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Effects of ACE inhibition during fetal development on cardiac microvasculature in adult spontaneously hypertensive rats
International Journal of Cardiology 101 (2005) 237 – 242 www.elsevier.com/locate/ijcard
Effects of ACE inhibition during fetal development on cardiac microvasculature in adult spontaneously hypertensive rats Alfredo de Souza Bomfim, Carlos Alberto Mandarim-de-Lacerda * Laboratory of Morphometry and Cardiovascular Morphology, Biomedical Center, State University of Rio de Janeiro, Rio de Janeiro, Brazil Received 9 September 2003; received in revised form 22 January 2004; accepted 3 March 2004 Available online 18 May 2004
Abstract Background: Early angiotensin-converting enzyme (ACE) inhibition is able to re-program spontaneously hypertensive rats (SHR) to express an attenuated form of disease in adulthood. Methods: Three groups of animals (n = 5 each) were studied: Wistar male rats, SHR males, and SHR males obtained from dams treated with enalapril maleate (15 mg/kg/day) during gestation. Animals were sacrificed 180 days after birth, and hearts were removed for stereological quantification. Volume [Vv] (myocytes, cardiac interstitium and intramyocardial vessels), length [Lv] (intramyocardial vessels), surface [Sv] densities (myocyte and intramyocardial vessels), and the mean cross-sectional area [a¯] (myocyte) were estimated. Results: Blood pressure (BP) was lower in Wistar group, higher in SHR group, and intermediate in SHR – enalapril group (respectively: 122 F 8.4, 194 F 11.4, and 158 F 7.6 mm Hg, p < 0.0001). Increased Vv ( p = 0.016), Lv ( p < 0.01), and Sv ( p < 0.01) of intramyocardial vessels were observed in SHR – enalapril group when compared to untreated SHR. A small but significant reduction was observed in a¯ of myocytes ( p = 0.045). Conclusion: Prenatal ACE inhibition resulted in partial hypertension attenuation as well as left ventricular hypertrophy (LVH). The positive impact on the vascular compartment came along with little or no difference in myocytes and interstitium, suggesting the involvement of a direct mechanism. D 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Myocardium; Angiotensin-converting enzyme; Enalapril maleate; Hypertension; Stereology
1. Introduction Over the past few years, a new concept for understanding the mechanisms of hypertension and left ventricular hypertrophy (LVH) has arisen from observations of permanent or long-term changes in morphology occurring in response to an insult or stimulus at a critical period of the development [1,2] and became known as the ‘‘programming’’ hypothesis. Animal models of programmed hypertension have been used to investigate the role of the renin – angiotensin – aldosterone system (RAAS) in the start and maintenance of high blood pressure (BP). The identification and quantification of angiotensin II (Ang II) receptors in developing rat heart [3,4] suggest an active role for the RAAS during early cardiac development. Renal renin activity in sponta-
* Corresponding author. Laborato´rio de Morfometria e Morfologia Cardiovascular, Centro, Biome´dico, Universidade do Estado do Rio de Janeiro (UERJ), Av 28 de Setembro 87 (fds)-20551-030 Rio de Janeiro, RJ, Brazil. Tel./Fax: +55-21-2587-6416. E-mail address: [email protected] (C.A. Mandarim-de-Lacerda). URL: http://www2.uerj.br/~lmmc. 0167-5273/$ - see front matter D 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2004.03.029
neously hypertensive rat (SHR) rapidly increases during the late stages of gestation and the early postnatal period, while renal AT1 receptor mRNA levels rise [5]. Therefore, early pharmacological intervention in RAAS results in short- and long-term changes in cardiac physiology and morphology. Ang II receptor blockade during gestation in rats results in newborns with decreased cardiac collagen content [6]. In SHR, early brief inhibition of RAAS with captopril results in lower adult BP [7]. In such a manner, it seems clear that fetal intervention in cardiovascular physiology can target on the opposite direction: prevention of cardiovascular disease in otherwise predisposed animals. The structural remodeling process following sustained hypertension and LVH is characterized by a disproportional compartments growth of the myocardium with fibrosis and adverse changes in microcirculation [8,9] and cardiac myocyte apoptosis [10]. In general, capillary growth in the socalled heterogeneous or pathological LVH does not pace with the rise in cardiac mass and accounts for cardiac functional impairment [11]. The clinical importance of LVH relates to the mortality associated with the remodeling process, and increased risk for coronary heart disease, stroke
238
A. de Souza Bomfim, C.A. Mandarim-de-Lacerda / International Journal of Cardiology 101 (2005) 237–242
and heart failure [12]. Previous studies concerning intramyocardial vessels in hypertension models have demonstrated a decrease in its densities as hypertrophic remodeling develops [13,14], contrarily to the increased capillary length seen in the physiological growth of the myocardium [15]. Therefore, preventing or attenuating microvascular remodeling in hypertension is a goal for keeping the myocardial functional integrity. The present study was designed to test the hypothesis that a treatment of pregnant SHR with an angiotensinconverting enzyme (ACE) inhibitor during gestational period can alter the course of hypertension and cause permanent positive myocardial changes in adult SHR. Morphological aspects were focused and a quantitative approach adopted.
