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International Journal of Morphology

versión On-line ISSN 0717-9502

Int. J. Morphol. v.21 n.1 Temuco mar. 2003

http://dx.doi.org/10.4067/S0717-95022003000100002 

Int. J. Morphol.,
21(1):9-14, 2003.

ENALAPRIL AND VERAPAMIL ATTENUATE THE AORTIC WALL
REMODELING IN NITRIC OXIDE DEFICIENCY1

ENALAPRIL Y VERAPAMIL ATENUANTES DE LA REMODELACION
DE LA PARED AÓRTICA EN DIFICIENCIA DE ÓXIDO NÍTRICO

Leila Maria Meirelles Pereira; Daniele Gonçalves Bezerra & Carlos Alberto Mandarim-de-Lacerda2


PEREIRA, L. M. M.; BEZERRA, D. G. & MANDARIM-DE-LACERDA, C. A. Enalapril and verapamil attenuate the aortic wall remodeling in nitric oxide deficiency. Int. J. Morphol., 21(1):9-14, 2003.

SUMMARY: Forty mature male rats (Wistar strain) were maintained alive for 40 days, separated in four groups of five rats each (Control, L-NAME, L-NAME + Enalapril, L-NAME + Verapamil). Three aortic wall parameters were analyzed, as follows: the QA(SMN) (the number of the tunica media smooth muscle nuclear profiles per area), the SV(lamellae) (surface density of the aortic lamellae), and the aortic wall thickness (AWT). Rats of the L-NAME group showed high blood pressure and cardiac hypertrophy. The AWT, the QA(SMN) and the SV(lamellae) showed the same tendency among the groups. The increased AWT, or the reduced QA(SMN) and the SV(lamellae) in l-NAME rats were not completely prevented by the administration of enalapril or verapamil. Hypertension causes the AWT increase by hypertrophy of the lamellar units of the media layer (SV(lamellae) decrease) and the following rarefaction of the smooth muscle nuclei (the QA(SMN) decrease). The relative number of the smooth muscle cells (SMC) nuclei in L-NAME rats was not confirmed when this number was corrected by the arterial wall area. This does not support the SMC intense proliferation in animals with NO deficiency with the L-NAME dose and the duration used in this experimentation.

KEY WORDS: 1. Nitric oxide; 2. Aorta; 3. Enalapril; 4. Verapamil; 5. Stereology.


INTRODUCTION

The nitric oxide (NO) is considered the main vasodilator produced in the endothelium, and the restriction of its production is associated with attenuation of vascular relaxation in different parts of the vascular tree (Holécyová et al., 1996). Mechanical properties of large arteries are primarily determined by the composition of the arterial wall. Hypertension is associated with morphological and functional alterations of large capacitance arteries, inducing increased thickness and stiffness of these arteries tunica media (Bézie et al., 1998; Folkow et al., 1973; Karam et al., 1996; Safar & Frohlich, 1995). In hypertension, the lumen diameter is decreased, causing an increase of the peripheral resistance which is regulated by the quantity and organization of smooth muscle cells (SMC) and extracellular matrix (ECM) of these arteries tunica (McGuffee & Little, 1996; Mulvany & Aalkjaer, 1990). It was speculated that the impairment in the vascular smooth muscle NO production precedes the development of hypertension in SHR and may play a pathophysiologic role in the early BP elevation in genetically hypertensive rats (Cruzado et al., 2002).

Treatment with angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor subtype 1 antagonist and long-acting Ca2+ channel blockers has corrected changes in large and small arteries in hypertension. Recent trials suggest that these different drugs may provide similar benefits essentially through blood pressure lowering, although some minor differences between the drugs were noted (Pereira & Mandarim-de-Lacerda, 1998; Pereira & Mandarim-de-Lacerda, 2001a-b; Schiffrin, 2001). Since the aortic stiffness is strongly related to the organization of the ECM, the question arising is whether treatment with ACE inhibitor, Ca2+ channel blocker and their combination is able to prevent arterial stiffness by acting on the ECM composition and organization (Koffi et al., 1999).

