assessed with quantitative autoradiography. Results: Compared to placebo, only irbesartan lowered BP (P<0.001) and prevented LV and small resistance artery hypertrophy. The aorta of placebo-treated TGR showed an increase in foetal-type smooth muscle cell content and fibrillar collagen staining, compared to controls. These changes were blunted by irbesartan, which increased ETA receptors in the arterial wall, enhanced by BMS-182874 and unaffected by bosentan. Nifedipine also blunted both the VSMC and collagen changes despite having no effect on BP and ETA receptors. Conclusions: In TGRen2, vascular hypertrophy entails both smooth muscle cell phenotypic modulation and collagen deposition. These alterations do not follow closely the BP changes and seem to imply the dihydropyridine-sensitive calcium channels.

KEYWORDS Ang II, Angiotensin II; AT-1, Angiotensin II type 1 receptor; BP, blood pressure; BW, body weight; CVD, cardiovascular damage; EC, endothelial cells; ET-1, endothelin-1; LV, left ventricle; TGR, Transgenic [mREN2] 27 rat; VSMC, Vascular Smooth Muscle Cell


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Increased vascular resistance is a hallmark of arterial hypertension and results from functional and structural changes in large (elastic-type) and small resistance arteries [1], which entail vascular wall hypertrophy. Molecular changes in the contractile machinery composition are involved in this process [2,3], but despite intensive research efforts the precise underlying mechanisms remain poorly understood. In a rabbit model of renovascular hypertension and in stroke-prone spontaneously hypertensive rats, the thickening of the media of large arteries implies an increase in foetal phenotype of VSMCs [4,5], i.e. a shift of VSMCs from the contractile to a synthetic phenotype and/or the expansion of a preexisting ‘immature’ medial VSMC population [3,6]. Likewise, an increased deposition of collagen and other proteins of the extracellular matrix can contribute to changing the viscodynamic properties of the vessel wall in hypertension [7]. The extracellular matrix (fibrillar collagen and fibronectin matrix assembly) can play a pivotal role in the regulation of arterial VSMC differentiation and proliferation [7–9]. Thus, it would appear that the process of vascular remodeling in hypertension involves coordinated and reciprocally regulated changes in extracellular matrix proteins and VSMC phenotypes

The TGR(mREN2)27 rat (TGR) is a monogenic model of severe hypertension created with the insertion of the mouse Ren2 gene into the rat genome [10]. The transgene is expressed in several tissues and causes increased local Ang II synthesis, which is mirrored by a twofold increase in Ang II content in the wall of the aorta [11]. The TGR is characterized by an early and prominent cardiovascular disease (CVD), and thus is a paradigm of endogenous Ang II-induced hypertension and excess CVD [10]. Accordingly, it is particularly useful to investigate the role played by Ang II and related mechanisms in hypertension-related changes in the arterial wall, including the process of VSMC phenotypic modulation and extracellular matrix deposition [12].

Compelling evidence indicates that Ang II can turn on the synthesis of the very potent vasoconstrictor peptide Endothelin(ET)-1 in different vascular cell types, including cultured VSMC and endothelial cells (EC), via AT-1 receptors linked to protein-1/kinase C-mediated mechanisms [13–15]. ET-1 was shown to be markedly activated in TGR [16], where it could mediate the hypertrophic response to Ang II and thus play an important role in CVD and vascular remodeling [13–15] (for review, see Ref. [12]). It is also likely that BP lowering per se might elicit beneficial effects independent of blockade of specific peptide pathways [17], but this contention remains controversial because it was difficult to prove.

