It Is Never Too Early to Start: Educating Health Science Students to Become Future Educators.

Teachers lead, learn, and live as they walk through the journey of education, experiencing humanity in and outside their classrooms. No task is small when it comes to teaching; it is a craftsmanship that takes years to develop, and never too early to get started. In this commentary, the authors extract thoughtful viewpoints from years of teaching experience regarding how to inspire and engage more students to become educators. After all, nothing is more exciting and rewarding for a teacher than to make more and better teachers.

The functional role of PI3K in maintenance of blood pressure and baroreflex suppression in (mRen2)27 and mRen2.Lewis rat.

The phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway in brain of spontaneously hypertensive rats, but not Wistar-Kyoto (WKY) rats, contributes to elevated mean arterial pressure (MAP). The role of PI3K in the regulation of blood pressure or autonomic function in the nucleus tractus solitarii (NTS) is yet to be established in other Ang II-dependent models of hypertension. Thus, we microinjected PI3K inhibitors, wortmannin or LY294002, into the NTS, and measured MAP, baroreflex sensitivity (BRS) for heart rate (HR) control, and HR variability (HRV) in mRen2.Lewis congenic and (mRen2)27 transgenic rats. Bilateral NTS microinjections of wortmannin (100 nmol/L; 50 nL) reduced MAP in (mRen2)27 and mRen2.Lewis rats (33 ± 5 mm Hg, n = 7, and 32 ± 6 mm Hg, n = 9, respectively) for approximately 90 minutes. Spectral and sequence analysis showed improvements in spontaneous BRS and HRV (50%-100%) after treatment in both hypertensive strains. Injections of wortmannin into NTS of Hannover Sprague-Dawley or Lewis control rats failed to alter MAP, BRS, or HRV. In mRen2.Lewis, but not in control Lewis rats, LY294002 (50 µmole/L) reduced MAP and increased BRS and HRV similar to wortmannin. Thus, the pharmacologic blockade of the PI3K signaling pathway in NTS reveals an important contribution to resting MAP and BRS in rats with overexpression of the Ren2 gene.

Acute AT(1)-receptor blockade reverses the hemodynamic and baroreflex impairment in adult sheep exposed to antenatal betamethasone.

To accelerate lung development and protect neonates from other early developmental problems, synthetic steroids are administered maternally in the third trimester, exposing fetuses that are candidates for premature delivery to them. However, steroid exposure at this point of gestation may lead to elevated blood pressure [mean arterial pressure (MAP)] during adolescence. We hypothesize that fetal exposure to steroids activates the renin-angiotensin system, inducing an elevation in blood pressure and attenuation of baroreflex sensitivity (BRS) that is angiotensin II dependent in early adulthood. To test this hypothesis, fetal sheep were exposed to betamethasone (Beta) or vehicle (control) administered to ewes at day 80 of gestation and delivered at full term. At 1.8 yr of age, male offspring were instrumented for conscious recording of MAP, heart rate, and measurement of BRS [as low-frequency-alpha, high-frequency-alpha, sequence (seq) UP, seq DOWN, and seq TOTAL]. Beta-exposed sheep (n = 6) had higher MAP than control sheep (n = 5) (93 + or - 2 vs. 84 + or - 2 mmHg, P < 0.01). Acute blockade of angiotensin type 1 receptors with candesartan (0.3 mg/kg iv) normalized MAP in Beta-exposed sheep (85 + or - 4 mmHg), with no effect in control sheep (82 + or - 3 mmHg). Before angiotensin type 1 blockade, BRS maximum gain was significantly lower in Beta-exposed vs. control sheep (11 + or - 3 vs. 26 + or - 3 ms/mmHg, P < 0.0.01). However, 45 min after candesartan injection, BRS was increased in Beta-exposed (21 + or - 5 ms/mmHg) and control (35 + or - 4 ms/mmHg) sheep. Heart rate variability (HRV) and blood pressure variability (BPV) revealed lower HRV (SD of beat-to-beat interval and root mean square of successive beat-to-beat differences in R-R interval duration) and higher BPV (SD of MAP, systolic arterial pressure in low-frequency range) in Beta-exposed sheep. Candesartan partially restored HRV in Beta-exposed sheep and fully corrected BPV. Thus, in utero exposure to synthetic glucocorticoids causes long-lasting programming of the cardiovascular system via renin-angiotensin system-dependent mechanisms.

Angiotensin-(1-7) and baroreflex function in nucleus tractus solitarii of (mRen2)27 transgenic rats.

