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1.  Chymase Mediates Angiotensin-(1-12) Metabolism in Normal Human Hearts 
Background
Identification of angiotensin-(1-12) [Ang-(1-12)] in forming Ang II by a non-renin dependent mechanism has increased knowledge on the paracrine/autocrine mechanisms regulating cardiac expression of Ang peptides. This study now describes in humans the identity of the enzyme accounting for Ang-(1-12) metabolism in the left ventricular (LV) tissue of normal subjects.
Methods and Results
Reverse phase HPLC characterized the products of 125I-Ang-(1-12) metabolism in plasma membranes (PMs) from human LV in the absence and presence of inhibitors for chymase (chymostatin), angiotensin converting enzyme (ACE) 1 (lisinopril) and 2 (MLN-4760) and neprilysin (SHC39370). In the presence of the inhibitor cocktail ≥ 98 ± 2% of cardiac 125I-Ang-(1-12) remained intact, whereas exclusion of chymostatin from the inhibitor cocktail led to significant conversion of Ang-(1-12) into angiotensin II. In addition, chymase-mediated hydrolysis of 125I-Ang I was higher compared to Ang-(1-12). Negligible Ang-(1-12) hydrolysis occurred by ACE, ACE2, and neprilysin. A high chymase activity was detected for both 125I-Ang-(1-12) and 125I-Ang I substrates.
Conclusions
Chymase accounts for the conversion of Ang-(1-12) and Ang I to Ang II in normal human LV. These novel findings expand knowledge of the alternate mechanism by which Ang-(1-12) contributes to the production of cardiac angiotensin peptides.
doi:10.1016/j.jash.2012.12.003
PMCID: PMC3605226  PMID: 23312967
ACE2; cardiac myocytes; angiotensin converting enzyme inhibitors; angiotensin II; heart disease; proangiotensin 12; angiotensin-(1-7)
2.  Hemodynamic and Hormonal Changes to Dual Renin Angiotensin System Inhibition in Experimental Hypertension 
Hypertension  2012;61(2):417-424.
We examined the antihypertensive effects of valsartan, aliskiren or both drugs combined on circulating, cardiac and renal components of the renin-angiotensin system (RAS) in congenic mRen2.Lewis hypertensive rats assigned to: vehicle (n=9), valsartan (via drinking water, 30 mg/kg/day; n=10), aliskiren (s.c. by osmotic mini-pumps, 50 mg/kg/day; n=10), or valsartan (30 mg/kg/day) combined with aliskiren (50 mg/kg/day; n=10). Arterial pressure and heart rate were measured by telemetry before and during two weeks of treatment; trunk blood, heart, urine and kidneys were collected for measures of RAS components. Arterial pressure and left ventricular weight/tibia length ratio were reduced by monotherapy of valsartan, aliskiren and further reduced by the combination therapy. Urinary protein excretion was reduced by valsartan and further reduced by the combination. The increases in plasma Ang II induced by valsartan were reversed by the treatment of aliskiren and partially suppressed by the combination. The decreases in plasma Ang-(1–7) induced by aliskiren recovered in the combination group. Kidney Ang-(1–12) was increased by the combination therapy while the increases in urinary creatinine mediated by valsartan were reversed by addition of aliskiren. The antihypertensive and antiproteinuric actions of the combined therapy were associated with marked worsening of renal parenchymal disease and increased peritubular fibrosis. The data show that despite improvements in the surrogate endpoints of blood pressure, ventricular mass and proteinuria, dual blockade of Ang II receptors and renin activity is accompanied by worsening of renal parenchymal disease reflecting a renal homeostatic stress response due to loss of tubuloglomerular feedback by Ang II.
doi:10.1161/HYPERTENSIONAHA.112.201889
PMCID: PMC3576205  PMID: 23232645
angiotensin-(1–12); aliskiren; arterial remodeling; direct renin inhibitors; urinary protein; valsartan
3.  Characterization of the Cardiac Renin Angiotensin System in Oophorectomized and Estrogen-Replete mRen2.Lewis Rats 
PLoS ONE  2013;8(10):e76992.