Thus, our aim was to investigate whether blockade of the dihydropyridine-sensitive calcium channel, the Ang II type 1 (AT-1), the ET-1 type A (ETA) and B (ETB) receptors, which might lower BP by different mechanisms, differentially affected VSMC phenotypic changes and collagen deposition in the arterial wall of TGR.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1 Animals and treatments
The study protocol and handling of animals followed our institutional guidelines for animal studies and were previously reported [18]. Four-week-old male heterozygous TGR were BW- and BP (tail-cuff method)-matched and randomly assigned to receive the following oral treatments: a placebo (n = 8), the dihydropyridine calcium entry blocker nifedipine (30 mg/kg BW, n = 6), the AT-1 receptor selective antagonist irbesartan (50 mg/kg BW, n = 6), the mixed ETA/ETB endothelin receptor antagonist bosentan (100 mg/kg BW, n = 6) or the ETA selective receptor antagonist BMS-182874 (52 mg/kg BW, n = 5). All these dosages were previously shown to provide maximal antihypertensive effects. At these dosages, nifedipine exerted a full-blown antihypertensive effect and normalized left ventricular (LV) mass in renovascular hypertensive rats [19]; bosentan abolished the pressor effect of exogenous ET-1 in TGR [20]; BMS-182874 exerted a maximal antihypertensive effect in rats [21] and blunted the pressor response to exogenous ET-1 [22]. The dosage of irbesartan was at least fourfold higher than that which prevented the pressor response to Ang II [23]. BMS-182874 and irbesartan were gifts of Dr James Powell of Bristol Myers Squibb (Princeton, NJ, USA) and bosentan of Dr Martine Clozel (Actelion Ltd, Allschwil, Switzerland). Nifedipine was prepared as a 0.1% stock solution in a solvent that consisted of 969 g of polyethylene-glycol 400 (Sigma), 60 g of glycerine (BDH), and 100 g of water. Since nifedipine undergoes UV destruction, the stock solution was prepared fresh every day, diluted by saline, and covered with aluminum foil. Each treatment was BW-tailored weekly and administered individually as chocolate-flavored tablets and lasted for 4 weeks; the last dosage was given 24 h before sacrifice [18]. The same chocolate-flavored tablets with no drug was given as a placebo.

2.2 Histomorphometric changes in arterioles and aorta
To assess the structural changes in the intra-myocardial arterioles, 5-µm-thick equatorial and serial sections of the LV tissue were stained with hematoxylin–eosin. All animals from each group were examined; for each rat four-to-six arterioles (i.d. 50–250 µm) cut across their transverse axis were examined at 20x magnification with a Leica DM photomicroscope equipped with QWin Standard LeicaTM image software (Leica, Wetzlar, Germany). The internal diameter, calculated as the mean value of the major and minor axis, and the media thickness, estimated as the mean value of at least four values in each arteriole, were used to calculate the media cross-sectional area (MCSA). The same segment of thoracic and abdominal aorta from all rats was isolated, washed-perfused with PSS, snap frozen in dry ice-cooled isopentane and then stored in liquid nitrogen or at –80 °C until assayed. Six-µm-thick sections were then cut in the Leitz 1720 cryostat (Leitz, Wetzlar, Germany) and processed as reported [4]. Internal diameter and media thickness, estimated as specified above at magnification 5x in each section of aorta, were used to calculate the MCSA.

2.3 Immunohistochemistry and VSMC immunophenotyping
Immunophenotyping of VSMC from normal and hypertensive rats was accomplished with the use of the monoclonal antibodies to VSMC lineage (SM {alpha}-actin) and SMC differentiation (SM and MyHC-Apla2 myosin; EIIIA-Fn) markers [3,6,24,25] which are shown in Table 1. In the abdominal rat aorta, coexpression of SM-MyHC, MyHC-Apla2 and EIIIA-Fn identifies the foetal-type VSMC, whereas down-regulation of the latter two markers in the presence of high SM immunoreactivity pertains to a well differentiated adult-type VSMC [5,6]. A commercially available polyclonal antibody to ETA receptors was also used. The antibodies were applied to freshly cut unfixed cryosections as described [4]; the secondary antibody was the swine IgG anti-mouse IgG, or goat IgG anti-rabbit IgG, coupled with horseradish peroxidase (Dako, Dakopatts a/s, Glostrup, Denmark). Non-immune rat or rabbit serum was added to the secondary antibody to saturate non-specific binding-sites. Bound IgG was revealed by incubation in amino-ethyl-carbazole solution. Counterstaining was carried out with Harris's hematoxylin. Non-immune IgG instead of the primary antibody were used in control experiments. Quantification of staining was carried out blindly by a single examiner, using the aforementioned photomicroscope and software. To minimise operator-dependent variability, a specific routine automatically detecting the red-stained areas corresponding to binding to the specific antigen was developed. All animals from each group were examined. The entire surface of aorta cryosections was measured at 40x magnification after careful exclusion of spoiled portions. At least five views were captured and analysed for each section. Results are expressed as percentage of tunica media area stained with chosen antibodies.