BACKGROUND: Endogenous angiotensin (Ang)-(1-7) enhances, while Ang II attenuates, baroreceptor sensitivity (BRS) for reflex control of heart rate (HR) in Sprague-Dawley (SD) rats. In (mRen2)27 renin transgenic rats [(mRen2)], there is overexpression of the mouse Ren2 gene in brain, leading to elevated Ang II and reduced Ang-(1-7) in brain medullary, and associated with hypertension and impaired BRS. METHODS: We therefore tested the contribution of endogenous Ang-(1-7) to BRS for control of HR and responses to cardiac vagal chemosensitive afferent fiber activation (CVA) with phenylbiguanide (PBG) in anesthetized SD and (mRen2) 27 rats before and after bilateral nucleus of the solitary tract (nTS) injection of the Ang-(1-7) receptor antagonist (D-Ala7)-Ang-(1-7). RESULTS: (mRen2) 27 rats exhibited a approximately 50% impairment in BRS as compared with SD (P < 0.05). (D-Ala7)-Ang-(1-7) attenuated BRS by approximately 50% in SD rats, but was without effect in (mRen2) 27 rats. (D-Ala7)-Ang-(1-7) did not alter the responses to CVA by PBG (iv bolus) in either strain. There were no differences in the depressor effects of Ang-(1-7) injected into the nTS, nor were levels of mRNA different for angiotensin-converting enzyme, angiotensin-converting enzyme 2, neprilysin, or the mas receptor in medullary tissue from SD versus (mRen2)27 rats. CONCLUSION: Endogenous Ang-(1-7) does not provide tonic input in the nTS to modulate BRS for control of HR in (mRen2)27 rats, which may contribute to impairment of BRS in these animals.

Reversal of vascular hypertrophy in hypertensive patients through blockade of angiotensin II receptors.

Angiotensin II (Ang II) has been linked to vascular dysfunction and target-organ damage. Blockade of the angiotensin II type 1 receptor (AT(1)) with an angiotensin receptor blocker may reverse vascular pathology independent of blood pressure (BP) lowering. Stage I hypertensive, nondiabetic patients (61% male; age 38 to 67 years) were randomized after a 4-week washout period to olmesartan medoxomil 20 to 40 mg or atenolol 50 to 100 mg plus additional agents (hydrochlorothiazide, amlodipine, or hydralazine) as needed for a goal BP <140/90 mm Hg. At baseline and after 1 year of treatment, subcutaneous gluteal resistance arteries were examined on a pressurized myograph to evaluate remodeling. Biopsies were available from 22 atenolol recipients, 27 olmesartan medoxomil recipients, and 11 normal volunteer controls. BP was reduced to a comparable degree by olmesartan medoxomil (from 149 +/- 11/92 +/- 8 mm Hg to 120 +/- 9/77 +/- 6 mm Hg; P < .05 [mean +/- standard deviation]) and atenolol (from 147 +/- 10/90 +/- 6 mm Hg to 125 +/- 12/78 +/- 7 mm Hg; P < .05 [mean +/- standard deviation]) from baseline for each arm (P = .08 for the 40-week treatment mean between arms). After one year's treatment, the wall-to-lumen ratio in arteries from patients treated with olmesartan medoxomil was significantly reduced (from 14.9% to 11.1%; P < .01), whereas no significant change was observed in arteries from atenolol-treated patients (from 16.0% to 15.5%; P = NS); the wall-to-lumen ratio in controls was 11.0%. Blockade of AT(1) receptors showed a superior corrective effect on the altered structure of resistance arteries in essential hypertension that was independent of the magnitude of BP reduction, and resulted in values similar to those in normotensive controls.

Primary role of angiotensin-converting enzyme-2 in cardiac production of angiotensin-(1-7) in transgenic Ren-2 hypertensive rats.

