| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Gynecology and Obstetrics (M.K., H.I., S.Y., H.Mo., S.F.), Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan; Departments of Etiology and Pathology (S.-I.S.) and Atherosclerosis and Diabetes (H.Ma., Y.M., Y.Y.), National Cardiovascular Center, Suita, Osaka 565-8565, Japan; Department of Obstetrics and Gynecology (N.S.), Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan; and Precursory Research for Embryonic Science and Technology (PRESTO) (S.Y.), Japan Science and Technology Agency (JST), Kawaguchi City, Saitama 332-0012, Japan
Address all correspondence and requests for reprints to: Hiroaki Itoh, Department of Gynecology and Obstetrics, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: ihiroaki{at}kuhp.kyoto-u.ac.jp.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The renin-angiotensin system (RAS) plays an important role in primary as well as secondary forms of hypertension in both animals and humans (3). More recently, components of the RAS, such as angiotensin-converting enzyme (ACE) and angiotensin II, were revealed to be produced locally in the cardiac tissues, and termed the local cardiac RAS (4), being primary candidates for the factors promoting cardiac remodeling, mainly cardiac myocyte hypertrophy and increased extracellular matrix fibrosis, thereby deteriorating cardiac function (5). Various experimental animal models have been developed to investigate the associations between fetal undernutrition and cardiovascular disease later in life (6, 7), and a possible commitment of a systemic RAS in the developmental origins of hypertension was reported (8). Therefore, the aim of the present study was to investigate whether the local cardiac RAS is associated with the developmental origins of cardiac remodeling in offspring exposed to undernutrition in utero.
Recently, we developed a mouse model of undernutrition in utero using maternal food restriction, in which the offspring (UN offspring) developed pronounced obesity when fed a high-fat diet, accompanied by impaired hypothalamic leptin sensitivity, as compared with normally nourished offspring (NN offspring) (9). Using this model, we investigated whether fetal undernutrition affects systolic blood pressure (SBP), cardiac remodeling, and expression of local cardiac RAS-associated bioactive substances. We found that undernutrition in utero caused a significant increase in SBP as well as cardiac remodeling, concomitant with a significant elevation in mRNA expression in angiotensinogen (Ang) and endothelin-1 (ET-1) in the left ventricle.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Measurement of SBP
At 4, 8, and 16 wk of age, SBP was measured at least five times in conscious mice (n = 8–10 for each group) using an indirect tail-cuff method (MK-2000; Muromachi Kikai Co. Ltd., Tokyo, Japan).
Neonatal leptin or monosodium glutamate treatment
Leptin (2.5 µg/g body weight·d) (PeproTech Inc., Rocky Hill, NJ) or vehicle saline was sc administered to NN offspring daily from 5.5 to 10.5 d of age, as a model of premature leptin surge (9), then SBP was measured at 8 wk. Monosodium glutamate (2 mg/g body weight·d) was sc administered to NN and UN offspring from 1.5 to 5.5 d of age, as previously described (9), for the purpose of permanent chemical injury of the arcuate nucleus of the hypothalamus (ARC) (10), then SBP was measured at 16 wk.
Morphological analysis of the kidney
For morphological analysis, whole kidneys were sampled at 8 and 16 wk, weighed and fixed in 10% formalin, and embedded in paraffin. The kidneys were cut into sections 2-µm thick and stained with hematoxylin and eosin, periodic-acid Schiff (PAS), or Masson trichrome. The stained sections were analyzed light microscopically.
Serum nitrite/nitrate (NOx) and plasma angiotensin II concentration
NOx concentration was determined by the Griess reaction using a commercial colorimetric assay kit (Cayman Chemical, Ann Arbor, MI).
The angiotensin II concentration was determined with an ELISA kit (Peninsula Laboratories, Belmont, CA), after extraction through C18 Sep-Pak columns (Waters Co., Milford, MA).
Urine microalbumin concentration
Urine was collected for 24 h using metabolic cages, and microalbuminuria was determined by the competitive ELISA method (Albuwell M assay kit; Exocell, Philadelphia, PA) at 16 wk of age. Urine creatinine values were assessed simultaneously by enzyme assay (MIZUHO MEDY Co., Ltd., Saga, Japan) and were used to calculate the albumin to creatinine ratio.
Morphometric analysis of the heart
The whole hearts were sampled, fixed in 10% formalin, and embedded in paraffin at 8 and 16 wk. The heart was cut into two subserial cross-sections 6-µm thick at intervals of 1 mm and stained with Sirius Red to evaluate the perivascular fibrosis of coronary arteries 100–200 µm in diameter. The perivascular fibrosis was assessed by analyses of digital images, calculating the ratio of the area of Sirius Red-stained fibrosis to the total vessel area using a KS400 image system (Zeiss, Oberkochen, Germany). To evaluate perivascular fibrosis in renal small arteries 100–200 µm in diameter, the kidneys were also sampled in the offspring at 16 wk and evaluated in the same manner as the coronary arteries.