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  		Table 1 Monoclonal antibodies used for the immunophenotypic characterization of VSMC

 
2.4 Endothelin receptor subtype density
Autoradiography was used to assess ET receptor density on iliac artery sections processed as reported [26]. All animals from each group were examined. In brief, ET-1 binding sites were labelled by incubation at room temperature for 120 min with 100 pmol/l [125I]ET-1 (Amersham Laboratories, Aylesbury, UK, specific activity 2000 Ci/mmol). Non-specific binding and selective displacement of [125I]ET-1 were studied by adding 1 µmol/l of unlabeled ET-1, or 100 nmol/l BQ-123 or BQ-788 (all from Neosystem Laboratories, Strasbourg, France), respectively. Washing thrice the samples in cold 50 nmol/l Tris–HCl buffer terminated the reaction. After rinsing in distilled water, the sections were rapidly dried, fixed in paraformaldehyde vapours at 80 °C for 120 min, and then coated with NTB2 Kodak Nuclear emulsion (Eastman Kodak, Rochester, NY). Exposure lasted for 2 weeks at 4 °C; undiluted D19 Kodak was used for development. ETA and ETB receptor density was quantified with computer-assisted image analysis software (Casti Imaging, Venice) coupled to a Leitz LaborluxTM microscope.

2.5 Effects of additional antihypertensive agents
The effect of BP-lowering on VSMC phenotypes in the abdominal aorta by different treatments was investigated in 5-week-old TGRen2 rats, which received a placebo (n = 6), the dual ACE and neutral vasopeptidase inhibitor MDL-100,240 (MDL, 40 mg/kg BW, n = 8), or ramipril (RAM, 5 mg/kg BW, n = 8), as reported in detail [27]. In brief, treatment lasted for 4 weeks at the end of which the abdominal aorta was collected and processed as described above for the immunohistochemistry experiments. We carried out the immunostaining for SM {alpha}-actin and EIIIA-Fn [3,5,6].

2.6 Statistical analysis
Results are expressed as mean±S.D., or S.E.M. One-way ANOVA with Bonferroni's post-hoc test and unpaired t-test was used for comparisons. Natural log-transformed values were used whenever Levene's test showed a non-gaussian distribution. Statistical significance was set at {alpha}<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1 Body weight, blood pressure and cardiac weight
Body weight was higher in the nifedipine than in the bosentan group but there were no other differences between groups (Table 2). BP was markedly lower in the irbesartan than in the other groups starting from the first week of treatment; by the end of the study period, it was 94±12, 107±13, 90±14, and 81±13 mmHg lower in the irbesartan group than in the Placebo, bosentan, BMS-182874, and nifedipine groups, respectively. The weight of the heart and LV were significantly lower in the irbesartan than in the other groups; they were significantly higher in BMS than in both the bosentan and the nifedipine groups.


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  		Table 2 Systolic BP, indices of structural changes in the abdominal aorta and the intramyocardial arterioles in the five experimental groups. Results of quantitative autoradiography performed with [125I]ET-1 binding to the wall iliac artery of TGR in the five experimental groups are also shown

 
3.2 Vascular structural changes
The thoracic aorta was examined for structural changes and the abdominal aorta also for histochemical and immunohistochemical changes. The irbesartan-treated TGR showed a lower mean cross-sectional area of intramyocardial arterioles than the other groups (Table 2). The BMS-182874 group showed the highest values of media cross-sectional area of all the four treatments.

3.3 VSMC phenotypes
In the aortas from normotensive Sprague–Dawley rats (BP=126±26 mmHg at 8 weeks of age) the immunostaining for SM myosin and SM {alpha}-actin did not change appreciably during the developmental stages (19 days foetus, 7 days, 4 and 8 weeks) that were examined. In contrast, the proportion of VSMCs stained for non-muscle myosin and EIIIA-Fn decreased progressively from foetal, to neonatal and to adult stage (Fig. 1). Thus, in adult Sprague–Dawley rats, VSMCs expressing these antigens were barely detectable and mainly confined to the subendothelial region [3]. Compared to age-matched Sprague–Dawley rats, TGR (Fig. 1G,H) exhibited increased percentages of non-muscle myosin- and EIIIA-Fn-stained VSMCs in the media, despite a similar proportion of SM {alpha}-actin positive cells (Fig. 2). A significant correlation (r = 0.835, P<0.0001) between EIIIA-Fn and MyHC-Apla2 immunostaining was found in these rats.