2. Methods This investigation conforms to the ‘‘Guide for the Care and Use of Laboratory Animals’’, published by the US National Institutes of Health (NIH publication No. 85-23, revised 1985). Fifteen animals obtained from colonies maintained in the State University of Rio de Janeiro were divided into three groups (n = 5 each, not consanguineous): Wistar normotensive male rats, SHR males untreated, and SHR males obtained from females treated with enalapril maleate (15 mg/kg/day), dissolved into drinking water. Treatment of pregnant female SHR started in coupling period and continued during pregnancy until delivery. To assure that total drug amount was administered, females were observed during 1 week prior to the experiment and the daily water intake was determined. After delivery, all animals were housed in polypropylene cages and received only food and water ad libitum. They were kept in temperature (21 F 2 jC) and humidity (60 F 10%) controlled conditions in a room with a 12-h light and dark cycle, and air exhaustion system (15 min/h). The BP was weekly verified in conscious rats through the non-invasive method of the tail-cuff plethysmography (Letica LE 5100, PanlabR). In the 180th day of experimentation, animals were deeply anaesthetized (intraperitonial Thiopental), and then sacrificed (cardiac injection of 10% KCl). Hearts were removed and dissected to separate the atria from the ventricles. Body mass, heart mass, and left ventricular mass (including the interventricular septum) were measured using an accurate digital scale. The left ventricular (LV) mass index was calculated as the LV mass/body mass ratio. The left ventricle was cleaved in order to obtain random and uniform isotropic sections [16]. Fragments were kept during 48 h at room temperature in fixative (freshly prepared 4% w/v formaldehyde in 0.1 M phosphate buffer pH 7.2) [17], embedded in Paraplast plusR (Sigma, St. Louis, MO) and sectioned at 3-Am thickness. Sections were stained with picro sirius red for light microscopy. For the stereological quantification, the myocardium was considered to consist of cardiac myocytes and cardiac interstitium (composed by
connective tissue and intramyocardial vessels). Five random fields per animal were analyzed. The analysis used a videomicroscopic system composed of a Leica DMRBE microscope ( 40 objective NA 1.25), a Kappa video camera and a Sony Triniton video monitor. A multipurpose 42-point frame was used for point and intersection count [18,19]. The volume density (myocyte, m, and intramyocardial vessels, ve) was estimated as Vv[structure]wPP[structure]/PT, where PP denotes the number of points hitting the structure and PT represents the total number of test points (w meaning it is an estimate). The length density (intramyocardial vessels) was estimated as Lv[ve]w2QA[ve], where QA denotes the outlines number of the structure in the frame. The intramyocardial vessels-to-myocyte ratio was estimated as ve/ mwVv[ve]/Vv[m]. The mean cross-sectional area (myocyte) was estimated as a¯[m]wVv[m]/2QA[m] [20,21]. Differences between groups considering biometry were analyzed by one-way ANOVA and Newman –Keuls post hoc test. Differences between groups considering stereology were analyzed by non-parametric Mann – Whitney test. The level of significance was fixed at 0.05 [22].
3. Results Figs. 1 – 5 summarize the results. Usually, Wistar rats and untreated SHR were put in the extremes of the groups, while
Fig. 1. Blood pressure alteration in adult Wistar rats, spontaneously hypertensive rats (SHR) and SHR from mothers treated with enalapril during pregnancy. Newman – Keuls test: in signaled cases, when compared, p = 0.05, if: (a) when compared with the Wistar group; (b) with the SHR group.