To determine the interstitial fibrosis of the heart at 16 wk of age, we randomly selected 20 fields in two different sections and calculated the ratio of the areas of Sirius Red-stained interstitial fibrosis to the total cross-sectional areas.
Cardiomegaly was assessed by whole-heart weight to body weight ratio at 8 and 16 wk. Cardiomyocyte enlargement was estimated by measuring shortest transverse diameter in nucleated transverse sections of the myocytes. In each sample at 16 wk, 8 fields were randomly selected, and 80 cells were measured.
Quantitative RT-PCR analysis
Total RNA was extracted from whole hearts of fetal mice at 18.5 dpc and from left ventricles of the mice at 3, 8, and 16 wk, as well as from kidneys at 16 wk. The mRNA expression was measured by real-time quantitative RT-PCR using Taqman technology (Model 7000 sequence detector; Applied Biosystems, Foster City, CA). The forward and reverse primers and Fam/Tamra or Fam/MGB probes used for the targeted amplification of part of the cDNAs of murine Ang, angiotensin II type 1 receptor (AT1R), angiotensin II type 2 receptor (AT2R), ACE, renin, ET-1, atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) are summarized in Table 1
. The forward and reverse primers and Joe/Tamra probes for the murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal RNA coding region were purchased from Applied Biosystems. Serial dilutions of total RNA sample, isolated from mouse left ventricles or kidneys, were used to construct the standard curve for each substance. The standard curves were calculated by linear regression analysis, and threshold cycle values were used to read off relative RNA amounts. An mRNA expression value was then obtained by dividing the value for the gene of interest by the value for the ribosomal RNA or GAPDH.
|
Immunohistochemistry of angiotensin II, ET-1, and renin
Six-micrometer-thick sections of the paraffin-embedded whole heart were incubated for overnight at 4 C with rabbit antiserum against angiotensin-II (1:500) (T-4007; Peninsula Laboratories), ET-1 (1:500) (T-4050; Peninsula Laboratories), or goat antiserum against renin (1:1600) (kindly donated by Professor Tadashi Inagami, Vanderbilt University School of Medicine, Nashville, TN) (11). Normal goat or rabbit serum (Dako Co., Carpinteria, CA) was used as negative controls. Staining was detected using an avidin-biotin-peroxidase method kit (ELITE ABC; Vector Laboratories, Burlingame, CA) with 3,3'-diaminobenzidine as previously described (12).
Statistical analysis
Values were expressed as means ± SEM. The significance of differences was assessed with Student’s t test. P values < 0.05 were regarded as significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
).
|
Serum NOx concentration and plasma angiotensin II concentration
The serum NOx concentration of UN offspring was significantly lower than that of NN offspring at 8 wk (P < 0.05) (Table 2). Such a tendency was also observed at 16 wk, but the difference was not statistically significant (Table 2).
|
). At 16 wk, the plasma angiotensin II concentration of UN offspring was higher than that of NN offspring, but the difference was not significant (Table 2).
Urine microalbuminuria
There was no significant difference in urine microalbumin concentration between UN and NN offspring at 16 wk (25.03 ± 2.06 µg/mg creatinine, n = 7 vs. 22.52 ± 1.65 µg/mg creatinine, n = 8).
Morphological analysis of the kidney
At 16 wk of age, the ratio of renal weight to body weight (mg/g) in UN offspring (5.58 ± 0.32, n = 20) was similar to that of in NN offspring (5.69 ± 0.27, n = 20).
Microscopic observation of hematoxylin and eosin (data not shown), PAS (Fig. 1Ba), and Masson trichrome (Fig. 1Bb) staining of kidneys from UN offspring at 8 and 16 wk showed no histological abnormalities as compared with NN offspring including nephron numbers.
Perivascular fibrosis of the coronary artery and renal small artery
At 8 wk of age, the ratio of coronary perivascular fibrosis to total vessel area in UN offspring had tended to increase as compared with that in NN offspring; however, the difference was not significant (Fig. 2B). At 16 wk of age, the ratio of coronary perivascular fibrosis to total vessel area was significantly higher in the UN offspring than NN offspring (P < 0.05) (Fig. 2, A and B). By contrast, the ratio of perivascular fibrosis to total vessel area in renal small arteries of UN offspring was similar to that in NN offspring at 16 wk of age (Fig. 2C).
|
).
|
), in parallel with the increased perivascular fibrosis of coronary artery (Fig. 2B). However, cardiomegaly was not detected in UN offspring at 8 wk (Table 3B).