Figure 1
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  		Fig. 1 Immunohistochemistry of aortic cryosections from 19-day-old fetus (A, B), day 2 (C, D) and 4 weeks (E,F) control Sprague–Dawley rats exposed to antibodies to EIIIA-Fn (A,C,E) and MyHC-Apla2 (B,D,F). Note that immunostaining of medial VSMC with both antibodies decreases during development. Asterisks indicate localization of antigens in the subendothelial portion of the media (m). Bars: 100 µm (panels A–D); 80 µm (panels E,F). Panels G and H show the exemplary results obtained with EIIIA-Fn (G) and MyHC-Apla2 (H) antibodies in an adult (8-week-old) male heterozygous TGR rat. Note the marked increase in VSMCs expressing the two antigens compared to an age- and sex-matched normotensive Sprague–Dawley rat (panels E and F). Bar: 100 µm (panels G,H); a, adventia; m, media; vv, vasa vasorum.

 

Figure 2
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  		Fig. 2 Bar graphs showing the percentage of abdominal aorta tunica media area immunostained with antibodies to SM-type {alpha}-actin, EIIIA-Fn and MyHC-Apla2 from 8-week-old male normotensive Sprague–Dawley rats (black bars) and placebo-treated sex- and age-matched heterozygous TGR (open bars). Note the similar percentage of SM-type {alpha}-actin and the significantly (P<0.05) lower percentages of EIIIA-Fn and MyHC-Apla2 foetal-type VSMC in the aortic media of normotensive animals compared to TGR rats.

 
The different antihypertensive agents elicited quite different effects on VSMC phenotypes. The proportion of SM myosin positive VSMCs was similar in all groups, including placebo, but that of SM {alpha}-actin was variable and lower in nifedipine (33.3±20.8%, P<0.05) and BMS-182874 (40.2±6.9%, P<0.01) than in Placebo (69.7±14.2%), bosentan (67.2±10.6%) and irbesartan groups (75.5±12.7%). The increase in non-muscle myosin and EIIIA-Fn positive VSMCs observed in TGR was blunted by nifedipine and irbesartan.

Contrasting results were seen with the endothelin antagonists. BMS-182874 enhanced non-muscle myosin and EIIIA-Fn positive VSMCs (Figs. 3 and 4Go), while bosentan had variable effects, both between rats and between different areas of the same section. Thus, the proportion of VSMCs expressing MyHC-Apla2 in the bosentan group did not differ from placebo, although it was significantly lower than in BMS-182874 group (Fig. 4).


Figure 3
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  		Fig. 3 Immunohistochemistry with antibodies to EIIIA-Fn (left panels) and MyHC-Apla2 (right panels) of aortic cryosections from 8-week-old male heterozygous TGR Placebo-treated rats (A,B) and matched TGR rats treated with bosentan (C,D), irbesartan (E,F), BMS-182874 (G,H) and nifedipine (I, J). Note the abundance of foetal-type VSMCs in the aortic media from BMS-182874-treated rats and the scarce immunostaining of VSMC from irbesartan- and nifedipine-treated rats. Bars: 45 µm (A,B); 50 µm (C–J); a, adventia; m, media.

 

Figure 4
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  		Fig. 4 Bar graph showing percentage of area of abdominal aorta tunica media stained for EIIIA-Fn (upper panel) and MyHC-Apla2 (lower panel) from heterozygous TGR treated for 4 weeks with Placebo, bosentan, irbesartan, BMS-182874 and nifedipine (*P<0.05, **P<0.01, ***P<0.001 vs. BMS-182874).

 
Immunohistochemistry with antibodies to ETA and EIIIA-Fn on serial aortic sections (Fig. 5) showed that the increase in foetal-type VSMC in placebo-treated TGR was paralleled by a heterogeneous pattern of VSMC ETA expression, while in irbesartan-treated TGR the ETA expression was similar to that of normotensive rats.


Figure 5
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  		Fig. 5 Immunohistochemistry with antibodies to ETA (left panels) and EIIIA-Fn (right panels) of aortic cryosections from an 8-week-old male Sprague–Dawley normotensive (A, B), and from heterozygous TGR rats treated with Placebo (C, D) and irbesartan (E, F). Note the homogenous pattern of immunostaining for ETA in both the normotensive and the irbesartan-treated rat, which contrasts with the patchy immunostaining of a placebo-treated TGR rat. This pattern of immunostaining was associated with a marked increase in EIIIA-Fn-positive VSMC in the aortic media from Placebo-treated TGR (D). Bars: 50 µm; a, adventia; m, media; vv, vasa vasorum.