The cardioprotective effects of estrogen are well recognized, but the mechanisms remain poorly understood. Accumulating evidence suggests that the local cardiac renin-angiotensin system (RAS) is involved in the development and progression of cardiac hypertrophy, remodeling, and heart failure. Estrogen attenuates the effects of an activated circulating RAS; however, its role in regulating the cardiac RAS is unclear. Bilateral oophorectomy (OVX; n = 17) or sham-operation (Sham; n = 13) was performed in 4-week-old, female mRen2.Lewis rats. At 11 weeks of age, the rats were randomized and received either 17 β-estradiol (E2, 36 µg/pellet, 60-day release, n = 8) or vehicle (OVX-V, n = 9) for 4 weeks. The rats were sacrificed, and blood and hearts were used to determine protein and/or gene expression of circulating and tissue RAS components. E2 treatment minimized the rise in circulating angiotensin (Ang) II and aldosterone produced by loss of ovarian estrogens. Chronic E2 also attenuated OVX-associated increases in cardiac Ang II, Ang-(1–7) content, chymase gene expression, and mast cell number. Neither OVX nor OVX+E2 altered cardiac expression or activity of renin, angiotensinogen, angiotensin-converting enzyme (ACE), and Ang II type 1 receptor (AT1R). E2 treatment in OVX rats significantly decreased gene expression of MMP-9, ACE2, and Ang-(1–7) mas receptor, in comparison to sham-operated and OVX littermates. E2 treatment appears to inhibit upsurges in cardiac Ang II expression in the OVX-mRen2 rat, possibly by reducing chymase-dependent Ang II formation. Further studies are warranted to determine whether an E2-mediated reduction in cardiac chymase directly contributes to this response in OVX rats.
doi:10.1371/journal.pone.0076992
PMCID: PMC3808369  PMID: 24204720
4.  Restoration of the Blood Pressure Circadian Rhythm By Direct Renin Inhibition and Blockade of Angiotensin II Receptors in mRen2.Lewis hypertensive rats 
Background
Alterations in the circadian arterial pressure (AP) rhythm predict cardiovascular mortality. We examined the circadian AP rhythm and the effect of renin-angiotensin system blockade in congenic mRen2.Lewis hypertensive rats, a renin-dependent model of hypertension derived from the backcross of transgenic hypertensive [mRen-2]27 rats with Lewis normotensive ones.
Methods
Twenty nine mRen2.Lewis hypertensive rats were randomly assigned to drink tap-water (vehicle; n=9), valsartan (30 mg/kg/day; n=10) or valsartan (30mg/kg/day) combined with aliskiren given s.c. (50 mg/kg/day; n=10) for two weeks treatment. AP, heart rate, and locomotive activity were recorded with chronically implanted radiotelemetry probes. The awake/asleep ratio was calculated as (awake mean AP (MAP) mean – asleep MAP mean)/ (awake MAP mean) × 100. Plasma renin activity (PRA) and concentration (PRC), and plasma and kidney angiotensin II (Ang II) were measured by radioimmunoassays.
Results
Untreated hypertensive rats showed an inverse AP rhythm, higher at day and lower at night, accompanied by normal rhythms of heart rate and locomotive activity. Treatment with valsartan or aliskiren/valsartan normalized both the elevated AP and the AP rhythm with the combination therapy being more effective both in reducing MAP and in restoring the awake/asleep ratio. While PRA and PRC increased with the treatments, addition of aliskiren to valsartan partially reversed the increases in plasma Ang II levels while both valsartan and aliskiren/valsartan markedly reduced renal cortical content of Ang II.
Conclusion
The altered circadian AP rhythm in this renin-dependent hypertension model uncovers a significant role of Ang II on the desynchronization of the circadian rhythm among AP, heart rate, and locomotive activity.
doi:10.1177/1753944711434039
PMCID: PMC3798054  PMID: 22222314
angiotensin II; aliskiren; blood pressure; circadian rhythm; direct renin inhibitors; hypertension; valsartan
5.  Low glial angiotensinogen improves body habitus, diastolic function, and exercise tolerance in aging male rats 
Cardiovascular endocrinology  2012;1(3):49-58.