A. de Souza Bomfim, C.A. Mandarim-de-Lacerda / International Journal of Cardiology 101 (2005) 237–242
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Fig. 2. Left ventricular mass index in adult Wistar rats, spontaneously hypertensive rats (SHR) and SHR from mothers treated with enalapril during pregnancy. Newman – Keuls test: in signaled cases, when compared, p = 0.05, if: (a) when compared with the Wistar group; (b) with the SHR group.
Fig. 4. Length density of intramyocardial vessel in adult Wistar rats, spontaneously hypertensive rats (SHR) and SHR from mothers treated with enalapril during pregnancy. Mann – Whitney test: in signaled cases, when compared, p = 0.05, if: (a) when compared with the Wistar group; (b) with the SHR group.
enalapril-treated SHR were intermediate between Wistar and untreated SHR. The BP levels became different among the groups around the 6th postnatal week, increasing differences in the following weeks (Fig. 1). At 24th week, BP level was 60% higher in SHR group than in Wistar group, and 30%
higher in SHR + enalapril group than in Wistar group. The LV mass index increased 130% comparing Wistar and untreated SHR groups, and increased 80% comparing Wistar and enalapril-treated SHR. Treated SHR showed a LV mass index around 25% smaller than untreated SHR (Fig. 2).
Fig. 3. Volume density of the intramyocardial vessels in adult Wistar rats, spontaneously hypertensive rats (SHR) and SHR from mothers treated with enalapril during pregnancy. Mann – Whitney test: in signaled cases, when compared, p = 0.05, if: (a) when compared with the Wistar group; (b) with the SHR group.
Fig. 5. The intramyocardial vessels-to-myocyte ratio in adult Wistar rats, spontaneously hypertensive rats (SHR) and SHR from mothers treated with enalapril during pregnancy. Mann – Whitney test: in signaled cases, when compared, p = 0.05, if: (a) when compared with the Wistar group; (b) with the SHR group.
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Fig. 6. Mean cross-sectional spontaneously hypertensive rats with enalapril during pregnancy. when compared, p = 0.05, if: (a) (b) with the SHR group.
myocyte area in adult Wistar rats, (SHR) and SHR from mothers treated Mann – Whitney test: in signaled cases, when compared with the Wistar group;
The myocardium showed differences in both the intramyocardial vessels (Vv[ve] and Lv[ve) content and the cross-sectional myocyte area (a¯[m]). Contrarily, no significant difference was seen in the cardiac interstitium. Vv[ve], Lv[ve] and the [ve]/[m] ratio showed the same intramyocardial vessels alteration, i.e. the reduction of 70% comparing Wistar with untreated SHR, while treated SHR showed these stereological parameters more than 100% greater than untreated SHR (Figs. 3 Figs. 4 Figs. 5). A 60% increase in untreated SHR a¯[m] and a 25% increase in treated SHR were observed in comparison with Wistar rats. Enalapril-treated SHR showed a¯[m] reduction of 20% in relation to untreated SHR (Fig. 6).
4. Discussion The consequence of the programming concept , according to which stimulus in early stages of fetal development result in long-term manifestations in the structure and in the function of an organ, is the search for an effective strategy of early intervention to prevent or attenuate possible diseases in potential populations. In the SHR model of hypertension, animals have a prehypertensive state for the first 6 – 8 weeks of life and develop hypertension in adulthood [23]. While this pattern of late development of disease resembles the one of undernutrition-programmed hypertension, it is reasonable to speculate whether fetal influence is present as an element in the pathogenesis of LVH in this model of experimental hypertension.