Angiotensin-converting enzyme-2 (ACE2) converts angiotensin II (ANG II) to angiotensin-(1-7) [ANG-(1-7)], and this enzyme may serve as a key regulatory juncture in various tissues. Although the heart expresses ACE2, the extent that the enzyme participates in the cardiac processing of ANG II and ANG-(1-7) is equivocal. Therefore, we utilized the Langendorff preparation to characterize the ACE2 pathway in isolated hearts from male normotensive Sprague-Dawley [Tg((-))] and hypertensive [mRen2]27 [Tg((+))] rats. During a 60-min recirculation period with 10 nM ANG II, the presence of ANG-(1-7) was assessed in the cardiac effluent. ANG-(1-7) generation from ANG II was similar in both the normal and hypertensive hearts [Tg((-)): 510 +/- 55 pM, n=20 vs. Tg((+)): 497 +/- 63 pM, n=14] with peak levels occurring at 30 min after administration of the peptide. ACE2 inhibition (MLN-4760, 1 microM) significantly reduced ANG-(1-7) production by 83% (57 +/- 19 pM, P<0.01, n=7) in the Tg((+)) rats, whereas the inhibitor had no significant effect in the Tg((-)) rats (285 +/- 53 pM, P>0.05, n=10). ACE2 activity was found in the effluent of perfused Tg((-)) and Tg((+)) hearts, and it was highly associated with ACE2 protein expression (r=0.78). This study is the first demonstration for a direct role of ACE2 in the metabolism of cardiac ANG II in the hypertrophic heart of hypertensive rats. We conclude that predominant expression of cardiac ACE2 activity in the Tg((+)) may be a compensatory response to the extensive cardiac remodeling in this strain.

Angiotensin metabolism in renal proximal tubules, urine, and serum of sheep: evidence for ACE2-dependent processing of angiotensin II.

Despite the evidence that angiotensin-converting enzyme (ACE)2 is a component of the renin-angiotensin system (RAS), the influence of ACE2 on angiotensin metabolism within the kidney is not well known, particularly in experimental models other than rats or mice. Therefore, we investigated the metabolism of the angiotensins in isolated proximal tubules, urine, and serum from sheep. Radiolabeled [(125)I]ANG I was hydrolyzed primarily to ANG II and ANG-(1-7) by ACE and neprilysin, respectively, in sheep proximal tubules. The ACE2 product ANG-(1-9) from ANG I was not detected in the absence or presence of ACE and neprilysin inhibition. In contrast, the proximal tubules contained robust ACE2 activity that converted ANG II to ANG-(1-7). Immunoblots utilizing an NH(2) terminal-directed ACE2 antibody revealed a single 120-kDa band in proximal tubule membranes. ANG-(1-7) was not a stable product in the tubule preparation and was rapidly hydrolyzed to ANG-(1-5) and ANG-(1-4) by ACE and neprilysin, respectively. Comparison of activities in the proximal tubules with nonsaturating concentrations of substrate revealed equivalent activities for ACE (ANG I to ANG II: 248 +/- 17 fmol x mg(-1) x min(-1)) and ACE2 [ANG II to ANG-(1-7): 253 +/- 11 fmol x mg(-1) x min(-1)], but lower neprilysin activity [ANG II to ANG-(1-4): 119 +/- 24 fmol x mg(-1) x min(-1); P < 0.05 vs. ACE or ACE2]. Urinary metabolism of ANG I and ANG II was similar to the proximal tubules; soluble ACE2 activity was also detectable in sheep serum. In conclusion, sheep tissues contain abundant ACE2 activity that converts ANG II to ANG-(1-7) but does not participate in the processing of ANG I into ANG-(1-9).

Baroreceptor reflex regulation in anesthetized transgenic rats with low glia-derived angiotensinogen.

Endogenous angiotensin (ANG) II and ANG-(1-7) act at the nucleus tractus solitarius (NTS) to differentially modulate neural control of the circulation. The role of these peptides endogenous to NTS on cardiovascular reflex function was investigated in transgenic rats with low brain angiotensinogen (Aogen) due to glial overexpression of an antisense to Aogen (ASrAOGEN) and in Sprague-Dawley (SD) rats. Arterial baroreceptor reflex sensitivity (BRS) for control of heart rate (HR) in response to increases in mean arterial pressure (MAP) was tested before and after bilateral microinjection of the angiotensin type 1 (AT(1)) receptor blocker candesartan or the ANG-(1-7) receptor blocker (d-Ala(7))-ANG-(1-7) into the NTS of urethane-chloralose-anesthetized ASrAOGEN and SD rats. Baseline MAP was higher in ASrAOGEN than in SD rats under anesthesia (P < 0.01). Injection of candesartan or (d-Ala(7))-ANG-(1-7) decreased MAP (P < 0.01) and HR (P < 0.05) in ASrAOGEN, but not SD, rats. The BRS at baseline was similar in ASrAOGEN and SD rats. Candesartan increased BRS by 41% in SD rats (P < 0.01) but was without effect in ASrAOGEN rats. In contrast, the reduction in BRS after (d-Ala(7))-ANG-(1-7) administration was comparable in SD (31%) and ASrAOGEN rats (34%). These findings indicate that the absence of glia-derived Aogen is associated with 1) an increase in MAP under anesthesia mediated via AT(1) and ANG-(1-7) receptors within the NTS, 2) the absence of an endogenous ANG II contribution to tonic inhibition of BRS, and 3) a continued contribution of endogenous ANG-(1-7) to tonic enhancement of BRS.

