Antenatal maternal low protein diet: ACE-2 in the mouse lung and sexually dimorphic programming of hypertension
© Goyal et al.; licensee BioMed Central. 2015
Received: 20 June 2013
Accepted: 4 May 2015
Published: 14 May 2015
Elevated blood pressure is an important global health problem, and in-utero under-nutrition may be an important factor in the pathogenesis of hypertension. In the present study, we tested the hypothesis that antenatal maternal low protein diet (MLPD) leads to sexually dimorphic developmental programming of the components of the pulmonary renin-angiotensin system. This may be important in the antenatal MLPD-associated development of hypertension. In pregnant mice, we administered normal (control) and isocaloric 50 % protein restricted diet, commencing one week before mating and continuing until delivery of the pups. From the 18th to 24th week postnatal, we measured blood pressure in the offspring by use of a non-invasive tail-cuff method. In the same mice, we examined the mRNA and protein expression of the key components of the pulmonary renin-angiotensin system. Also, we examined microRNA complementary to angiotensin converting enzymes (ACE) 2 in the offspring lungs. Our results demonstrate that as a consequence of antenatal MLPD: 1) pup birthweight was significantly reduced in both sexes. 2) female offspring developed hypertension, but males did not. 3) In female offspring, ACE-2 protein expression was significantly reduced without any change in the mRNA levels. 4) miRNA 429, which has a binding site on ACE-2 - 3’ UTR was significantly upregulated in the female antenatal MLPD offspring. 5) In males, ACE-2 mRNA and protein expression were unaltered. We conclude that in the mouse, antenatal MLPD-induced reduction of ACE-2 in the female offspring lung may be an important mechanisms in sexually dimorphic programming of hypertension.
KeywordsAngiotensin converting enzyme microRNA Epigenetics DOHaD Fetal programming Developmental origins Barker hypothesis
More than 150 years after its first description, high blood pressure has been listed as the primary cause of death of 61,005 Americans in 2008 (www.heart.org/statistics). In 90 to 95% of these cases, the cause of hypertension is not known (AHA, 2008). Importantly, a family history of hypertension is an important risk factor for its development ; which suggests a strong genetic or familial environmental factor in its genesis. Moreover, during recent years, accumulating evidence indicates that to a significant degree, hypertension may have a developmental origin. Epidemiological data from human studies , and experiments in several species of laboratory animals including mice [3–6], rats , guinea pigs , and sheep , have demonstrated the importance of maternal nutrition during gestation in the genesis of hypertension in the adult offspring.
In humans, evidence supports sexually dimorphic trends in the occurrence of hypertension , with males being more affected than females before the latter’s menopause; and males and females being affected equally following female menopause . Notably, with increasing age the blood pressure in females increases more rapidly than that of males . The mechanisms of these changes are not known, however.
Following the discovery of renin as a pressor molecule , the renin-angiotensin system (RAS) has been established as one of the major pathways involved in both the development of hypertension and in fluid homeostasis. RAS has received increased attention, and our understanding of this system has changed radically over the past several decades. The cascade starts with an α2-globulin angiotensinogen (AGT), which is produced constitutively and released into the circulation, chiefly by the liver. A substrate for renin (secreted by kidneys), AGT is converted into the decapeptide angiotensin I (Ang I), and subsequently by angiotensin converting enzyme 1 (ACE 1) to the octapeptide Ang II. By the action of ACE-2, Ang II activity is terminated by its conversion to Ang 1–7. Of note, both ACE-1 and ACE-2 are secreted by the lung . Importantly, a number of drugs (enalapril, lisinopril, etc.) used clinically [15–17] inhibit ACE1 and underscore the role of lungs in hypertension. Furthermore, studies have demonstrated that modulation of ACE from the lungs have important implications in the development of hypertension [18, 19]. Studies from our laboratory [3–5] and others  have demonstrated significant changes in the RAS in response to antenatal MLPD. However, the extent to which antenatal MLPD can lead to sexual dimorphic programing of the RAS in association with hypertension is not known. Also, the specific components of the RAS pathway that may be important in this programing are not well understood. Thus, we tested the hypothesis that antenatal MLPD leads to sexually dimorphic developmental programming of the components of the pulmonary renin-angiotensin system, which may be important in the antenatal MLPD-associated development of hypertension.