 
3.4 Fibrillar collagen
The results of staining for fibrillar collagen of abdominal aorta sections from adult TGR receiving the different treatments are shown in Fig. 7. The proportion of stained vessel wall did not differ between placebo and BMS-182874 groups, but was markedly lower in the nifedipine group. The decrease in the bosentan and irbesartan groups did not achieve statistical significance.


Figure 7
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  		Fig. 7 Bar graphs showing the percentage of Syrius red-stained abdominal aorta tunica media from heterozygous TGR treated for 4 weeks with placebo, bosentan, irbesartan, BMS-182874 and nifedipine (°P<0.05 vs. nifedipine; ***P<0.001 vs. placebo).

 
3.5 Endothelin receptor subtypes density
Autoradiography demonstrated specific [125I]ET-1 binding sites in the iliac artery wall; displacement with BQ-123 and BQ-788 showed the coexistence of both receptor subtypes. Table 2 shows the changes in ETA and ETB receptor subtype density in the five experimental groups. Irbesartan increased significantly ETA receptor density, compared to both placebo and BMS-182874; no differences between the other groups were seen. The ETB receptor density was similar in all treatment groups.

3.6 Effects of additional antihypertensive agents
The effects of MDL-100,240 and ramipril, compared to placebo, on BP and cardiac and vascular structure were recently reported in detail and therefore will be mentioned only briefly [26,27]. Both treatments proved to be similarly effective in lowering BP and in preventing the development of cardiac hypertrophy and vascular structural changes. At the end of the fourth week of treatment, the immunostaining of the abdominal aorta for EIIIA-Fn [3,5], expressed as percent of immunostained area [6], was significantly lowered by both MDL-100,240 (0.46±0.13%, P = 0.001) and ramipril (0.22±0.04%, P<0.001), compared to placebo (3.9±1.0%). Similar statistically significant differences between active treatments and placebo were seen when results were normalised for differences in SM {alpha}-actin immunostaining.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
One of the most relevant results of this study is the demonstration that the aorta, which has a twofold increase in Ang II content in this transgenic rat model [28], and the intramyocardial resistance artery wall developed marked hypertrophy, as indicated by increased media cross-sectional area. These changes were already evident at 8 weeks of age and are consistent with previous results in small mesenteric resistance arteries [18].

4.1 Mechanisms of vascular hypertrophy
Ang II-dependent hypertension has been linked with eutrophic remodelling of small resistance arteries and hypertrophy of elastic arteries in rats [1], but the underlying mechanisms remained to be clarified. According to one theory, VSMC hypertrophy would involve an increase in the ‘contractile’ protein content [29], whereas VSMC hyperplasia would be mirrored by a shift of VSMC from a contractile to a synthetic and/or proliferating phenotype [2,3]. Aortic rings from TGR were found to exhibit increased maximal developed tension responses to receptor and non-receptor mediated vasoconstricting stimuli, which indicated an enhanced efficiency of the contractile machinery via qualitative or quantitative changes in VSMC [18]. Thus, the present and previous findings collectively indicate that vascular hypertrophy occurs early in the life span of TGR and involves both elastic and resistance type arteries.

We also found that the increased MCSA of the abdominal aorta occurring with the development of severe hypertension implied two consequences: (1) significant alterations of the proportion of VSMC expressing antigens that are normally found during foetal development, but vanish soon after birth; (2) deposition of fibrillar collagen. Thus, it would appear that hyperplasia occurs in elastic-type arteries like the aorta. This finding accords well with the previous report of an accumulation of polyploid VSMC in the aortic media of TGR [17]. However, it remains unclear whether the same changes involve also resistance arteries, which develop wall hypertrophy but have different VSMC phenotypic composition [2,30].

The deposition of fibrillar collagen in the abdominal aorta tunica media (Fig. 6) was associated with expression of foetal VSMC phenotypes and was differently modulated by the different drug treatments (see later) (Figs. 6 and 7Go). Polymerised type I collagen can arrest VSMC in the G1 phase, via inhibition of phosphorylation of a cyclin-dependent kinase 2 (cdk2), thus inhibiting their proliferation [9]. Hence, the present results suggest that collagen deposition act as a modulator of VSMC growth.