Objectives
Long-term systemic blockade of the renin–angiotensin system (RAS) with either an angiotensin (Ang) II type 1 receptor antagonist or an angiotensin-converting enzyme inhibitor attenuates age-related cardiac remodeling and oxidative damage, and improves myocardial relaxation. However, the role of the brain RAS in mediating the development of diastolic dysfunction during aging is not known. We hypothesized that low brain RAS protects against the development of age-related diastolic dysfunction and left ventricular remodeling.
Methods
Sixty-week-old transgenic male ASrAOGEN rats (n =9), with normal circulating Ang II and functionally low brain Ang II, because of a GFAP promoter-linked angiotensinogen antisense targeted to glia, and age-matched and sex-matched Hannover Sprague–Dawley (SD; n= 9) rats, with normal levels of both circulating and brain Ang II, underwent echocardiograms to evaluate cardiac structure and function. Postmortem hearts were further compared for histological, molecular, and biochemical changes consistent with cardiac aging.
Results
ASrAOGEN rats showed preserved systolic and diastolic function at mid-life and this was associated with a lower, more favorable ratio of the phospholamban–SERCA2 ratio, reduced incidence of histological changes in the left ventricle, and increased cardiac Ang-(1–7) when compared with the in-vivo functional, and ex-vivo structural and biochemical indices from age-matched SD rats. Moreover, ASrAOGEN rats had lower percent body fat and a superior exercise tolerance when compared with SD rats of the same age.
Conclusion
Our data indicate that the central RAS plays a role in the maintenance of diastolic function and exercise tolerance in mid-life and this may be related to effects on body habitus.
doi:10.1097/XCE.0b013e32835a2159
PMCID: PMC3685869  PMID: 23795309
brain; cardiac aging; diastolic dysfunction; renin–angiotensin system; SERCA2; tissue Doppler
6.  Inhibition of Angiotensin-Converting Enzyme 2 Exacerbates Cardiac Hypertrophy and Fibrosis in Ren-2 Hypertensive Rats 
American journal of hypertension  2010;23(6):687-693.
Background
Emerging evidence suggests that cardiac angiotensin-converting enzyme 2 (ACE2) may contribute to the regulation of heart function and hypertension-induced cardiac remodeling. We tested the hypothesis that inhibition of ACE2 in the hearts of (mRen2)27 hypertensive rats may accelerate progression of cardiac hypertrophy and fibrosis by preventing conversion of angiotensin II (Ang II) into the antifibrotic peptide, angiotensin-(1–7) (Ang-(1–7)).
Methods
Fourteen male (mRen2)27 transgenic hypertensive rats (12 weeks old, 401 ± 7 g) were administered either vehicle (0.9% saline) or the ACE2 inhibitor, MLN-4760 (30 mg/kg/day), subcutaneously via mini-osmotic pumps for 28 days.
Results
Although ACE2 inhibition had no effect on average 24-h blood pressures, left ventricular (LV) Ang II content increased 24% in rats chronically treated with the ACE2 inhibitor (P < 0.05). Chronic ACE2 inhibition had no effect on plasma Ang II or Ang-(1–7) levels. Increased cardiac Ang II levels were associated with significant increases in both LV anterior, posterior, and relative wall thicknesses, as well as interstitial collagen fraction area and cardiomyocyte hypertrophy in the transgenic animals chronically treated with the ACE2 inhibitor. Cardiac remodeling was not accompanied by any further alterations in LV function.