The determination of the quantitative alterations in the aortic wall in a NO deficiency situation and the efficiency of the enalapril (ACE inhibitor) and verapamil (Ca2+ channel blocker) are the aims of this study.

MATERIAL AND METHOD

All procedures were carried out in accordance with conventional guidelines for experimentation with animals (NIH Publication No. 85-23, revised 1996). The experimental protocols used in this study were approved by the Ethics Committee for Animal Experimentation at the State University of Rio de Janeiro. Forty mature male rats (Wistar strain) were obtained from colonies maintained at the Laboratory of Morphometry & Cardiovascular Morphology (www2.uerj.br/~lmmc). At the beginning of the study, rats had body mass (BM) of 270±22g and systolic blood pressure (BP) of 117±6mmHg (mean±SD). Rats were individually housed in temperature-controlled (21±1C) and humidity-controlled (60±10%) room submitted to a 12h-dark/light cycle (artificial lights, 7:00­19:00h) and to air exhaustion cycle (15min/h). Rats were given food (Nuvilab®, Rio de Janeiro, Brazil) and water ad libitum.

After a 7-day equilibration period the average daily water intake per animal was determined and this volume was used to dissolve drugs and guarantee the total intake of the planned daily drug dosage. The BP and the BM were verified weekly in conscious rats. The BP was verified through the non-invasive method of the tail-cuff plethysmography. Animals were maintained alive for 40 days and separated in four groups of five rats each. Control group rats received water and food ad libitum; L-NAME group rats received L-NAME 50 mg.kg-1.day-1 dissolved in drinking water (Sigma Chemical Co., St. Louis, Lot 67H0876; L-NAME +Enalapril group rats received L-NAME combined to enalapril maleate 15 mg.kg-1.day-1 dissolved in drinking water (Sigma Chemical Co., St. Louis, Lot 38H0500; L-NAME +Verapamil group rats received L-NAME associated with verapamil hydrochloride 15 mg.kg-1.day-1 dissolved in drinking water (Sigma Chemical Co., St. Louis, Lot 56H0925).

At the time of euthanasia, the BM and the BP were measured and animals were then deeply anesthetized (Thiopental 15mg.Kg-1 intraperitonial) and sacrificed by exsanguination. The heart was removed and the left ventricle with the interventricular septum (LVS) was dissected. The LVS mass was recorded in the day of the sacrifice and the LVS mass/body mass (LVSM/BM) ratio was determined.

Arterial rings (5mm in length) of the thoracic aorta proximal to the 1st intercostal artery were immediately isolated and cleaned free of adherent adipose and connective tissue, and then immersed in freshly prepared 4% w/v formaldehyde in 0.1M phosphate buffer pH 7.2 for 48 hours. The arterial rings were processed according to the routine histological procedures embedded with Paraplast plus® (Sigma Chemical Co., St. Louis, USA) and vertically cut 3µm thick. Sections were stained by hematoxylin-eosin and Masson's trichrome.

Stereology analyzed 25 fields per group by a video-microscopy (Leica DMRBE microscope and video-camera Kappa CF 15/5). A test-system of cycloid arcs was put upon the screen of the monitor and calibrated with a micrometer 1/100mm. Two parameters were analyzed, as follows: the QA(SMN) (the number of the tunica media smooth muscle nuclear profiles per area) which was determined by the use of a 4000µm_ frame, and the SV(lamellae) (surface density of the

lamellae intersection with the cycloid arcs and the LT is the test-line length (Mandarim-de-Lacerda, 1999). The tunica media was defined as the region between the internal and external elastic laminae, the tunica intima was defined as the region between the endothelium and the internal elastic laminae. The aortic wall thickness was measured including both the tunica intima and the tunica media.

Differences in BP levels comparing day 1 and day 41 were tested through the paired t-test and differences among groups in day 41 were tested by analysis of variance and Newman-Keuls multiple comparison test. Stereological differences comparing the control group and the experimental groups were tested through the Mann-Whitney non-parametric test. The significant level of 0.05 was considered statistically significant (Zar, 1999).