Figure 6
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  		Fig. 6 Syrius red-stained aortic cryosections from 8-week-old male Sprague–Dawley rats (panel A) and from age- and sex-matched heterozygous TGR rats treated with placebo (B), bosentan (C), irbesartan (D), BMS-182874 (E) and nifedipine (F). Note the weak staining in the subendothelial region of Sprague–Dawley rats (asterisk) and the more marked and randomly dispersed staining in the media of the Placebo group. A marked decrease in Syrius red-stained areas is visible in nifedipine and irbesartan and to a lesser extent in bosentan groups, compared to the placebo group, which was similar to the BMS-182874 group. Bar: 50 µm; a, adventia; m, media.

 
4.2 Effects of blockade of the dihydropyridine L-type calcium channel
We used nifedipine on the assumption that it could lower BP by mechanisms different from those of AT-1 and ET-1 antagonists. We unexpectedly found that nifedipine, used at dosages shown to be maximally effective on BP in Ang II-dependent hypertension in rats [19], did not hinder the development of hypertension. This is in contrast with data with amlodipine [31] and manidipine [32], but in these latter studies amlodipine was up-titrated to a high dosage that lowered telemetrically measured BP [31], while manidipine decreased BP when given in combination with atenolol [32]. We used only one dose of nifedipine, which was lower than that of amlodipine [31]; furthermore, we did not use telemetry, which is a more accurate technique compared to the tail-cuff method. Therefore we cannot totally exclude that a slight antihypertensive effect of nifedipine might have been missed. However, the lack of prevention of LV hypertrophy argues against a major BP lowering effect of nifedipine [32] since practically in all studies where dihydropyridine calcium antagonists did lower BP, a decrease in LV mass concurred [19,31]. The lack of BP-lowering might be due to the fact that the pressor role of Ang II greatly exceeds that of L-type calcium channel-mediated mechanisms in this model, as indicated by the consistent prevention of hypertension and CVD also observed with MDL-100,240 and ramipril, e.g. with Ang II blockade [27]. Alternatively, it might be that in TGR the VSMC phenotypic modulation leads, by increasing the proportion of foetal type VSMC, to a decreased expression of L-type calcium channel and thus to some resistance to calcium channel blockade, as indicated by an in vitro study [33].

Of much interest, nifedipine strikingly decreased collagen deposition in the media (Figs. 6 and 7Go) and reduced the foetal-type VSMC population in favour of the adult-type VSMC counterpart (Figs. 3 and 4Go), even though this latter effect attained statistical significance only compared to the BMS-182874 group (Fig. 4). These findings strongly support the contention of the existence of a nifedipine-sensitive VSMC population in the aortic media [34]. Additionally, as both effects occurred despite the lack of prevention of hypertension, vascular MCSA and LV mass increase, they seem to be BP-independent. However, the possibility that a higher dose of nifedipine might have lowered BP and induced significant prevention also of VSMC phenotypic modulation remains to be investigated.

4.3 Effects of AT-1 receptor blockade
Up-regulation of non-muscle myosin and/or Fn in VSMC might be related to hypertension-induced mechanical stretch [29] and/or to paracrine effects of Ang II, which may occur via ET-1 [35,36]. To investigate the relative importance of the two peptides, we used an AT-1 antagonist and drugs interfering with ET-1 pressor mechanisms (see later). Irbesartan hindered the development of both hypertension and cardiac hypertrophy, a finding that accords with data with telmisartan [37] and with results with MDL-100,240 and ramipril [27]. However, although cultured VSMC from arteries of TGR showed hyperplastic changes and increased Ang II content [38], neither ACE nor AT-1 receptor blockade prevented DNA synthesis and cell proliferation in vitro. Furthermore, mesangial cells from TGR exhibited blunted [Ca2+]i responses to Ang II [39]. Thus, it would appear that the VSMC from TGR are resistant to Ang II effects and thereby to AT-1 blockade. Accordingly, the favourable effects of irbesartan on VSMC phenotypic modulation and collagen deposition might be predominantly due to the BP lowering rather than to blockade of the autocrine–paracrine effects of Ang II. The fact that MDL-100,240 and ramipril, which lowered BP, accomplished a prevention of VSMC phenotypic modulation similar to that seen with irbesartan does not clarify this issue.