Conclusions
These studies demonstrate that chronic inhibition of ACE2 causes an accumulation of cardiac Ang II, which exacerbates cardiac hypertrophy and fibrosis without having any further impact on blood pressure or cardiac function.
doi:10.1038/ajh.2010.51
PMCID: PMC3739444  PMID: 20300067
angiotensin-converting enzyme 2; angiotensin II; angiotensin-(1-7); blood pressure; cardiac hypertrophy; hypertension
7.  Decreased cardiac Ang-(1–7) is associated with salt-induced cardiac remodeling and dysfunction 
Objective
Angiotensin II has a critical role in the regulation of blood pressure and cell growth and excess activity of the peptide is implicated in the pathogenesis of salt-induced cardiovascular injury. On the other hand, the role of counteracting angiotensin-(1–7) in cardiac structural and functional responses to high salt diet has not been elucidated. Therefore, the present study examined the changes in cardiac angiotensin-(1–7), its forming enzyme angiotensin converting enzyme 2 (ACE2) and receptor mas in response to a high salt diet in spontaneously hypertensive rats (SHR).
Methods
Eight-week-old male spontaneously hypertensive rats (SHR) were given an 8% salt diet for 5weeks (n = 8). Age- and gender-matched controls received standard chow (n = 6).
Results
Salt excess increased arterial pressure (p< 0.05) and plasma renin and angiotensin II concentrations (p<0.05). Salt-induced left ventricular remodeling and diastolic dysfunction were associated with diminished levels of angiotensin-(1–7) in the heart (p<0.05) and no changes in cardiac angiotensin II levels. Exposure to high salt intake decreased cardiac ACE2 mRNA and protein level (p< 0.05). There was no difference in the protein levels of angiotensin II type 1 and mas receptors between the two experimental groups.
Conclusion
The adverse cardiac effects of excessive salt intake may result not only from the undesirable action of angiotensin II but may also be a consequence of diminished protective effects of the angiotensin-(1–7).
doi:10.1177/1753944709353337
PMCID: PMC3733447  PMID: 19946038
ACE2; angiotensin II; angiotensin-(1–7); hypertension; salt
8.  The COSEHC™ Global Vascular Risk Management quality improvement program: first follow-up report 
The Global Vascular Risk Management (GVRM) Study is a 5-year prospective observational study of 87,863 patients (61% females) with hypertension and associated cardiovascular risk factors began January 1, 2010. Data are gathered electronically and cardiovascular risk is evaluated using the Consortium for Southeastern Hypertension Control™ (COSEHC™)-11 risk score. Here, we report the results obtained at the completion of 33 months since study initiation. De-identified electronic medical records of enrolled patients were used to compare clinical indicators, antihypertensive medication usage, and COSEHC™ risk scores across sex and diabetic status subgroups. The results from each subgroup, assessed at baseline and at regular follow-up periods, are reported since the project initiation. Inference testing was performed to look for statistically significant differences between goal attainments rates between sexes. At-goal rates for systolic blood pressure (SBP) were improved during the 33 months of the study, with females achieving higher goal rates when compared to males. On the other hand, at-goal control rates for total and low-density lipoprotein (LDL) cholesterol (chol) were better in males compared to females. Diabetic patients had lower at-goal rates for SBP and triglycerides but higher rates for LDL-chol. The LDL-chol at-goal rates were higher for males, while high-density lipoprotein (HDL)-chol rates were higher for females. Utilization of antihypertensive medications was similar during and after the baseline period for both men and women. Patients taking two or more antihypertensive medications had higher mean COSEHC™-11 scores compared to those on monotherapy. With treatment, hypertensive patients can reach SBP and cholesterol goals; however, population-wide improvement in treatment goal adherence continues to be a challenge for physicians. The COSEHC™ GVRM Study shows, however, that continuous monitoring and feedback to physicians of accurate longitudinal data is an effective tool in achieving better control rates of cardiovascular risk factors.
doi:10.2147/VHRM.S44950
PMCID: PMC3724686  PMID: 23901282
cardiovascular risk; coronary heart disease; dyslipidemia; electronic medical records; hypertension; metabolic syndrome
9.  Combination Direct Renin Inhibition with Angiotensin Type 1 Receptor Blockade improves Aldosterone but does not improve Kidney Injury in the Transgenic Ren2 rat 
Regulatory Peptides  2012;176(1-3):36-44.