A possible target for an early intervention in SHR is the RAAS. In rats exposed to low-protein diet in utero, both renin and ACE activities are increased, and a brief treatment with ACE inhibitor resulted in lower BP comparable to controls [24], and permanent lowering of BP with brief ACE inhibition was seen in SHR [25,26]. It has been suggested that SHR had increased placental concentrations of glucocorticoids that can, in turn, potentiate the action of Ang II [27]. In the present study, significant differences in systolic BP and biometric body and heart parameters occurred among the three groups, with animals born from treated SHR in an intermediate position between usually hypertensive adult untreated SHR with myocardial hypertrophy and Wistar normotensive controls with normal hearts. The differences in BP became more pronounced from 18 to 24 weeks that correspond to a period of BP increase in untreated SHR from our colony. These results indicate that at least an attenuation of hypertension can be achieved by ACE inhibition early in cardiac fetal development in SHR. With stereological tools, three-dimensional quantitative parameters of morphological structures can be estimated from bi-dimensional counts leading to unbiased results [20]. In the present study, the partial but significant attenuation of hypertension in genetically predisposed animals came along with an improvement of stereological parameters of myocardial hypertrophy, especially concerning the vascular compartment. Significant increases were observed in volume, length, and surface intramyocardial vessels densities in SHR – enalapril-treated animals when compared with untreated ones. At first sight, such results can be interpreted as a paradox, once Ang II has angiogenic properties [28]. However, a positive impact in the vascular compartment after ACE inhibition has been reported by other investigators. In SHR, ACE inhibition resulted in increased capillary density, while total capillary length remained unchanged, suggesting a relative improvement due to decreased cardiac mass [29]. On the other hand, low-dose ACE inhibition at a dose that did not affect BP and cardiac hypertrophy caused increased capillary density as well, suggesting a direct effect of ACE inhibitors on coronary intramyocardial vessels [30]. The ACE inhibitor lisinopril has shown to improve the coronary reserve, increase in length density of intramyocardial vessels in experimental hypertension [31]. Ang II may be involved in microvascular growth via a non-AT1 receptor-mediated mechanism, or other vasoactive peptides degraded by ACE may contribute to the effects of the ACE inhibitor [32]. Further studies employing Ang II receptor blockers could provide information to contribute to elucidate all mechanisms involved. Possible mechanisms for ACE inhibitors-induced stimulation of vascular growth include increased levels of bradykinin [33], mediated by B(2)-receptor pathway [34] and decreased Ang II AT2 receptors-mediated inhibition of
A. de Souza Bomfim, C.A. Mandarim-de-Lacerda / International Journal of Cardiology 101 (2005) 237–242
endothelial cell proliferation [35]. In the present study, the positive impact of fetal ACE inhibition was greater in the vascular compartment than in the cardiac myocytes and interstitium. This points to a direct beneficial effect on microcirculation, but since BP was lower and the myocytes mean cross-sectional area was smaller in treated than in untreated SHR, a partial effect mediated by attenuation of hypertrophic process cannot be ruled out. A major concern on the clinical use of ACE inhibitors during human pregnancy refers to their teratogenic potential. Adverse effects on fetus from ACE inhibition are thought to be due to fetal hypotension [36], with skull hypoplasia and renal dysfunction as the most consistent abnormalities seen [37]. In the present study, however, treatment was well tolerated. A possible explanation for this finding is that nephrogenesis in rats occur late in fetal development until the first days after birth [38]. In the rat, the critical period for the induction of irreversible abnormalities by enalapril after birth comprises the first 13 days [39], a period when our animals received nothing but water and food ad libitum. So, even though some damage from intrauterine exposure cannot be ruled out, it was not apparent in the light of the parameters analyzed. Besides, some reports on the use of ACEI inhibitors in human pregnancy suggest that these agents can be safe during the first trimester and sometimes during the whole gestation [40]. Nevertheless, when weighing risk versus benefit, the current recommendation is to avoid ACEI in pregnant women, except in rare instances where there is no other therapeutic option is available [41], e.g. Bartter’s syndrome. On the other hand, as a strategy for experimental investigation on hypertension, ACE inhibition is useful for understanding the role of angiotensin II in cardiac development and physiology, providing the basis for an effective pharmacological approach for the prevention or attenuation of hypertension in susceptible individuals. In conclusion, our results indicate that an early pharmacological intervention in RAAS during the fetal development is able to change the natural history of animals otherwise programmed to develop hypertension and myocardial hypertrophy, and re-program them to express an attenuated form of the disease. Morphological quantification has demonstrated a positive impact on vascular compartments, with significant improvement in all stereological parameters for the analyzed intramyocardial vessels. A moderate, but significant reduction in BP came along with attenuation, although not a complete regression, of myocardial hypertrophy. A possible limitation of this study is that the exact amount of enalapril reaching the fetus during pregnancy could not be determined. The dose administered was chosen according to previous reports in literature, in which the pharmacological intervention showed significant results. A more precise determination of enalapril kinetics across the placenta barrier is needed to better understand such a complex mechanism.
