Objective
Examine temporal alterations in vascular angiotensin II receptors (AT 1 R and AT 2 R) and determine vascular response to angiotensin II in growth-restricted offspring.
Study Design
Offspring of pregnant rats fed low-protein (6%) and control (20%) diet were compared.
Results
Prenatal protein restriction reprogrammed AT 1a R messenger RNA expression in male rats’ mesenteric arteries to cause 1.7- and 2.3-fold increases at 3 and 6 months of age associated with arterial pressure increases of 10 and 33 mm Hg, respectively; however, in female rats, increased AT 1a R expression (2-fold) and arterial pressure (15 mm Hg) occurred only at 6 months. Prenatal protein restriction did not affect AT 2 R expression. Losartan abolished hypertension, suggesting that AT 1a R plays a primary role in arterial pressure elevation. Vasoconstriction to angiotensin II was exaggerated in all protein-restricted offspring, with greater potency and efficacy in male rats.
Conclusion
Prenatal protein restriction increased vascular AT 1 R expression and vasoconstriction to angiotensin II, possibly contributing to programmed hypertension.
Perturbation of nutrition during critical periods of fetal growth induces long-term structural and functional defects in the developing organism, predisposing it to increased risk for development of hypertension and cardiovascular disease later in life. Animal studies support the concept of developmental programming of hypertension, although the mechanisms are still not completely understood.
Evidence suggests that the renin angiotensin system (RAS), a regulatory system important in the long-term control of blood pressure, is altered in the offspring of protein-restricted dams and may contribute to the cause of programmed hypertension. The blocking of angiotensin II (ANG II) formation with angiotensin converting enzyme blocker or inhibition of ANG II action at ANG II type-1 receptor (AT 1 R) with antagonist prevents elevation of blood pressure in the offspring of protein-restricted dams. RAS components are highly expressed in the brain and kidneys, and numerous investigators have associated the changes in central and intrarenal RAS to increases in arterial pressure. Increased expression of AT 1 R is observed in cardiovascular regulating regions of the brain in adult male protein-restricted offspring. Further, intracerebroventricular injection of the ACE inhibitor enalaprilat or of the AT 1 R antagonist losartan is shown to normalize blood pressure in the offspring of protein-restricted dams. Offspring of rat dams fed a low-protein diet during gestation display significantly altered messenger RNA (mRNA) and protein levels of AT 1 R and AT 2 R in kidneys. Renal AT 1 R and AT 2 R receptor protein expression was significantly lower in prenatal protein-restricted rats from fetal day 18 to postnatal day 10. In 4-week-old protein-restricted offspring, the renal AT 1 R protein level was greater. In adult 16-week-old protein-restricted rats, AT 1 R protein was upregulated whereas AT 2 R protein was downregulated, suggesting that the ontogeny of the intrarenal RAS is altered throughout the perinatal and early postnatal period and through adult life. Vehaskari and colleagues reported that plasma renin activity is increased in prenatally protein-restricted adult rats, suggesting inappropriate activation of the peripheral RAS. Circulating ANG II, in addition to acting at the central and intrarenal levels, can impact the vascular system to increase blood pressure. However, information on expression of vascular AT 1 R and AT 2 R and the functional effect of ANG II in the resistance mesenteric arteries, the major determinant of systemic blood pressure in prenatally protein-restricted adult rats, is lacking.
Studies examining the role of RAS in hypertension induced by maternal protein restriction are focused in men, as women were found to be relatively protected. Because cardiovascular dysfunction in women is rapidly increasing, mechanistic studies in women, as well as men, are critical to understanding the sex differences in the pathogenesis of hypertension. We use a rat model of maternal dietary protein restriction (6% protein diet compared with 20% in controls) that leads to hypertension in both sexes, with more pronounced effects in adult males than females, similar to effects observed in population settings. Therefore, the purpose of this study was to examine the male and female offspring of control and protein-restricted groups to determine (1) whether the expression of vascular AT 1 R and AT 2 R mRNA is altered in the resistance arteries and if they relate to onset and magnitude of hypertension and (2) whether peripheral vascular response (mesenteric arteries) to ANG II is altered in adult male and female protein-restricted offspring compared with respective control offspring.