The protective effects of angiotensin II blockade with olmesartan medoxomil on resistance vessel remodeling (The VIOS study): rationale and baseline characteristics.

BACKGROUND: The VIOS (Vascular Improvement with Olmesartan medoxomil Study) study is a randomized, parallel study to determine the relative effects of suppressing the renin-angiotensin system (RAS) with the angiotensin receptor antagonist olmesartan medoxomil versus suppressing sympathetic drive with the beta-adrenoceptor antagonist atenolol on remodeling of the subcutaneous small resistance vessel. Remodeling of small resistance vessels may be the earliest pathologic finding associated with hypertension. It may predate the onset of clinically apparent hypertension. METHODS: In this study, 100 patients with stage I hypertension are characterized at baseline before being treated for 1 year to obtain a goal BP of less than 140/90 mm Hg as defined by Joint National Committee (JNC)-7. Resistance vessel remodeling is determined using the gluteal fat biopsy technique in the hypertensive patients and a group of normotensive healthy volunteers. Additionally, efforts will be made to define whether noninvasive hemodynamic parameters, retinal vessel measurement changes, or biologic markers may predict and track the underlying vascular morphologic and physiologic changes induced by either regimen during the 12-month treatment period. RESULTS: The primary endpoint will be the degree of vascular remodeling as obtained from percutaneous biopsy of gluteal subcutaneous resistance vessels in each of two treatment arms compared with the normal volunteers. The design of the study and the pertinent baseline characteristics of these patients with uncomplicated essential hypertension are presented. CONCLUSION: The suppression of the RAS by the blockade of angiotensin II type 1 (AT(1)) receptors may demonstrate remodeling effects on the ubiquitous small resistance vessels similar to that seen in the myocardium and renal glomeruli, thus affording more complete end-organ protection.

Effect of angiotensin II blockade on a new congenic model of hypertension derived from transgenic Ren-2 rats.

The generation of the Lew.Tg(mRen2) congenic hypertensive rat strain, developed through a backcross of the hypertensive (mRen2)27 transgenic rat with normotensive Lewis rats, provides a new model by which primary hypertension can be studied without the genetic variability found in the original strain. The purpose of this study was to characterize the Lew.Tg(mRen2) rats by dually investigating the effects of type 1 angiotensin II (ANG II) receptor (AT(1)) blockade and angiotensin-converting enzyme (ACE) activity inhibition on the ANG-(1-7)/ACE2 axis of the renin-angiotensin system in this new hypertensive model. The control of blood pressure elicited by 12-day administration of either lisinopril (mean difference change = 92 +/- 2, P < 0.05) or losartan (mean difference change = 69 +/- 2, P < 0.05) was associated with 54% and 33% increases in cardiac ACE2 mRNA and 54% and 43% increases in cardiac ACE mRNA, respectively. Lisinopril induced a 3.1-fold (P < 0.05) increase in renal cortical expression of ACE2, whereas losartan increased ACE2 mRNA 3.5-fold (P < 0.05). Both treatment regimens increased renal ACE mRNA 2.6-fold (P < 0.05). The two therapies augmented ACE2 protein activity, as well as increased cardiac and renal AT(1) receptor mRNAs. ACE inhibition reduced plasma ANG II levels (81%, P < 0.05) and increased plasma ANG-(1-7) (265%, P < 0.05), whereas losartan had no effect on the peptides. In contrast with what had been shown in normotensive rats, ACE inhibition decreased renal ANG II excretion and transiently decreased ANG-(1-7) excretion, whereas losartan treatment was associated with a consistent decrease in ANG-(1-7) urinary excretion rates. In response to the treatments, the expression of both renal cortical renin and angiotensinogen mRNAs was significantly augmented. The paradoxical effects of blockade of ANG II synthesis and activity on urinary excretion rates of the peptides and plasma angiotensins levels suggest that, in Lew.Tg(mRen2) congenic rats, a failure of compensatory ACE2 and ANG-(1-7)-dependent vasodepressor mechanisms may contribute both to the development and progression of hypertension driven by increased formation of endogenous ANG II.

Differential expression of nuclear AT1 receptors and angiotensin II within the kidney of the male congenic mRen2. Lewis rat.