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Acknowledgements Authors wish to thank Mrs. Marinho T., Soares A. and Ferreira G. for their technical assistance. This work was partially supported by Brazilian grants from CNPq and Faperj.
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Effects of ACE inhibition during fetal development on cardiac microvasculature in adult spontaneously hypertensive rats Alfredo de Souza Bomfim, Carlos Alberto Mandarim-de-Lacerda * Laboratory of Morphometry and Cardiovascular Morphology, Biomedical Center, State University of Rio de Janeiro, Rio de Janeiro, Brazil Received 9 September 2003; received in revised form 22 January 2004; accepted 3 March 2004 Available online 18 May 2004
Abstract Background: Early angiotensin-converting enzyme (ACE) inhibition is able to re-program spontaneously hypertensive rats (SHR) to express an attenuated form of disease in adulthood. Methods: Three groups of animals (n = 5 each) were studied: Wistar male rats, SHR males, and SHR males obtained from dams treated with enalapril maleate (15 mg/kg/day) during gestation. Animals were sacrificed 180 days after birth, and hearts were removed for stereological quantification. Volume [Vv] (myocytes, cardiac interstitium and intramyocardial vessels), length [Lv] (intramyocardial vessels), surface [Sv] densities (myocyte and intramyocardial vessels), and the mean cross-sectional area [a¯] (myocyte) were estimated. Results: Blood pressure (BP) was lower in Wistar group, higher in SHR group, and intermediate in SHR – enalapril group (respectively: 122 F 8.4, 194 F 11.4, and 158 F 7.6 mm Hg, p < 0.0001). Increased Vv ( p = 0.016), Lv ( p < 0.01), and Sv ( p < 0.01) of intramyocardial vessels were observed in SHR – enalapril group when compared to untreated SHR. A small but significant reduction was observed in a¯ of myocytes ( p = 0.045). Conclusion: Prenatal ACE inhibition resulted in partial hypertension attenuation as well as left ventricular hypertrophy (LVH). The positive impact on the vascular compartment came along with little or no difference in myocytes and interstitium, suggesting the involvement of a direct mechanism. D 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Myocardium; Angiotensin-converting enzyme; Enalapril maleate; Hypertension; Stereology
1. Introduction Over the past few years, a new concept for understanding the mechanisms of hypertension and left ventricular hypertrophy (LVH) has arisen from observations of permanent or long-term changes in morphology occurring in response to an insult or stimulus at a critical period of the development [1,2] and became known as the ‘‘programming’’ hypothesis. Animal models of programmed hypertension have been used to investigate the role of the renin – angiotensin – aldosterone system (RAAS) in the start and maintenance of high blood pressure (BP). The identification and quantification of angiotensin II (Ang II) receptors in developing rat heart [3,4] suggest an active role for the RAAS during early cardiac development. Renal renin activity in sponta-
* Corresponding author. Laborato´rio de Morfometria e Morfologia Cardiovascular, Centro, Biome´dico, Universidade do Estado do Rio de Janeiro (UERJ), Av 28 de Setembro 87 (fds)-20551-030 Rio de Janeiro, RJ, Brazil. Tel./Fax: +55-21-2587-6416. E-mail address: [email protected] (C.A. Mandarim-de-Lacerda). URL: http://www2.uerj.br/~lmmc. 0167-5273/$ - see front matter D 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2004.03.029
neously hypertensive rat (SHR) rapidly increases during the late stages of gestation and the early postnatal period, while renal AT1 receptor mRNA levels rise [5]. Therefore, early pharmacological intervention in RAAS results in short- and long-term changes in cardiac physiology and morphology. Ang II receptor blockade during gestation in rats results in newborns with decreased cardiac collagen content [6]. In SHR, early brief inhibition of RAAS with captopril results in lower adult BP [7]. In such a manner, it seems clear that fetal intervention in cardiovascular physiology can target on the opposite direction: prevention of cardiovascular disease in otherwise predisposed animals. The structural remodeling process following sustained hypertension and LVH is characterized by a disproportional compartments growth of the myocardium with fibrosis and adverse changes in microcirculation [8,9] and cardiac myocyte apoptosis [10]. In general, capillary growth in the socalled heterogeneous or pathological LVH does not pace with the rise in cardiac mass and accounts for cardiac functional impairment [11]. The clinical importance of LVH relates to the mortality associated with the remodeling process, and increased risk for coronary heart disease, stroke
238
A. de Souza Bomfim, C.A. Mandarim-de-Lacerda / International Journal of Cardiology 101 (2005) 237–242
and heart failure [12]. Previous studies concerning intramyocardial vessels in hypertension models have demonstrated a decrease in its densities as hypertrophic remodeling develops [13,14], contrarily to the increased capillary length seen in the physiological growth of the myocardium [15]. Therefore, preventing or attenuating microvascular remodeling in hypertension is a goal for keeping the myocardial functional integrity. The present study was designed to test the hypothesis that a treatment of pregnant SHR with an angiotensinconverting enzyme (ACE) inhibitor during gestational period can alter the course of hypertension and cause permanent positive myocardial changes in adult SHR. Morphological aspects were focused and a quantitative approach adopted.