Experimental animal and tissues
The present study was in compliance with the Animal Welfare Act, guidelines of the American Physiological Society, and was approved by the Institutional Animal Care and Use Committee (IACUC) of Loma Linda University. We have described all these methods in our previous publications [3–6]. Briefly, we obtained FVB/NJ mice (~8 weeks of age) from Jackson Laboratories (Bar Harbor, ME), and housed them in the Animal Research Facility, Loma Linda University under conditions of 14 h light, 10 h darkness, ambient temperature of 20 °C, and relative humidity of 30 to 60 %. At 16 weeks of age, the mice were bred, by keeping the males and females together for 12 h (overnight). In the morning, mating was confirmed by examination of vaginal plugs, and considered 0.5 day post coitum (DPC). We started the study with 16 animals in each group. However, following overnight mating, the mice dams without vaginal plug and significant weight gain on day 7 post coitum were excluded from the study. This reduced the number of mice dams to 8 to 10 dams in each group. Following birth, 8 pups per group from 8 different mice dams were used to conduct the blood pressure measurement study. Molecular biology study (real-time PCR) were conducted on 4 animals from each group.
Protein restricted chow was obtained from Newco Distributors Inc. (Rancho Cucamonga, CA). Mice dams were divided in two groups: control protein diet (18 g/100 g, 100 % protein content), and antenatal MLPD (9 g/100 g, 50 % protein diet). To avoid the stress of food change during gestation and to include the peri-conceptual period, diet administration was started one week before mating. To maintain an isocaloric diet with low protein, we replaced the proteins with carbohydrates. The normal diet contained 18 g protein/100 g food, as described (additional file 1) [3–6]. We measured the amount of food (g/day) consumed by the mice on both normal and isocaloric low protein diets; and these were similar, [being 3 ± 0.5 g for females (n = 16) and 4 ± 0.3 g for males (n = 16) in the control group and 3 ± 0.4 (n = 16) for females and 4 ± 0.3 g for males (n = 16) in the antenatal MLPD group, respectively].
We measured body weight on the morning after the pups were delivered (0.5 Days). Intact placentas were collected from the cage and placental weight was determined. After birth, all the mice dams were maintained on the control diet to examine the effect of antenatal developmental programming per se. Blood pressure was measured non-invasively, weekly by determining the tail blood volume, flow, and pressure with a volume pressure recording sensor and an occlusion tail-cuff (CODA System, Kent Scientific, Torrington, CT). This system is significantly different from the plethysmographic based tail-cuff measurement system, which measures only systolic blood pressure . This is a highly accurate system with the capability of measuring systolic and diastolic blood pressures with the heart rate simultaneously and non-invasively [22, 23]. Moreover, in numerous other studies, the tail cuff method has been shown to measure the blood pressure non-invasively and accurately in mice and rats [24, 25]. Before commencing our studies, mice were placed on a warming plate at 37° centigrade until the temperature of the tail-region measured 37° by an infrared thermometer. Following the warming, the mice were trained for three 15-min sessions each day for three days, or until we obtained stable blood pressure recordings. Blood pressure was measured 20 times on a fixed day (Tuesday) and time (~11:30 AM), once weekly from 18 to 24 weeks of age.
mRNA and protein quantification
At 32 weeks of age the mice were euthanized by cervical dislocation and the lungs were isolated. The isolated lungs were snap frozen in liquid nitrogen and stored at −80° centigrade for later analysis. For each experiment lung tissue from 4 different mice from different mothers were used. Real-time PCR and western immunoblot assays were conducted, as described and validated previously in our laboratory [3–5]. We isolated and quantified RNA and protein by Allprep DNA/RNA Mini Kit according to the manufacturer’s instructions (Qiagen Inc, Valencia, CA Cat # 80204). Isolated mRNA was analyzed using a NanoDrop1000 Spectrophotometer (Thermo Scientific, Waltham, MA) at 260/280 wavelength UV rays to check for quality and quantity. The 260/280 ratio of 1.8 to 2 was accepted for quantification with real-time PCR. Real-time PCR was performed on Light Cycler 1.5 (Roche Inc., Indianapolis, IN) using hydrolysis Taqman probes and primers, designed using the Universal Probe Library, a web based software (Roche Inc.), and the Quantfast Real-Time PCR Kit (Qiagen). Total RNA (1 ug per reaction) was reverse transcribed using Quantitect reverse transcriptase kit (Qiagen, Valencia, CA). Relative expression was normalized to 18S RNA and fold-changes were calculated using the ΔΔCt method with normalization of individual PCR efficiencies .