The mRNA expression of local cardiac RAS-associated bioactive substances in the left ventricles at 3, 8, and 16 wk
There were no significant changes in Ang, ACE, AT1R, AT2R, ET-1, ANP, or BNP mRNA expression between NN and UN offspring at 3 wk (Figs. 3 and 4).
|
|
). By contrast, a significant increase was detected in AT2R (P < 0.01), ET-1 (P < 0.01), and BNP (P < 0.01) (Figs. 3 and 4) at 8 wk; whereas ANP mRNA expression had a tendency to increase, but not significantly (Fig. 4).
At 16 wk, a significant increase was observed in the mRNA expression of Ang (P < 0.05), AT2R (P < 0.05), and ET-1 (P < 0.01), but not in that of other substances (Figs. 3 and 4).
The renin mRNA expression in the left ventricles at 3, 8, and 16 wk was less than detection sensitivity of quantitative RT-PCR analysis (<0.00024-fold, compared with the whole kidney as a positive control).
Immunohistochemistry of angiotensin II, ET-1, and renin in the left ventricle
Immunostaining of both angiotensin II and ET-1 were mainly observed in cardiomyocytes of the left ventricle at 16 wk (Fig. 5, A and B). There occurred a tendency to increase in immunostaining for angiotensin II as well as ET-1 in UN offspring, as compared with NN offspring (Fig. 5, A and B).
|
). There was no apparent difference in the renin staining between NN and UN offspring at 16 wk (Fig. 5C).
The mRNA expression of local cardiac RAS-associated bioactive substances in the whole fetal heart at 18.5 dpc
A significant increase was observed in the mRNA expression of Ang (P < 0.05), ACE (P < 0.01), and ET-1 (P < 0.05) in the whole fetal heart at 18.5 dpc, but not in that of other substances (Table 4).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) nor the microalbumin concentration in UN offspring produced significant changes, although basal plasma angiotensin II concentration at 16 wk was higher than other reports (13). A significant decrease in the plasma NOx concentration of UN offspring was observed at 8 wk, as compared with that of NN offspring (Table 2). The decrease in the plasma NOx concentration of UN offspring was also observed at 16 wk, although it was not significant (Table 2). These observations suggested a possible involvement of endothelial dysfunction in the elevation of blood pressure in UN offspring, which is relevant to previous reports (14, 15). Histological examinations detected no abnormal findings in the renal tissues of UN offspring at 8 and 16 wk, although some investigators have demonstrated a possible involvement of small nephron numbers and/or a small number and size of glomeruli in increases in blood pressure during adulthood (16, 17). Using the same animal model, we recently reported pronounced obesity in UN offspring on a high-fat diet compared with NN offspring (9). We found premature onset of the neonatal leptin surge, i.e. a transient increase in serum leptin levels during the neonatal period, in UN offspring. We also demonstrated that the premature leptin surge programs hypothalamic low sensitivity to circulating leptin, a potent anti-obesity hormone, causatively contributing to pronounced obesity on a high-fat diet in adulthood, by showing that an artificial premature leptin surge model produced hypothalamic low sensitivity to circulating leptin and pronounced obesity on a high-fat diet (9). However, in the present study, an artificial premature leptin surge did not increase SBP in NN offspring. Moreover, artificial premature leptin surge did not augment cardiac remodeling (Kawamura, M., and H. Itoh, unpublished observations). We also revealed that chemical injury of the ARC by neonatal monosodium glutamate treatment during the neonatal period cancelled the acceleration of obesity on the high-fat diet in UN offspring (9). However, a significant increase in SBP was not blocked by monosodium glutamate treatment in the present study. The mechanisms leading to increased blood pressure in adult UN offspring with undernutrition in utero are currently not entirely clear.