2. Methods This investigation conforms to the ‘‘Guide for the Care and Use of Laboratory Animals’’, published by the US National Institutes of Health (NIH publication No. 85-23, revised 1985). Fifteen animals obtained from colonies maintained in the State University of Rio de Janeiro were divided into three groups (n = 5 each, not consanguineous): Wistar normotensive male rats, SHR males untreated, and SHR males obtained from females treated with enalapril maleate (15 mg/kg/day), dissolved into drinking water. Treatment of pregnant female SHR started in coupling period and continued during pregnancy until delivery. To assure that total drug amount was administered, females were observed during 1 week prior to the experiment and the daily water intake was determined. After delivery, all animals were housed in polypropylene cages and received only food and water ad libitum. They were kept in temperature (21 F 2 jC) and humidity (60 F 10%) controlled conditions in a room with a 12-h light and dark cycle, and air exhaustion system (15 min/h). The BP was weekly verified in conscious rats through the non-invasive method of the tail-cuff plethysmography (Letica LE 5100, PanlabR). In the 180th day of experimentation, animals were deeply anaesthetized (intraperitonial Thiopental), and then sacrificed (cardiac injection of 10% KCl). Hearts were removed and dissected to separate the atria from the ventricles. Body mass, heart mass, and left ventricular mass (including the interventricular septum) were measured using an accurate digital scale. The left ventricular (LV) mass index was calculated as the LV mass/body mass ratio. The left ventricle was cleaved in order to obtain random and uniform isotropic sections [16]. Fragments were kept during 48 h at room temperature in fixative (freshly prepared 4% w/v formaldehyde in 0.1 M phosphate buffer pH 7.2) [17], embedded in Paraplast plusR (Sigma, St. Louis, MO) and sectioned at 3-Am thickness. Sections were stained with picro sirius red for light microscopy. For the stereological quantification, the myocardium was considered to consist of cardiac myocytes and cardiac interstitium (composed by
connective tissue and intramyocardial vessels). Five random fields per animal were analyzed. The analysis used a videomicroscopic system composed of a Leica DMRBE microscope ( 40 objective NA 1.25), a Kappa video camera and a Sony Triniton video monitor. A multipurpose 42-point frame was used for point and intersection count [18,19]. The volume density (myocyte, m, and intramyocardial vessels, ve) was estimated as Vv[structure]wPP[structure]/PT, where PP denotes the number of points hitting the structure and PT represents the total number of test points (w meaning it is an estimate). The length density (intramyocardial vessels) was estimated as Lv[ve]w2QA[ve], where QA denotes the outlines number of the structure in the frame. The intramyocardial vessels-to-myocyte ratio was estimated as ve/ mwVv[ve]/Vv[m]. The mean cross-sectional area (myocyte) was estimated as a¯[m]wVv[m]/2QA[m] [20,21]. Differences between groups considering biometry were analyzed by one-way ANOVA and Newman –Keuls post hoc test. Differences between groups considering stereology were analyzed by non-parametric Mann – Whitney test. The level of significance was fixed at 0.05 [22].
3. Results Figs. 1 – 5 summarize the results. Usually, Wistar rats and untreated SHR were put in the extremes of the groups, while
Fig. 1. Blood pressure alteration in adult Wistar rats, spontaneously hypertensive rats (SHR) and SHR from mothers treated with enalapril during pregnancy. Newman – Keuls test: in signaled cases, when compared, p = 0.05, if: (a) when compared with the Wistar group; (b) with the SHR group.