RESULTS

Results are shown in Table I and Fig 1. The BP and the LVSM/BM ratio had a similar alteration among the groups. In rats with NO deficiency (L-NAME group) the BP and the LVSM/BM ratio showed higher values than the other groups whereas no difference was observed among the other groups.

Light microscopy of the aortic wall showed alterations in the L-NAME group tunica media thicker than the untreated control rats, probably due to an enlargement of the SMC in this group. This can be easily observed by comparing the distances between each concentric elastic laminae intermingled with the SMC layers. The aortic wall in rats of the L-NAME group was characterized by an enlargement of endothelial cells lying over thick lamellae, foci of the intimal thickening, and disarray and disruption of the lamellae. Treatment with enalapril or verapamil resulted in clearly attenuated aortic wall alterations (Fig. 1).

Fig. 1. Photomicrographs of the aortic wall sections stained with Masson's trichrome and taken with the same magnification (bar = 50µm). All pictures have the endothelium at the left side. (a) Control group, (b) L-NAME group, (c) L-NAME+Enalapril group group, and (d) L-NAME+Verapamil group. The minor structural alterations are clearly seen in a, and the major alterations are seen in b. In c and in d altered tunica media exhibits the enlarged and disrupted elastic lamellae segments, more pronounced in the tunica media outer layer.

The aortic wall thickness, the QA(SMN) and the SV(lamellae) were significantly different when Control group and other groups were compared. The aortic thickness and the SV(lamellae) was different between the L-NAME and the L-NAME+verapamil groups, and it was also different between the L-NAME+enalapril and the L-NAME+verapamil groups, but not between the L-NAME and the L-NAME + enalapril groups. Compared to the Control group, the aortic thickness was more than 20% greater than the other groups, and both the QA(SMN) and the SV(lamellae) were more than 20% lower than the other groups. The combined administration of L-NAME and enalapril or L-NAME and verapamil did not modify the QA(SMN) concerning the simple administration of L-NAME. However, the number of SMC nuclei in an area equal to the square aortic wall thickness in square micrometers (a number of the tunica media smooth muscle nuclear profiles corrected per area by multiplying the QA(SMN) and Thickness (T) showed no difference among the groups.

DISCUSSION

This study confirmed the well known hypertensive effect of the inhibition of the NO synthesis and the efficiency in reducing this hypertension through the administration of enalapril or verapamil (Pereira & Mandarim-de-Lacerda, 1998; Pereira & Mandarim-de-Lacerda, 2001a-b). The inhibition of the NO synthesis also caused LV hypertrophy probably via pressure overload and was demonstrated by the significant increase of the LVSM/BM ratio in the L-NAME group in relation to the other groups. This result agrees with previous studies (Devlin et al., 1998; Moreno et al., 1996; Numaguchi et al., 1995; Pereira et al., 1998) but it contrasts with the fact that a prolonged use of a small dose of L-NAME (7.5 mg. kg-1. day-1 during 6 months) caused the reduction in LV weight, as well as in cardiac myocyte size (Oliveira et al., 2000). In this study we confirmed the efficiency of the treatment with enalapril or verapamil preventing the LV hypertrophy in rats with NO deficiency (Mazzadi et al., 1998; Pereira & Mandarim-de-Lacerda, 2001a-b).

The thickness of the aortic wall, the QA(SMN) and the SV(lamellae) showed a same tendency among the groups. The increased aortic wall thickness, or the reduced QA(SMN) and the SV(lamellae) in animals taking L-NAME was not completely recovered through the administration of enalapril or verapamil. The arterial wall thickening caused by hypertension is mainly due to the hypertrophy of the lamellar units of the media layer (confirmed here by the decrease of the SV(lamellae)) and it caused a rarefaction of the smooth muscle nuclei (measured by the decrease of the QA(SMN)).