It might be proposed that the changes elicited by irbesartan on BP and VSMC could be ascribed to a different mechanism. As irbesartan, at the dosage used in this study, was shown to cross the blood–brain barrier in an acute study of spontaneously hypertensive rats [40], it might accomplish blockade of angiotensin receptors located in the central nervous system [41,42] and thereby reduce peripheral efferent sympathetic nerve activity [42]. However, this contention needs further investigation on the central effects of oral chronic irbesartan administration in TGR since the sympathetic inhibitory effect of irbesartan was not confirmed in all studies [43].

4.4 Effects of blockade of the endothelin receptor subtypes
ET-1 is a likely mediator of the pressor and growth-promoting effects of Ang II [13–15] and was shown to be markedly activated in TGR [16], where it could mediate the hypertrophic response to Ang II and thus play an important role in CVD and vascular remodeling (for review, see Ref. [12]). To investigate the role of ET-1 and of its receptor subtypes we used the mixed ETA/ETB antagonist bosentan and an ETA antagonist. This strategy was preferred to a head-to-head comparison of an ETA- and an ETB-selective agent, because experience with AT-1 and AT-2 antagonist indicated that blockade of a single receptor subtype was associated with enhanced activation of the other, thus making enigmatic the interpretation of results. The information on ET-1 antagonist in experimental hypertension is conflicting (for review, see Refs. [12,18]). In rats treated with either infusion of exogenous Ang II [44] or administration of mineralocorticoid and/or salt [45], ETA antagonists lowered BP and protected from CVD [46]. In contrast, neither a mixed nor an ETA-selective receptor antagonist [20–22,47] elicited such effects in this and previous studies in TGR [20]. Furthermore, ETA blockade not only failed to prevent hypertension and cardiac hypertrophy, but worsened aortic hypertrophy, VSMC phenotypic modulation and fibrillar collagen deposition. In this regard the most striking of our findings was the increase in foetal-type VSMC population in BMS-182874 treated animals (Figs. 3G,H and 4Go), which was accompanied by a high (Figs. 6E and 7Go) level of fibrillar collagen deposition in the abdominal aorta tunica media. These nefarious effects accord with findings of impaired renal function in TGR [18], and of increased mortality following administration of an ETA antagonist in spontaneously hypertensive rats [48]. Unlike BMS-182874, bosentan did not adversely affect VSMC phenotypic modulation (Figs. 2 and 3GoC,D) and collagen deposition (Figs. 6C and 7Go), possibly because the detrimental effect of ETA blockade was counterbalanced by a favourable outcome of ETB blockade.

The density of ETA and ETB receptors in the artery wall was similar in all groups with the exception of irbesartan that significantly increased the density of ETA receptor, compared to placebo (Table 2), possibly because it prevented the down-regulation of ETA receptor seen in placebo-treated TGR. The increase in foetal-type VSMC population in the latter TGR was associated with a patchy expression of ETA immunostaining (Fig. 5C). Thus, collectively these results indicate that severe Ang II-dependent hypertension implies a decreased and heterogeneous expression of ETA receptors within the arterial tunica media. The lack of BP lowering effect and the detrimental outcome on CVD of ETA blockade in TGR might be explained on this ground.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
The development of severe hypertension in TGR is associated with early and prominent vascular structural changes that involve both elastic-type and small resistance arteries. In the aortic tunica media, these changes imply both an increase in foetal VSMC phenotypes and an increased fibrillar collagen deposition. AT1 receptor blockade prevented hypertension and the vascular structural changes. Blockade of the dihydropyridine-sensitive calcium channels also protected TGR from collagen deposition even despite the lack of any BP lowering effect. In contrast, both VSMC phenotypic modulation and fibrillar collagen deposition were enhanced by an ETA selective receptor antagonist, possibly because of the shift of endogenous ET-1 on the ETB receptors. Thus, in this model of Ang II-dependent hypertension, ET-1 intervenes in the autocrine–paracrine processes leading to vascular changes.

Time for primary review 31 days.


   Acknowledgements
 
We are grateful to Dr A. Sacchetto and Giuseppe Gottardo for technical assistance and to Abigail Johnson for editorial assistance. This study was supported by grants from the Italian Ministry of University and Scientific Research (MURST) (9906193152-001/06), from the University of Padua to Professor Rossi and by an A.S.I. grant to Professor Sartore.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

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