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Fig. 2. Left ventricular mass index in adult Wistar rats, spontaneously hypertensive rats (SHR) and SHR from mothers treated with enalapril during pregnancy. Newman – Keuls test: in signaled cases, when compared, p = 0.05, if: (a) when compared with the Wistar group; (b) with the SHR group.
Fig. 4. Length density of intramyocardial vessel in adult Wistar rats, spontaneously hypertensive rats (SHR) and SHR from mothers treated with enalapril during pregnancy. Mann – Whitney test: in signaled cases, when compared, p = 0.05, if: (a) when compared with the Wistar group; (b) with the SHR group.
enalapril-treated SHR were intermediate between Wistar and untreated SHR. The BP levels became different among the groups around the 6th postnatal week, increasing differences in the following weeks (Fig. 1). At 24th week, BP level was 60% higher in SHR group than in Wistar group, and 30%
higher in SHR + enalapril group than in Wistar group. The LV mass index increased 130% comparing Wistar and untreated SHR groups, and increased 80% comparing Wistar and enalapril-treated SHR. Treated SHR showed a LV mass index around 25% smaller than untreated SHR (Fig. 2).
Fig. 3. Volume density of the intramyocardial vessels in adult Wistar rats, spontaneously hypertensive rats (SHR) and SHR from mothers treated with enalapril during pregnancy. Mann – Whitney test: in signaled cases, when compared, p = 0.05, if: (a) when compared with the Wistar group; (b) with the SHR group.
Fig. 5. The intramyocardial vessels-to-myocyte ratio in adult Wistar rats, spontaneously hypertensive rats (SHR) and SHR from mothers treated with enalapril during pregnancy. Mann – Whitney test: in signaled cases, when compared, p = 0.05, if: (a) when compared with the Wistar group; (b) with the SHR group.
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Fig. 6. Mean cross-sectional spontaneously hypertensive rats with enalapril during pregnancy. when compared, p = 0.05, if: (a) (b) with the SHR group.
myocyte area in adult Wistar rats, (SHR) and SHR from mothers treated Mann – Whitney test: in signaled cases, when compared with the Wistar group;
The myocardium showed differences in both the intramyocardial vessels (Vv[ve] and Lv[ve) content and the cross-sectional myocyte area (a¯[m]). Contrarily, no significant difference was seen in the cardiac interstitium. Vv[ve], Lv[ve] and the [ve]/[m] ratio showed the same intramyocardial vessels alteration, i.e. the reduction of 70% comparing Wistar with untreated SHR, while treated SHR showed these stereological parameters more than 100% greater than untreated SHR (Figs. 3 Figs. 4 Figs. 5). A 60% increase in untreated SHR a¯[m] and a 25% increase in treated SHR were observed in comparison with Wistar rats. Enalapril-treated SHR showed a¯[m] reduction of 20% in relation to untreated SHR (Fig. 6).
4. Discussion The consequence of the programming concept , according to which stimulus in early stages of fetal development result in long-term manifestations in the structure and in the function of an organ, is the search for an effective strategy of early intervention to prevent or attenuate possible diseases in potential populations. In the SHR model of hypertension, animals have a prehypertensive state for the first 6 – 8 weeks of life and develop hypertension in adulthood [23]. While this pattern of late development of disease resembles the one of undernutrition-programmed hypertension, it is reasonable to speculate whether fetal influence is present as an element in the pathogenesis of LVH in this model of experimental hypertension.