We established a new congenic model of hypertension, the mRen(2). Lewis rat and assessed the intracellular expression of angiotensin peptides and receptors in the kidney. The congenic strain was established from the backcross of the (mRen2)27 transgenic rat that expresses the mouse renin 2 gene onto the Lewis strain. The 20-wk-old male congenic rats were markedly hypertensive compared with the Lewis controls (systolic blood pressure: 195 +/- 2 vs. 107 +/- 2 mmHg, P < 0.01). Although plasma ANG II levels were not different between strains, circulating levels of ANG-(1-7) were 270% higher and ANG I concentrations were 40% lower in the mRen2. Lewis rats. In contrast, both cortical (CORT) and medullary (MED) ANG II concentrations were 60% higher in the mRen2. Lewis rats, whereas tissue ANG I was 66 and 84% lower in CORT and MED. For both strains, MED ANG II, ANG I, and ANG-(1-7) were significantly higher than CORT levels. Intracellular ANG II binding distinguished nuclear (NUC) and plasma membrane (PM) receptor using the ANG II radioligand 125I-sarthran. Isolated CORT nuclei exhibited a high density (Bmax >200 fmol/mg protein) and affinity for the sarthran ligand (KD<0.5 nM); the majority of these sites (>95%) were the AT1 receptor subtype. CORT ANG II receptor Bmax and KD values in nuclei were 75 and 50% lower, respectively, for the mRen2. Lewis vs. the Lewis rats. In the MED, the PM receptor density (Lewis: 50 +/- 4 vs. mRen2. Lewis: 21 +/- 5 fmol/mg protein) and affinity (Lewis: 0.31 +/- 0.1 vs. 0.69 +/- 0.1 nM) were lower in the mRen2. Lewis rats. In summary, the hypertensive mRen2. Lewis rats exhibit higher ANG II in both CORT and MED regions of the kidney. Evaluation of intracellular ANG II receptors revealed lower CORT NUC and MED PM AT1 sites in the mRen2. Lewis. The downregulation of AT1 sites in the mRen2. Lewis rats may reflect a compensatory response to dampen the elevated levels of intrarenal ANG II.

Effects of renin-angiotensin system blockade on renal angiotensin-(1-7) forming enzymes and receptors.

BACKGROUND: Angiotensin-converting enzyme (ACE)2, a homologue of ACE, which is insensitive to ACE inhibitors and forms angiotensin-(1-7) [Ang-(1-7)] from angiotensin II (Ang II) with high efficiency was investigated in response to chronic blockade with lisinopril, losartan, and both drugs combined. METHODS: Thirty-six adult Lewis rats were assigned to receive these medications in their drinking water for 2 weeks while their arterial pressure, water intake, and urine volume were recorded throughout the study. Measures of renal excretory variables included assessing excretion rates of angiotensin I (Ang I), Ang II and Ang-(1-7) while blood collected at the completion of the study was used for measures of plasma angiotensin concentrations. Samples from renal cortex were assayed for renin, angiotensinogen (Aogen), neprilysin, angiotensin types 1 and 2 (AT(1) and AT(2)) and mas receptor mRNAs by semiquantitative reverse transcriptase (RT) real-time polymerase chain reaction (PCR). ACE2 activity was determined as the rate of Ang II conversion into Ang-(1-7). RESULTS: Comparable blood pressure reductions were obtained in rats medicated with either lisinopril or losartan, whereas both drugs produced a greater decrease in arterial pressure. Polyuria was recorded in all three forms of treatment associated with reduced osmolality but no changes in creatinine excretion. Lisinopril augmented plasma levels and urinary excretion rates of Ang I and Ang-(1-7), while plasma Ang II was reduced with no effect on urinary Ang II. Losartan produced similar changes in plasma and urinary Ang-(1-7) but increased plasma Ang II without changing urinary Ang II excretion. Combination therapy mimicked the effects obtained with lisinopril on plasma and urinary Ang I and Ang-(1-7) levels. Renal cortex Aogen mRNA increased in rats medicated with either lisinopril or the combination, whereas all three treatments produced a robust increase in renal renin mRNA. In contrast, ACE, ACE2, neprilysin, AT(1), and mas receptor mRNAs remained unchanged with all three treatments. Renal cortex ACE2 activity was significantly augmented in rats medicated with lisinopril or losartan but not changed in those given the combination. CONCLUSION: Our data revealed a role for ACE2 in Ang-(1-7) formation from Ang II in the kidney of normotensive rats as primarily reflected by the increased ACE2 activity measured in renal membranes from the kidney of rats given either lisinopril or losartan. The data further indicate that increased levels of Ang-(1-7) in the urine of animals after ACE inhibition or AT(1) receptor blockade reflect an intrarenal formation of the heptapeptide.