Antibodies used in the study
Swant Inc. Switzerland
Santa Cruz Biotechnologies
Cell Signaling Technology
Santa Cruz Biotechnology
MicroRNAs (miRNA) were identified for the 3’ UTR of the ACE-2 mRNAs, using the web based bio-informatics software – TargetScan 4.2 (http://www.targetscan.org). Of 10 miRNA suggested by the bio-informatics software, we chose mmu-mir-429 miRNA for the present study, by the use of the Context Score of more than 90th percentile, as described by Grimson et al. . The identified miRNA levels were measured by the use of Real-Time Taqman microRNA PCR assays, according to manufacturer’s instructions (Life Technologies, Grand Island, NY).
We analyzed the data using repeated measure (RM) two-way Univariate Analysis of Variance (ANOVA) with Bonferroni’s and Tukey’s post-hoc analysis as well as linear and non-linear regression to determine statistically significant differences between groups, by the use of GraphPad Prism software (GraphPad Software Inc., San Diego, CA) and IBM SPSS (IBM Corp., NY). The hypothesis was accepted at P < 0.05. For the measurement of blood pressure one male or female offspring was included from one mice dam and considered n = 1.
Offspring mice weight in response to antenatal protein restriction
Litter size and placental weight
Neither litter size nor placental weights differ significantly. The average litter size was 7 ± 2 and 6 ± 3 in the control and antenatal MLPD group, respectively (P > 0.05). The pups sex distribution was between 40 to 60 % per mice dam between control and MLPD group (P > 0.05).
Renin angiotensin system expression
Protein levels of un-altered components of the pulmonary renin-angiotensin system in response to antenatal maternal low-protein diet
0.82 ± 0.07
0.92 ± 0.05
0.7 ± 0.1
0.7 ± 0.15
0.94 ± 0.04
0.81 ± 0.03
0.28 ± 0.15
0.16 ± 0.01
0.15 ± 0.01
0.14 ± 0.03
0.19 ± 0.01
0.21 ± 0.03
0.08 ± 0.01
0.08 ± 0.02
0.1 ± 0.2
0.9 ± 0.02
ACE-2 complementary miRNA expression
Accumulating evidence suggests that antenatal maternal protein deprivation can lead to the development of hypertension in the offspring [6, 7, 9, 28, 29]. Of note, in a manner similar to humans, mice also show an increase in blood pressure with aging . The mechanisms of this phenomenon are not known, however. In the present study, we identified that in females, antenatal MLPD-induced hypertension is associated with a reduction in the level of ACE-2 enzyme (Fig. 3). Importantly, female mice offspring were affected with hypertension to a significantly greater extent than males (Fig. 2), which may relate to the ACE-2 gene being located on the X chromosome . Further, studies in three different rat strains have demonstrated that reduced ACE-2 expression is associated with hypertension . In addition, ACE-2 gene maps to a defined quantitative trait locus (QTL) for increased blood pressure on the X chromosome in these hypertensive rats .
Several other reports also have suggested an important role of ACE-2 in hypertension [33, 34]. In the present study, as a consequence of antenatal MLPD, we observed that ACE-2 was reduced only in female, and not male offspring (Fig. 3A). Importantly, only female offspring from the antenatal MLPD group were significantly hypertensive (Fig. 2). It appears that, as a consequence of antenatal MLPD, in females ACE-2 is differentially regulated, and may play an important role in the developmental programming of hypertension. Being located on an X chromosome, ACE-2 gene is susceptible for sexually dimorphic programming, as the chromosome is well known for other sexually dimorphic gene expression and disorders . Additionally, ACE-2 is known to be regulated in a sexually dimorphic manner by estrogen . Also, a high fat diet in rats is known to cause sexually dimorphic regulation of ACE-2 and the development of hypertension . The present study, adds to the accumulating evidence that ACE-2 can be developmentally programmed by antenatal stressors during gestation, and may be a critical factor in the development of hypertension.
Developmental programming of adult health and disease is an important factor in the genesis of hypertension. The present study, demonstrates that sexual dimorphism of the development of hypertension may be mediated through miRNA-mediated mechanisms and alterations in the ACE-2 gene. The questions remain, by what mechanism miRNA expression is regulated and programmed by in-utero environment? What is the role of estrogen in this signaling? We shall pursue thesein future studies.
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