Materials and Methods
Animals
The animal protocol was approved by the Animal Care Committee of University of Texas Medical Branch and is in accordance with the National Institutes of Health guidelines (NIH publication no. 85-23, revised 1996). Timed pregnant Sprague-Dawley rats at gestational day 3 were purchased from Harlan Inc (Houston, TX). The rats were allocated to ad libitum isocaloric diet containing either 20% (control, n = 8) or 6% (protein-restricted, n = 9) casein, as in our previous studies. After the delivery of pups, dams were returned to regular chow. Pups were weaned at 3 weeks of age to regular chow, and male and female rats were housed separately and examined at 1, 3, and 6 months of age for arterial pressure, mesenteric vascular reactivity and expression of ANG II receptors (AT 1a R and AT 2 R). At each time period, blood pressure was measured and the animals were killed, mesenteric arteries were collected, RNA was isolated, and expression of ANG II receptors was determined. In the 6-month-old offspring, a portion of the mesenteric arteries was separated for vascular reactivity studies, and the remaining was quickly frozen for RNA isolation. Unless specified otherwise, 1 animal per litter in each sex was used for the different studies.
Experimental procedures
Mean arterial pressure
Mean arterial pressure in conscious free-moving male and female offspring of control and protein-restricted dams was determined at 1, 3, and 6 months of age using indwelling carotid arterial catheters as described in our previous publications. Briefly, rats under anesthesia (ketamine—45 mg/kg; xylazine—5 mg/kg; Burns Veterinary Supply, Westbury, NY) were surgically instrumented with flexible catheters (PE 50 tubing) in the left carotid artery. The catheters were tunneled to the nape of the neck and exteriorized. After 24-hour recovery period, when the animals are fully conscious and in free moving state, arterial catheter was connected to a pressure transducer and arterial blood pressure was obtained using a data acquisition system (DBP001 direct BP system and Workbench for Windows software; both from Kent Scientific, Litchfield, CT). After a 30-minute stabilization period, the arterial pressure was monitored continuously for 30 minutes and averaged to determine the baseline values.
Losartan treatment
Another set of male and female offspring at 6 months of age were gavaged with AT 1 R antagonist losartan (20 mg·kg –1 ·day –1 ) for 1 week. After losartan treatment, changes in arterial pressure were recorded as above using an indwelling arterial catheter.
Quantitative reverse transcription-polymerase chain reaction
Immediately after the measurements of baseline blood pressure the whole mesenteric arteries were collected from 1-, 3-, and 6-month-old animals. Mesenteric arteries were instantly frozen in liquid nitrogen and then processed for the total RNA extraction (TRIZOL; Invitrogen, Carlsbad, CA). All RNA isolates were made DNA free by treatment with DNAse and further purified with RNeasy clean up kit (QIAGEN Inc, Valencia, CA). Total RNA concentration and integrity were determined using an ND-1000 Nanodrop spectrophotometer (Thermo Fisher Scientific, Newark, DE). One microgram of total RNA was reverse transcribed using a modified Maloney murine leukemia virus-derived reverse transcriptase (New England Biolabs Inc, Ipswich, MA) and a blend of oligo (dT) and random hexamer primers (Invitrogen). The reaction was carried out at 28°C for 15 minutes and 42°C for 50 minutes, then stopped by heating at 94°C for 5 minutes, followed by 4°C before storage at −20°C until further analysis. One microliter of the diluted cDNA corresponding to 100 ng RNA was amplified by reverse transcription-polymerase chain reaction using FAM (Invitrogen) as the fluorophore in a CFX96 real-time thermal cycler (Bio-Rad, Hercules, CA). Polymerase chain reaction conditions used were 2 minutes at 50°C for 1 cycle; 10 minutes at 95°C, 15 seconds at 95°C, and 1 minute at 60°C for 40 cycles; and a final dissociation step (0.05 seconds at 65°C and 0.5 seconds at 95°C). Results were calculated using the 2 –▵▵CT method and expressed in folds increase/decrease of the gene of interest in protein-restricted vs control rats. All reactions were performed in duplicate, and 18S was used as an internal control. The following TaqMan assays were done in 10 μL volume for reverse transcription-polymerase chain reaction at a final concentration of 250 nM TaqMan probe and 900 nM of each primer; Assays-on-Demand for AT 1a R (Rn01435427_m1), AT 1b R (Rn02132799_s1), and AT 2 R (Rn00560677_s1) were obtained from Applied Biosystems.