Role of blood pressure reduction in prevention of cardiac and vascular hypertrophy.

We investigated whether prevention of cardiac and vascular remodeling associated with inhibition of angiotensin II is independent of the blood pressure (BP)-lowering action of angiotensin II type 1 (AT1) receptor blockade. Spontaneously hypertensive rats, 8 weeks old, were treated with olmesartan, atenolol, or vehicle in their drinking water for 56 days. At the end of each treatment, arterial pressure and heart rate were measured, the ratio of heart weight to body weight was calculated, collagen deposition in the heart was determined histochemically using picrosirius red staining, and wall-to-lumen ratio in isolated mesenteric arteries was measured by a videographic approach. At 3 weeks after the initiation of treatment, rats medicated with olmesartan showed lower values of systolic BP compared with rats given atenolol or vehicle, whereas no difference in directly measured BP were observed at the end of study in anesthetized rats given olmesartan or atenolol. Rats given atenolol showed sustained bradycardia, whereas cardiac hypertrophy and collagen deposition was prevented only in spontaneously hypertensive rats given olmesartan. Olmesartan or atenolol reduced arteriolar wall-to-lumen ratio (olmesartan: 11.5+/-0.4%; atenolol: 13.3+/-0.6%; vehicle: 18.4%+/-1.1); however, this effect was greatest in rats medicated with the angiotensin II type 1 antagonist. Although control of BP is a factor in the prevention of cardiac and vascular hypertrophy, our studies suggest that blockade of angiotensin II receptors may attenuate the structural changes in the heart and blood vessels of hypertensive animals independent of a reduction in BP.

Impaired heart rate baroreflex in older rats: role of endogenous angiotensin-(1-7) at the nucleus tractus solitarii.

Age-related baroreflex reductions in function may originate from central neural dysregulation as well as vascular structural/functional changes. We determined the role of 2 angiotensin (Ang) peptides at the nucleus tractus solitarii in age-related baroreflex impairment. Baroreflex sensitivity control of heart rate in response to increases in blood pressure was tested in younger (3 to 5 months) and older (16 to 20 months) anesthetized male Sprague-Dawley rats before and after bilateral solitary tract injections of the Ang II type 1 (AT1) receptor antagonist candesartan (24 pmol) or the Ang-(1-7) antagonist (D-Ala7)-Ang-(1-7) (144 fmol or 24 pmol). Basal reflex sensitivity of older rats was significantly lower than younger rats. In younger rats, the reflex was facilitated by bilateral candesartan injections and attenuated by bilateral (D-Ala7)-Ang-(1-7) injections. In older rats, the reflex was facilitated by AT1 blockade; however, (D-Ala7)-Ang-(1-7) injected into the solitary tract nucleus had no effect. Neprilysin mRNA in the medulla was lower in older rats compared with younger rats, whereas angiotensin-converting enzyme (ACE), ACE2, and mas receptor mRNA levels of older rats did not differ from values of younger rats. Thus, opposing actions of endogenous Ang II and Ang-(1-7) in the solitary tract nucleus contribute to baroreflex function in response to increases in mean arterial pressure of younger rats. The attenuated counterbalancing effect of Ang-(1-7) on baroreflex function is lost in older rats, which may be attributable to diminished production of the peptide from neprilysin.

Effect of angiotensin-converting enzyme inhibition and angiotensin II receptor blockers on cardiac angiotensin-converting enzyme 2.