Enhanced renin-angiotensin-aldosterone system (RAAS) activation contributes to proteinuria and chronic kidney disease by increasing glomerular and tubulointerstitial oxidative stress, promotion of fibrosis. Renin activation is the rate limiting step in angiotensin (Ang II) and aldosterone generation, and recent work suggests direct renin inhibition improves proteinuria comparable to that seen with Ang type 1 receptor (AT1R) blockade. This is important as, even with contemporary use of AT1R blockade, the burden of kidney disease remains high. Thereby, we sought to determine if combination direct renin inhibition with AT1R blockade in vivo, via greater attenuation of kidney oxidative stress, would attenuate glomerular and proximal tubule injury to a greater extent than either intervention alone. We utilized the transgenic Ren2 rat with increased tissue RAS activity and higher serum levels of aldosterone, which manifests hypertension and proteinuria. Ren2 rats were treated with renin inhibition (aliskiren), AT1R blockade (valsartan), the combination (aliskiren+valsartan), or vehicle for 21 days. Compared to Sprague-Dawley controls, Ren2 rats displayed increased systolic pressure (SBP), circulating aldosterone, proteinuria and greater urine levels of the proximal tubule protein excretory marker beta-N-acetylglucosaminidase (β-NAG). These functional and biochemical alterations were accompanied by increases in kidney tissue NADPH oxidase subunit Rac1 and 3-nitrotyrosine (3-NT) content as well as fibronectin and collagen type III. These findings occurred in conjunction with reductions in the podocyte-specific protein podocin as well as the proximal tubule-specific megalin. Further, in transgenic animals there was increased tubulointerstitial fibrosis on light microscopy as well as ultrastructural findings of glomerular podocyte foot-process effacement and reduced tubular apical endosomal/lysosomal activity. Combination therapy led to greater reductions in SBP and serum aldosterone, but did not result in greater improvement in markers of glomerular and tubular injury (ie. β-NAG) compared to either intervention alone. Further, combination therapy did not improve markers of oxidative stress and podocyte and proximal tubule integrity in this transgenic model of RAAS-mediated kidney damage despite greater reductions in serum aldosterone and BP levels.
doi:10.1016/j.regpep.2012.03.002
PMCID: PMC3348429  PMID: 22465166
Aldosterone; Combination; Renin inhibition; AT1R blockade; Podocyte; β-NAG; Oxidative Stress
10.  Addressing the theoretical and clinical advantages of combination therapy with inhibitors of the renin-angiotensin-aldosterone system: antihypertensive effects and benefits beyond BP control 
Life sciences  2009;86(9-10):289-299.
Aims
This article reviews the importance of the renin-angiotensin-aldosterone system (RAAS) in the cardiometabolic continuum; presents the pros and cons of dual RAAS blockade with angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs); and examines the theoretical and practical benefits supporting the use of direct renin inhibitors (DRIs) in combination with ACEIs or ARBs.
Main methods
The author reviewed the literature for key publications related to the biochemical physiology of the RAAS and the pharmacodynamic effects of ACEIs, ARBs, and DRIs, with a particular focus on dual RAAS blockade with these drug classes.
Key findings
Although ACEI/ARB combination therapy produces modest improvement in BP, it has not resulted in the major improvements predicted given the importance of the RAAS across the cardiorenal disease continuum. This may reflect the fact that RAAS blockade with ACEIs and/or ARBs leads to exacerbated renin release through loss of negative-feedback inhibition, as well as ACE/aldosterone escape through RAAS and non–RAAS-mediated mechanisms. Plasma renin activity (PRA) is an independent predictor of morbidity and mortality, even for patients receiving ACEIs and ARBs. When used alone or in combination with ACEIs and ARBs, the DRI aliskiren effectively reduces PRA. Reductions in BP are greater with these combinations, relative to the individual components alone.