Ex vivo vascular reactivity studies
Freshly excised third-order mesenteric arteries from 6-month-old male and female offspring were placed in ice cold modified Krebs bicarbonate solution (KBS) of the following composition (in mM): 118 NaCl, 4.7 KCl, 25 NaHCO 3 , 2.5 CaCl 2 , 1.2 MgSO 4 , 1.2 KH 2 PO 4 , and 11 dextrose. The mesenteric arteries were cleaned of adherent connective tissue and precisely cut into rings of same length (2 mm). Two to 4 rings from 1 rat were used for 1 experiment. The n presented with each figure represents the number of animals studied. In the presence of endothelium, ANG II does not elicit a stable contraction ex vivo, presumably because of the AT 2 receptor-mediated vasodilatory effects. Hence, studies were done in endothelium-denuded vessels. Further, our study shows that AT 2 receptor expression in the mesenteric arteries was unchanged, and endothelium denudation will help focus on the role of AT 1 receptors. Furthermore, we have previously shown that protein-restricted offspring have endothelial dysfunction ; hence, studies were performed in endothelium-denuded arterial rings to avoid confounding and to assess the AT 1 receptor-mediated effects on the smooth muscle. Endothelium was denuded by gently rubbing with tungsten wires. Two 25-μm tungsten wires were threaded through the lumen, and the rings were mounted in an isometric wire myograph system (model 610M wire myography; Danish Myotechniques, Aarhaus, Denmark). The rings were bathed in 6 mL KBS, gassed with 95% oxygen and 5% carbon dioxide, maintained at a temperature of 37°C, and allowed to equilibrate for 30 minutes before normalization to an internal diameter of 0.9 of L 13.3 kPa by using a normalization software package (Myodata; Danish Myotechnologies). This corresponds to a transmural pressure of ∼90 mm Hg. After normalization, rings were repeatedly exposed to KCl (80 mM) to test their viability and to determine a standard contractile response for each of them. The rings were contracted with phenylephrine (3 μM; Sigma-Aldrich, St. Louis, MO), and when responses were stable, endothelium-denudation was confirmed by absence of relaxation to acetylcholine (10 μM; Sigma-Aldrich). Rings were then allowed to recover for 60 minutes, after which cumulative concentration-response curves were generated with ANG II (10 −13 to 10 −8 M; Sigma-Aldrich) in the presence and absence of losartan (10 μM; AT 1 R antagonist; Sigma-Aldrich). Antagonists were added to the bath 30 minutes before cumulative concentration-response curves. Cumulative concentration-response curves were also generated with phenylephrine (10 −9 to 10 −5 M) and serotonin (10 −9 to 10 −5 M; Sigma-Aldrich).
Statistical analysis
For the comparison of arterial pressure, analysis was performed using analysis of variance (ANOVA), with adjustments for multiple comparisons. For comparison of genes expressed between the control and protein-restricted groups, unpaired Student t test was used. Cumulative concentration-response curves were analyzed by computer fitting to a 4-parameter sigmoid curve using the Prism 5 program (GraphPad, San Diego, CA) to evaluate the half-maximal effective concentration (EC 50 ) and E max , the maximum asymptote of the curve. All values are expressed as means ± standard error (SE). A P < .05 was considered significant.
Results
Mean arterial pressure
Mean arterial pressure measured in conscious free-moving rats via indwelling carotid catheter was comparable between control and protein-restricted male rats at 1 month of age, but protein-restricted male rats had higher arterial pressure than controls at 3 months (10 mm Hg higher; n = 8; P < .05) and 6 months of age (33 mm Hg higher; n = 8; P < .05; Figure 1 , A). In the female offspring, arterial pressure was similar between control and protein-restricted groups at 1 and 3 month of age, but protein-restricted female rats had significantly higher arterial pressure at 6 months of age (15 mm Hg higher; n = 8; P < .05; Figure 1 , B). Changes in both systolic and diastolic blood pressures were similar to that in mean arterial blood pressure; therefore, this data is not presented to reduce redundancy.