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) has emerged as a novel regulator of cardiac function and arterial pressure by converting angiotensin II (Ang II) into the vasodilator and antitrophic heptapeptide, angiotensin-(1-7) [Ang-(1-7)]. As the only known human homolog of ACE, the demonstration that ACE2 is insensitive to blockade by ACE inhibitors prompted us to define the effect of ACE inhibition on the ACE2 gene. METHODS AND RESULTS: Blood pressure, cardiac rate, and plasma and cardiac tissue levels of Ang II and Ang-(1-7), together with cardiac ACE2, neprilysin, Ang II type 1 receptor (AT1), and mas receptor mRNAs, were measured in Lewis rats 12 days after continuous administration of vehicle, lisinopril, losartan, or both drugs combined in their drinking water. Equivalent decreases in blood pressure were obtained in rats given lisinopril or losartan alone or in combination. ACE inhibitor therapy caused a 1.8-fold increase in plasma Ang-(1-7), decreased plasma Ang II, and increased cardiac ACE2 mRNA but not cardiac ACE2 activity. Losartan increased plasma levels of both Ang II and Ang-(1-7), as well as cardiac ACE2 mRNA and cardiac ACE2 activity. Combination therapy duplicated the effects found in rats medicated with lisinopril, except that cardiac ACE2 mRNA fell to values found in vehicle-treated rats. Losartan treatment but not lisinopril increased cardiac tissue levels of Ang II and Ang-(1-7), whereas none of the treatments had an effect on cardiac neprilysin mRNA. CONCLUSIONS: Selective blockade of either Ang II synthesis or activity induced increases in cardiac ACE2 gene expression and cardiac ACE2 activity, whereas the combination of losartan and lisinopril was associated with elevated cardiac ACE2 activity but not cardiac ACE2 mRNA. Although the predominant effect of ACE inhibition may result from the combined effect of reduced Ang II formation and Ang-(1-7) metabolism, the antihypertensive action of AT1 antagonists may in part be due to increased Ang II metabolism by ACE2.

Vascular responses to Angiotensin-(1-7) during the estrous cycle.

Mesenteric arteries (230-290 microm) were isolated from virgin female rats at diestrous and proestrous phases of the estrous cycle and from ovariectomized (OVX) rats with or without estrogen (E2) replacement (17beta-estradiol, 7.5 + 5 mg pellets, 21 d release). Arteries were mounted in a pressurized myograph system. Angiotensin-(1-7) [Ang-(1-7)] concentration-dependent responses (10(-10)-10(-5) M) were determined in arteries preconstricted with endothelin-1 (10(-7) M). Mesenteric arteries were pretreated with the specific Ang-(1-7) antagonist, D-[Ala7]-Ang-(1-7) (10(-7) M) to assess the Ang-(1-7) receptor-mediated dilator effect. Ang-(1-7) did not dilate mesenteric arteries from virgin rats at diestrus and placebo-treated OVX female rats as compared to the time control; however, Ang-(1-7) elicited a modest dilation at proestrus as compared to diestrus, which reached statistical significance at 10(-8) M concentrations. Ang-(1-7) caused a concentration-dependent vasodilation in mesenteric arteries of females with E2 replacement, with an EC50 of 21 nM. D-[Ala7]-Ang-(1-7) blocked the vasodilator effect of Ang-(1-7). Our results demonstrate that during proestrus Ang-(1-7) elicits modest vasodilation as compared to diestrus, but lacks vasodilatory properties in vessels from diestrous and ovariectomized rats. Estrogen replacement restores a significant dilator response to Ang-(1-7) in OVX rats that is mediated by a D-[Ala7]-Ang-(1-7) sensitive site.

Upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin II receptors.

We investigated in Lewis normotensive rats the effect of coronary artery ligation on the expression of cardiac angiotensin-converting enzymes (ACE and ACE 2) and angiotensin II type-1 receptors (AT1a-R) 28 days after myocardial infarction. Losartan, olmesartan, or the vehicle (isotonic saline) was administered via osmotic minipumps for 28 days after coronary artery ligation or sham operation. Coronary artery ligation caused left ventricular dysfunction and cardiac hypertrophy. These changes were associated with increased plasma concentrations of angiotensin I, angiotensin II, angiotensin-(1-7), and serum aldosterone, and reduced AT1a-R mRNA. Cardiac ACE and ACE 2 mRNAs did not change. Both angiotensin II antagonists attenuated cardiac hypertrophy; olmesartan improved ventricular contractility. Blockade of the AT1a-R was accompanied by a further increase in plasma concentrations of the angiotensins and reduced serum aldosterone levels. Both losartan and olmesartan completely reversed the reduction in cardiac AT1a-R mRNA observed after coronary artery ligation while augmenting ACE 2 mRNA by approximately 3-fold. Coadministration of PD123319 did not abate the increase in ACE 2 mRNA induced by losartan. ACE 2 mRNA correlated significantly with angiotensin II, angiotensin-(1-7), and angiotensin I levels. These results provide evidence for an effect of angiotensin II blockade on cardiac ACE 2 mRNA that may be due to direct blockade of AT1a receptors or a modulatory effect of increased angiotensin-(1-7).

Cardiac angiotensin-(1-7) in ischemic cardiomyopathy.