Significance
It is possible that aliskiren plus either an ACEI or ARB may provide greater RAAS blockade than monotherapy with ACEIs or ARBs, and lead to additive improvement in BP and clinically important outcomes.
doi:10.1016/j.lfs.2009.11.020
PMCID: PMC2853260  PMID: 19958778
hypertension; renin-angiotensin-aldosterone system (RAAS) inhibition; angiotensin II; angiotensin-(1–7); combination therapy; aliskiren; prorenin/renin receptor
12.  Role of olmesartan in combination therapy in blood pressure control and vascular function 
Angiotensin receptor blockers have emerged as a first-line therapy in the management of hypertension and hypertension-related comorbidities. Since national and international guidelines have stressed the need to control blood pressure to <140/90 mmHg in uncomplicated hypertension and <130/80 mmHg in those with associated comorbidities such as diabetes or chronic kidney disease, these goal blood pressures can only be achieved through combination therapy. Of several drugs that can be effectively combined to attain the recommended blood pressure goals, fixed-dose combinations of angiotensin receptor blockers and the calcium channel blocker amlodipine provide additive antihypertensive effects associated with a safe profile and increased adherence to therapy. In this article, we review the evidence regarding the beneficial effects of renin–angiotensin system blockade with olmesartan medoxomil and amlodipine in terms of blood pressure control and improvement of vascular function and target organ damage.
PMCID: PMC2941783  PMID: 20859541
amlodipine; angiotensin receptor blockers; angiotensin-converting enzyme 2; hypertension; renin–angiotensin system
13.  Nebivolol reduces cardiac angiotensin II, associated oxidative stress and fibrosis but not arterial pressure in salt-loaded spontaneously hypertensive rats 
Journal of hypertension  2012;30(9):1766-1774.
Objectives
Increased sympathetic outflow, renin–angiotensin system (RAS) activity, and oxidative stress are critical mechanisms underlying the adverse cardiovascular effects of dietary salt excess. Nebivolol is a third-generation, highly selective β1-receptor blocker with RAS-reducing effects and additional antioxidant properties. This study evaluated the hypothesis that nebivolol reduces salt-induced cardiac remodeling and dysfunction in spontaneous hypertensive rats (SHRs) by suppressing cardiac RAS and oxidative stress.
Methods
Male SHRs (8 weeks of age) were given an 8% high salt diet (HSD; n = 22), whereas their age-matched controls (n = 10) received standard chow. In a subgroup of HSD rats (n = 11), nebivolol was given at a dose of 10 mg/kg per day by gastric gavage.
Results
After 5 weeks, HSD exacerbated hypertension as well as increased left-ventricular weight and collagen deposition while impairing left-ventricular relaxation. Salt-induced cardiac remodeling and dysfunction were associated with increased plasma renin concentration (PRC), cardiac angiotensin II immunostaining, and angiotensin-converting enzyme (ACE)/ACE2 mRNA and activity ratio. HSD also increased cardiac 3-nitrotyrosine staining indicating enhanced oxidative stress. Nebivolol treatment did not alter the salt-induced increase in arterial pressure, left-ventricular weight, and cardiac dysfunction but reduced PRC, cardiac angiotensin II immunostaining, ACE/ACE2 ratio, oxidative stress, and fibrosis.
Conclusions
Our data suggest that nebivolol, in a blood pressure-independent manner, ameliorated cardiac oxidative stress and associated fibrosis in salt-loaded SHRs. The beneficial effects of nebivolol may be attributed, at least in part, to the decreased ACE/ACE2 ratio and consequent reduction of cardiac angiotensin II levels.
doi:10.1097/HJH.0b013e328356766f
PMCID: PMC3567851  PMID: 22895019
β1-adrenergic receptors; angiotensin II; blood pressure; cardiac fibrosis; nebivolol; salt
14.  Nebivolol Improves Insulin Sensitivity in the TGR(Ren2)27 Rat 
Metabolism: clinical and experimental  2011;60(12):1757-1766.