The aortic wall became thicker in response to the hypertension (Matsumoto & Hayashi, 1996), but the underlying mechanisms of vascular structural alterations are still poorly understood. Vasoactive substances and hormones, such as Angiotensin II, have been implicated, and studies in culture suggest that the mechanical deformation of endothelial cells and SMC is important (Chien et al., 1998; Hu et al., 1998). Supporting this we know that the NO deficiency causes vascular lesions, such as perivascular fibrosis, medial thickening and increase in intima/media or wall/lumen ratios in the mesenteric microvascular beds, arterioles, coronary arteries, and aorta (Fitch et al., 2001; Zhao et al., 1999), and may result in increased monocyte and platelet adhesion, and accompanying release of growth factors which would contribute to the thickening of the vascular wall (Babál et al.,1997; Kristek et al.,1995; Numaguchi et al., 1995; Sventek et al.,1996).

The NO is able to blockade the vascular SMC proliferation in vitro and in vivo (Garg & Hassid, 1989; Kolpakov et al., 1995; MacNamara et al., 1993), whereas a positive correlation between hypoxia and SMC proliferation has been demonstrated (Cooper & Beasley, 1999). The SMC proliferation has been considered a major mechanism responsible for the greater arterial hypertrophy in the hypertensive animals with reduced blood flow (Ueno et al., 2000). In the present study, the apparent reduction of the relative number of the SMC nuclei in animals that had taken L-NAME was not confirmed when this number was corrected by the arterial wall area, the number of the SMC nuclei was unalterable among the groups. These findings do not support the tunica media SMC intense proliferation in animals submitted to NO synthesis blockade probably due to the used L-NAME dose and the duration of the experimentation.

Acknowledgements: The authors would like to thank Mrs. Thatiany Marinho and Ana Claudia Souza for their technical assistance.


PEREIRA, L. M. M.; BEZERRA, D. G. & MANDARIM-DE-LACERDA, C. A. Enalapril y verapamil atenuantes de la remodelación de la pared aórtica en deficiencia de óxido nítrico. Int. J. Morphol., 21(1):9-14, 2003.

Cuatro grupos de 10 ratas cada uno, adultas, machos (Wistar) se mantuvieron vivas por 40 días, divididas en: Control, L-NAME, L-NAME + Enalapril, L-NAME + Verapamil. Tres parámetros de la pared de la aorta fueron analizados: el QA(SMN) (número de núcleos de células musculares lisas de la túnica media por área), la SV(lamellae) (densidad de superficie de las lamelas de la aorta), y AWT ( grosor de la pared de la aorta). Estos tres parámetros mostraron la misma tendencia entre los grupos. Las ratas del grupo L-NAME tuvieron elevación de la presión arterial e hipertrofia cardiaca. El aumento de AWT, o la reducción del QA(SMN) y la SV(lamellae) en las ratas L-NAME no fue completamente prevenido por la administración de enalapril o verapamil. La hipertensión causó aumento del AWT por hipertrofia de las unidades lamelares de la túnica media (SV(lamellae) disminuyó) y la rarefacción de los núcleos de músculo liso (QA(SMN)) disminuyó. El número relativo de núcleos de células musculares lisas (SMC) en ratas L-NAME no fue confirmado cuando este número fue corregido por el área de la pared arterial. La hipótesis de intensa proliferación de SMC en animales con deficiencia de ON, no está confirmada con la dosis de L-NAME usada y la duración del tratamiento de esta investigación.

PALABRAS CLAVE: 1. Oxido Nítrico; 2. Aorta; 3. Enalapril; 4. Verapamil; 5. Estereología.


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_______________

1 Research supported by Faperj and CNPq agencies from Brazil.

2 Laboratory of Morphometry & Cardiovascular Morphology, Biomedical Center, Instituto of Biology, State University of Rio de Janeiro, Brazil.

Correspondence to:
Prof. Dr. Carlos Alberto Mandarim-de-Lacerda
Laboratório de Morfometria e Morfologia Cardiovascular,
Centro Biomédico, Instituto de Biologia,
Departamento de Anatomia,
Universidade do Estado do Rio de Janeiro (UERJ).
Av 28 de Setembro, 87 (fds). 20551-030
Rio de Janeiro, RJ, BRASIL

Tel/Fax: (+55.21) 2587-6416.
E-mail: mandarim@uerj.br. URL: www2.uerj.br/~lmmc.

Received: 06-12-2002
Accepted: 17-01-2003

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