There were no significant changes in the mRNA expression of cardiac RAS-associated bioactive substances at 3 wk (Figs. 3 and 4). On the other hand, at 8 wk, the mRNA expression of ET-1, a factor promoting cardiac remodeling (18, 19), was significantly elevated in the left ventricles of UN offspring (Fig. 4A). However, several anticardiac remodeling phenomena were observed at the same time in the left ventricles as follows. The Ang mRNA expression was significantly decreased (Fig. 3A), concomitantly with the significant increase of AT2R (Fig. 3D), which suppresses cardiac remodeling (20). ANP and BNP are secreted from the heart and antagonize RAS through a decrease in blood pressure, diuresis, anticardiac hypertrophy, and anticardiac fibrosis, etc. (21, 22). The significant elevation of BNP mRNA expression in the left ventricles of UN offspring at 8 wk, in parallel with a tendency for an increase in ANP mRNA expression, suggested protective effects on cardiac tissues against the acceleration of cardiac remodeling. Therefore, changes that both promote and suppress cardiac remodeling are simultaneously observed in the left ventricles of UN offspring at 8 wk. These findings lead us to speculate that a kind of compensatory mechanism might be operating, thereby protecting the heart from ominous cardiac transformation at 8 wk, which was relevant to the finding that neither cardiac hypertrophy (Table 3B) nor augmentation of perivascular fibrosis (Fig. 2) was observed with a significant increase in SBP (Fig. 1).
At 16 wk, a significant augmentation of cardiac remodeling, i.e. cardiac hypertrophy (Table 3B) and perivascular fibrosis (Fig. 2), was observed in UN offspring. It is a further aim of the study to assess the movement and/or thickness of the ventricular wall by ultrasound examination.
In the present study, we first demonstrated that undernutrition in utero significantly increased the mRNA expression of both Ang (Fig. 3A) and ET-1 (Fig. 4A) in the left ventricles of UN offspring at 16 wk, concomitantly with the augmentation of cardiac hypertrophy and perivascular fibrosis. Angiotensin II is derived from Ang and plays a central role in the local cardiac RAS in the augmentation of cardiac remodeling (4, 5). ET-1 has been found to induce hypertrophy of cardiomyocytes (18), as well as cardiac fibrosis (19). ET-1 has a close association with the local cardiac RAS in the process of cardiac remodeling (23, 24). In the present study, the significant elevation of both Ang and ET-1 mRNA levels in the left ventricle of UN offspring was observed at 16 wk. The immunostaining of both angiotensin II and ET-1 showed a tendency to increase in UN offspring compared with NN offspring at 16 wk. These findings suggested a possible decompensation of cardiac homeostasis in response to various portentous factors, as a result of fetal undernutrition, including an increase in blood pressure. A significant elevation in the AT2R mRNA expression, which suppresses cardiac remodeling by antagonizing the effects of signaling through the AT1R (20), was observed in UN offspring at 8 and 16 wk, but the increase relative to NN offspring was much lower at 16 wk than at 8 wk (Fig. 3D). Long-term observations are necessary to prove that 16 wk is the beginning of decompensation of cardiac homeostasis in this animal model. Nevertheless, these findings suggested a possible involvement of local cardiac RAS activation in the developmental origins of cardiac remodeling.
Rather stable expression was observed in ACE and AT1R after birth in UN offspring. Ang mRNA expression decreased at 8 wk and increased at 16 wk. More detailed molecular investigation is necessary to clarify the regulatory mechanism of each substance.
A few renin positive cells were detected in the left ventricle at 16 wk (Fig. 5C), although mRNA expression was below detection sensitivity of quantitative RT-PCR. This discrepancy was relevant to the recent observation that cardiac renin was predominantly derived from circulation (25). There was no apparent difference in cardiac renin immunostaining between NN and UN offspring at 16 wk. It is an interesting study to investigate whether cardiac renin uptake is involved in developmental origins of cardiac remodeling.
A significant augmentation of mRNA expression of Ang, ACE, and ET-1 was observed in the whole fetal heart at 18.5 dpc (Table 4). A possible association of these changes with local cardiac RAS activation in adulthood is a future aim of the study.
In summary, using a mouse model of fetal undernutrition, we here demonstrated the possible involvement of the local cardiac RAS in the developmental origins of cardiac disorders, represented by cardiac remodeling, by a longitudinal assessment of the expression of local cardiac RAS-associated bioactive substances from the fetal to adult periods. This study also highlighted the local cardiac RAS as a promising target for prophylactic intervention in the developmental origins of cardiovascular disease.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Disclosure Statement: The authors have nothing to disclose.
First Published Online November 30, 2006
Abbreviations: ACE, Angiotensin-converting enzyme; Ang, angiotensinogen; ANP, atrial natriuretic peptide; ARC, arcuate nucleus of the hypothalamus; AT1R, angiotensin II type 1 receptor; AT2R, angiotensin II type 2 receptor; BNP, brain natriuretic peptide; dpc, d postcoitum; ET-1, endothelin-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NN offspring, normally nourished offspring; NOx, nitrite/nitrate; PAS, periodic-acid Schiff; RAS, renin-angiotensin system; SBP, systolic blood pressure; UN offspring, offspring of undernutrition in utero.
Received May 25, 2006.
Accepted for publication November 15, 2006.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