A possible target for an early intervention in SHR is the RAAS. In rats exposed to low-protein diet in utero, both renin and ACE activities are increased, and a brief treatment with ACE inhibitor resulted in lower BP comparable to controls [24], and permanent lowering of BP with brief ACE inhibition was seen in SHR [25,26]. It has been suggested that SHR had increased placental concentrations of glucocorticoids that can, in turn, potentiate the action of Ang II [27]. In the present study, significant differences in systolic BP and biometric body and heart parameters occurred among the three groups, with animals born from treated SHR in an intermediate position between usually hypertensive adult untreated SHR with myocardial hypertrophy and Wistar normotensive controls with normal hearts. The differences in BP became more pronounced from 18 to 24 weeks that correspond to a period of BP increase in untreated SHR from our colony. These results indicate that at least an attenuation of hypertension can be achieved by ACE inhibition early in cardiac fetal development in SHR. With stereological tools, three-dimensional quantitative parameters of morphological structures can be estimated from bi-dimensional counts leading to unbiased results [20]. In the present study, the partial but significant attenuation of hypertension in genetically predisposed animals came along with an improvement of stereological parameters of myocardial hypertrophy, especially concerning the vascular compartment. Significant increases were observed in volume, length, and surface intramyocardial vessels densities in SHR – enalapril-treated animals when compared with untreated ones. At first sight, such results can be interpreted as a paradox, once Ang II has angiogenic properties [28]. However, a positive impact in the vascular compartment after ACE inhibition has been reported by other investigators. In SHR, ACE inhibition resulted in increased capillary density, while total capillary length remained unchanged, suggesting a relative improvement due to decreased cardiac mass [29]. On the other hand, low-dose ACE inhibition at a dose that did not affect BP and cardiac hypertrophy caused increased capillary density as well, suggesting a direct effect of ACE inhibitors on coronary intramyocardial vessels [30]. The ACE inhibitor lisinopril has shown to improve the coronary reserve, increase in length density of intramyocardial vessels in experimental hypertension [31]. Ang II may be involved in microvascular growth via a non-AT1 receptor-mediated mechanism, or other vasoactive peptides degraded by ACE may contribute to the effects of the ACE inhibitor [32]. Further studies employing Ang II receptor blockers could provide information to contribute to elucidate all mechanisms involved. Possible mechanisms for ACE inhibitors-induced stimulation of vascular growth include increased levels of bradykinin [33], mediated by B(2)-receptor pathway [34] and decreased Ang II AT2 receptors-mediated inhibition of
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endothelial cell proliferation [35]. In the present study, the positive impact of fetal ACE inhibition was greater in the vascular compartment than in the cardiac myocytes and interstitium. This points to a direct beneficial effect on microcirculation, but since BP was lower and the myocytes mean cross-sectional area was smaller in treated than in untreated SHR, a partial effect mediated by attenuation of hypertrophic process cannot be ruled out. A major concern on the clinical use of ACE inhibitors during human pregnancy refers to their teratogenic potential. Adverse effects on fetus from ACE inhibition are thought to be due to fetal hypotension [36], with skull hypoplasia and renal dysfunction as the most consistent abnormalities seen [37]. In the present study, however, treatment was well tolerated. A possible explanation for this finding is that nephrogenesis in rats occur late in fetal development until the first days after birth [38]. In the rat, the critical period for the induction of irreversible abnormalities by enalapril after birth comprises the first 13 days [39], a period when our animals received nothing but water and food ad libitum. So, even though some damage from intrauterine exposure cannot be ruled out, it was not apparent in the light of the parameters analyzed. Besides, some reports on the use of ACEI inhibitors in human pregnancy suggest that these agents can be safe during the first trimester and sometimes during the whole gestation [40]. Nevertheless, when weighing risk versus benefit, the current recommendation is to avoid ACEI in pregnant women, except in rare instances where there is no other therapeutic option is available [41], e.g. Bartter’s syndrome. On the other hand, as a strategy for experimental investigation on hypertension, ACE inhibition is useful for understanding the role of angiotensin II in cardiac development and physiology, providing the basis for an effective pharmacological approach for the prevention or attenuation of hypertension in susceptible individuals. In conclusion, our results indicate that an early pharmacological intervention in RAAS during the fetal development is able to change the natural history of animals otherwise programmed to develop hypertension and myocardial hypertrophy, and re-program them to express an attenuated form of the disease. Morphological quantification has demonstrated a positive impact on vascular compartments, with significant improvement in all stereological parameters for the analyzed intramyocardial vessels. A moderate, but significant reduction in BP came along with attenuation, although not a complete regression, of myocardial hypertrophy. A possible limitation of this study is that the exact amount of enalapril reaching the fetus during pregnancy could not be determined. The dose administered was chosen according to previous reports in literature, in which the pharmacological intervention showed significant results. A more precise determination of enalapril kinetics across the placenta barrier is needed to better understand such a complex mechanism.
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Acknowledgements Authors wish to thank Mrs. Marinho T., Soares A. and Ferreira G. for their technical assistance. This work was partially supported by Brazilian grants from CNPq and Faperj.
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