BACKGROUND: Accumulating evidence suggests that angiotensin-(1-7) (Ang-[1-7]) may play an important role in counteracting the pressor, proliferative, and profibrotic actions of angiotensin II in the heart. Thus, we evaluated whether Ang-(1-7) is expressed in the myocardium of normal rats and those in which myocardial infarction was produced 4 weeks beforehand. METHODS AND RESULTS: The left coronary artery in 10-week-old Lewis rats was either ligated (n=5) or exposed but not occluded in age-matched controls (sham; n=5). Left ventricular end-diastolic pressures were significantly elevated 4 weeks after myocardial infarction (25+/-1 versus 5+/-1 mm Hg for sham; P<0.001), whereas left ventricular systolic pressures were significantly reduced (ligated 86+/-4 versus sham 110+/-5 mm Hg; P<0.01). Hemodynamic effects of coronary artery ligation were accompanied by significant cardiac hypertrophy (heart weight to body weight: ligated 4.3+/-0.1 versus sham 2.9+/-0.1 mg/g; P<0.001). In both ligated and sham rats, Ang-(1-7) immunoreactivity was limited to cardiac myocytes and absent in interstitial cells and coronary vessels. Ang-(1-7) immunoreactivity was significantly augmented in ventricular tissue surrounding the infarct area in the heart of rats with myocardial infarction. CONCLUSIONS: Development of heart failure subsequent to coronary artery ligation leads to increased expression of Ang-(1-7),which was restricted to myocytes.

Enhanced renal immunocytochemical expression of ANG-(1-7) and ACE2 during pregnancy.

Previously we demonstrated that kidney concentration and urinary excretion of angiotensin-(1-7) are increased during normal pregnancy in rats. Since this finding may reflect local kidney production of angiotensin-(1-7), we determined the immunocytochemical distribution of angiotensin-(1-7) and its newly described processing enzyme, ACE2, in kidneys of virgin and 19-day-pregnant Sprague-Dawley rats. Sprague-Dawley rats were killed at the 19th day of pregnancy, and tissues were prepared for immunocytochemical by using a polyclonal antibody to angiotensin- (1-7) or a monoclonal antibody to ACE2. Angiotensin-(1-7) immunostaining was predominantly localized to the renal tubules traversing both the inner cortex and outer medulla. ACE2 immunostaining was localized throughout the cortex and outer medulla and was visualized in the renal tubules of both virgin and pregnant rats. The quantification of angiotensin-(1-7) and ACE2 immunocytochemical staining showed that in pregnant animals, the intensity of the staining increased by 56% and 117%, respectively (P<0.05). This first demonstration of the immunocytochemical distribution of angiotensin-(1-7) and ACE2 in kidneys of pregnant rats shows that pregnancy increases angiotensin-(1-7) immunocytochemical expression in association with increased ACE2 intensity of staining. The findings suggest that ACE2 may contribute to the local production and overexpression of angiotensin-(1-7) in the kidney during pregnancy.

Estrogen or the AT1 antagonist olmesartan reverses the development of profound hypertension in the congenic mRen2. Lewis rat.

The influence of estrogen on the regulation of cardiovascular function remains a controversial and complex area of investigation. We assessed the effects of estrogen depletion in the congenic mRen(2). Lewis rat, established from the back-cross of the original (mRen2)-27 transgenic onto the Lewis inbred strain. Ovariectomy of heterozygous mRen(2). Lewis at 4 to 5 weeks resulted in a progressive increase in blood pressure compared with the sham surgery congenics at weeks 6 to 11. At 11 weeks, the ovariectomized mRen(2). Lewis (OVX) systolic blood pressure averaged 195+/-3.7 mm Hg versus 141+/-4.0 mm Hg for sham. Plasma Angiotensin (Ang) II, serum ACE activity, plasma renin concentration, as well as urinary excretion of Ang II, 8-isoprostane F2alpha, and endothelin-1 were elevated; however, renal mRNA levels of eNOS were suppressed after ovariectomy. Estrogen replacement reduced blood pressure below both the sham and OVX by 11 weeks (125+/-2.9 mm Hg, n=7, P<0.01 versus OVX and sham). Moreover, the AT1 receptor antagonist olmesartan (CS866; week 12 to 16) essentially normalized blood pressure to 113+/-5.4 mm Hg (n=6, P<0.01 versus OVX and sham). The attenuation of the hypertension was still evident 7 weeks after complete withdrawal of treatment (124+/-4.1 mm Hg at week 23). In summary, the OVX mRen.2. Lewis exhibited a rapid and sustained increase in blood pressure. Estrogen or olmesartan lowered pressure by a similar extent. We conclude that the ovary exerts considerable influence on the regulation of the blood pressure in the mRen2. Lewis strain, possibly by limiting activation of the renin-angiotensin system.