Effect of AT 1 R blockade on mean arterial pressure
Losartan treatment significantly decreased the blood pressure in protein-restricted male (mean decrease of 34 mm Hg; n = 5; P < .05; Figure 1 , A) and female (mean decrease of 27 mm Hg; n = 8; P < .05; Figure 1 , B) offspring. Mean arterial pressure did not significantly differ in losartan-treated control male and female offspring (n = 5 each; Figure 1 ).
Changes in mRNA of AT 1 and AT 2 receptors in the mesenteric artery
At the mRNA level, rodents possess 2 AT 1 receptor isoforms, designated AT 1a R and AT 1b R. AT 1b R was undetectable in the rat mesenteric arteries similar to previous studies. The mesenteric vascular expression of AT 1a R mRNA was comparable between control and protein-restricted male rats at 1 month of age (n = 6 in each group; Figure 2 , A). Expression of AT 1a R mRNA in the mesenteric arteries was significantly increased by 1.7-fold in protein-restricted male offspring (n = 7) relative to control male offspring (n = 8; P < .05; Figure 2 , B) at 3 months. At 6 months of age, a 2.3-fold increase in expression of AT 1a R in mesenteric arteries was observed in protein-restricted male vs control male offspring (n = 8 in each group; P < .05; Figure 2 , C). AT 2 R mRNA expression in the mesenteric arteries was similar between control and protein-restricted male rats at 1 (n = 8; Figure 2 , D), 3 ( Figure 2 , E), and 6 months of age ( Figure 2 , F). In protein-restricted male rats, the changes in vascular AT 1a R/AT 2 R ratio followed a trend similar to the changes in AT 1a R. The AT 1 R/AT 2 R ratio in protein-restricted male rats was similar to controls at 1 month of age (n = 7 in each group; Figure 2 , G), but significantly increased by 2.1- and 3.2-fold at 3 (n = 6; P < .05; Figure 2 , H) and 6 months of age (n = 6; P < .05; Figure 2 , I ), respectively.
In female rats, the mesenteric vascular expression of AT 1a R mRNA was comparable between control and protein-restricted female rats at 1 and 3 months of age (n = 8; Figure 3 , A and B), but protein-restricted female rats have 2-fold higher AT 1a R mRNA expression at 6 months of age (n = 5; P < .05; Figure 3 , C). AT 2 R mRNA was similar between control and protein-restricted female rats at 1, 3, and 6 months of age (n = 6 in each group; Figures 3 , D-F). The vascular AT 1 R/AT 2 R ratio was comparable between control and protein-restricted female rats at 1 and 3 months of age ( Figures 3 , G and H) but was significantly higher by 2.0-fold at 6 months of age in protein-restricted female rats compared with control female rats (n = 5; P < .05; Figure 3 , I).
Ex vivo vasomotor responses
ANG II induced a dose-dependent increase in contractile responses in mesenteric arterial rings in both male and female rats. However, the ANG II-induced contractile responses were exaggerated with a leftward shift in the dose-response curves as well as an increase in maximal responses in the protein-restricted compared with control rats. The magnitude of leftward shift, compared with respective controls, was greater in the protein-restricted male rats (n = 5; P < .05; Figure 4 , A, and the Table ) than protein-restricted female rats ( Figure 4 , B, and the Table 1 ). Similarly, the ANG II-induced maximal responses were greater in the protein-restricted male rats (n = 5; P < .05; Figure 4 , C, and the Table 1 ) than protein-restricted female rats (n = 5; P < .05; Figure 4 , D, and the Table 1 ). Pretreatment of the vascular rings with losartan inhibited the ANG II-induced vasoconstriction (n = 5; P < .05; Figure 4 , A and B). On the other hand, contractile responses to phenylephrine (n = 5 in each group; Figure 5 and the Table 1 ) and serotonin (n = 5 in each group; Figure 6 and the Table 1 ) were not increased in protein-restricted male rats and female rats compared with respective controls. Indeed, the contractile response to serotonin was reduced in protein-restricted female rats compared with control female rats (n = 5 in each group; P < .05; Figure 6 , B, and the Table ).