Objective
Hypertension is often associated with increased oxidative stress and systemic insulin resistance. Use of β adrenergic receptor blockers in hypertension is limited due to potential negative influence on insulin sensitivity and glucose homeostasis. We sought to determine the impact of nebivolol, a selective vasodilatory β1adrenergic blocker, on whole-body insulin sensitivity, skeletal muscle oxidative stress, insulin signaling and glucose transport in the transgenic TG(mRen2)27rat (Ren2). This rodent model manifests increased tissue renin angiotensin expression, excess oxidative stress, and whole-body insulin resistance.
Research design and methods
Young (age 6-9 wks) Ren2 and age-matched Sprague-Dawley control rats were treated with nebivolol 10 mg/kg/day or placebo for 21 days. Basal measurements were obtained for glucose and insulin to calculate the Homeostasis Model Assessment (HOMA–IR). Additionally, insulin metabolic signaling, NADPH oxidase activity, reactive oxygen species (ROS), and ultrastructural changes as evaluated by transmission electron microscopy were examined ex vivo in skeletal muscle tissue.
Results
The Ren2 rat demonstrated systemic insulin resistance as examined by HOMA-IR, along with impaired insulin metabolic signaling in skeletal muscle. This was associated with increased oxidative stress and mitochondrial remodeling. Treatment with nebivolol was associated with improvement in insulin resistance and decreased NADPH oxidase activity/levels ROS in skeletal muscle tissue.
Conclusions
Nebivolol treatment for 3 weeks reduces NADPH oxidase activity and improves systemic insulin resistance, in concert with reduced oxidative stress in skeletal muscle in a young rodent model of hypertension, insulin resistance and enhanced tissue RAS expression.
doi:10.1016/j.metabol.2011.04.009
PMCID: PMC3170670  PMID: 21640361
Insulin resistance; oxidative stress; skeletal muscle
15.  Angiotensin II AT1 Receptor Blockade Normalizes CD11b+ Monocyte Production in Bone Marrow of Hypercholesterolemic Monkeys 
Atherosclerosis  2007;196(2):624-632.
The enhanced production of monocytes expressing pro-inflammatory markers such as the integrin CD11b in patients with hypercholesterolemia may promote vascular inflammation and exacerbate atherogenesis. The objective of the present study was to determine whether hypercholesterolemia stimulates the production of CD11b+ monocytes in bone marrow, and whether the renin-angiotensin system participates in this process and thus provides a target for therapeutic intervention. The dietary induction of hypercholesterolemia in adult male cynomolgus monkeys was accompanied by increased bone marrow cellularity and elevated peripheral blood and bone marrow monocyte CD11b expression. Isolated bone marrow CD34+ hematopoietic stem cells (HSCs) evaluated by in vitro functional assays exhibited enhanced myeloproliferative capacity and differentiation into CD11b+ monocytes. Treatment of hypercholesterolemic monkeys with the angiotensin II AT1 receptor blocker losartan for 15 weeks reduced bone marrow cellularity, suppressed peripheral blood and bone marrow monocyte CD11b expression, and normalized CD34+ cell function assays. All variables returned to pretreatment levels 6 weeks after discontinuation of losartan treatment. Hypercholesterolemia was associated with increased CD34+ cell AT1 receptor expression and an exaggerated in vitro myeloproliferative response to angiotensin II stimulation that positively correlated to plasma LDL concentrations. In vitro exposure to native low-density lipoproteins (LDL) also increased CD34+ cell AT1 receptor expression and the myeloproliferative response to angiotensin II stimulation in a dose-dependent and receptor-mediated manner. Our data provide support for a positive regulatory role of plasma LDL on AT1 receptor-mediated HSC differentiation and the production of pro-atherogenic monocytes. LDL-regulated HSC function may explain in part hypercholesterolemia-induced inflammation as well as the anti-inflammatory and anti-atherosclerotic effects of AT1 receptor blockers.
doi:10.1016/j.atherosclerosis.2007.06.024
PMCID: PMC2265080  PMID: 17692319
Hypercholesterolemia; LDL; monocyte; CD34; angiotensin; hematopoietic stem cell; hematopoiesis
16.  Chymase-Dependent Generation of Angiotensin II from Angiotensin-(1-12) in Human Atrial Tissue 
PLoS ONE  2011;6(12):e28501.
Since angiotensin-(1-12) [Ang-(1-12)] is a non-renin dependent alternate precursor for the generation of cardiac Ang peptides in rat tissue, we investigated the metabolism of Ang-(1-12) by plasma membranes (PM) isolated from human atrial appendage tissue from nine patients undergoing cardiac surgery for primary control of atrial fibrillation (MAZE surgical procedure). PM was incubated with highly purified 125I-Ang-(1-12) at 37°C for 1 h with or without renin-angiotensin system (RAS) inhibitors [lisinopril for angiotensin converting enzyme (ACE), SCH39370 for neprilysin (NEP), MLN-4760 for ACE2 and chymostatin for chymase; 50 µM each]. 125I-Ang peptide fractions were identified by HPLC coupled to an inline γ-detector. In the absence of all RAS inhibitor, 125I-Ang-(1-12) was converted into Ang I (2±2%), Ang II (69±21%), Ang-(1-7) (5±2%), and Ang-(1-4) (2±1%). In the absence of all RAS inhibitor, only 22±10% of 125I-Ang-(1-12) was unmetabolized, whereas, in the presence of the all RAS inhibitors, 98±7% of 125I-Ang-(1-12) remained intact. The relative contribution of selective inhibition of ACE and chymase enzyme showed that 125I-Ang-(1-12) was primarily converted into Ang II (65±18%) by chymase while its hydrolysis into Ang II by ACE was significantly lower or undetectable. The activity of individual enzyme was calculated based on the amount of Ang II formation. These results showed very high chymase-mediated Ang II formation (28±3.1 fmol×min−1×mg−1, n = 9) from 125I-Ang-(1-12) and very low or undetectable Ang II formation by ACE (1.1±0.2 fmol×min−1×mg−1). Paralleling these findings, these tissues showed significant content of chymase protein that by immunocytochemistry were primarily localized in atrial cardiac myocytes. In conclusion, we demonstrate for the first time in human cardiac tissue a dominant role of cardiac chymase in the formation of Ang II from Ang-(1-12).
doi:10.1371/journal.pone.0028501
PMCID: PMC3236741  PMID: 22180785
17.  Salt-Induced Renal Injury in Spontaneously Hypertensive Rats: Effects of Nebivolol 
American Journal of Nephrology  2010;32(6):557-566.
Background
We investigated renal effects of nebivolol, a selective β1-receptor blocker with additional antioxidative ability, in spontaneously hypertensive rats (SHR) where increased salt intake induces oxidative stress and worsens renal function as a result of further activation of the renin-angiotensin and sympathetic nervous systems.
Methods
Male SHR were given an 8% salt diet (HS; n = 22) for 5 weeks; their age-matched controls (n = 9) received standard chow. Nebivolol was given at a dose of 10 mg/kg/day for 5 weeks in 11 HS rats.
Results
HS increased blood pressure, plasma renin concentration, urinary protein excretion, and renal nitroxidative stress while decreasing renal blood flow and angiotensin 1–7 receptor (mas) protein expression. There was no change in angiotensin II type 1 receptor expression among the experimental groups. Nebivolol did not alter the salt-induced increase in blood pressure but reduced urinary protein excretion, plasma renin concentration, and nitroxidative stress. Nebivolol also increased neuronal NOS expression while preventing the salt-induced decrease in renal blood flow and mas protein expression.
Conclusion
Nebivolol prevented salt-induced kidney injury and associated proteinuria in SHR through a blood pressure-independent mechanism. Its protective effects may be related to reduction in oxidative stress, increases in neuronal NOS and restoration of angiotensin II type 1/mas receptor balance.
doi:10.1159/000321471
PMCID: PMC2992650  PMID: 21042014
Salt; Hypertension; Kidney; Oxidative stress; Nitric oxide; β1-Receptor antagonism
18.  Uptake and Metabolism of the Novel Peptide Angiotensin-(1-12) by Neonatal Cardiac Myocytes 
PLoS ONE  2011;6(1):e15759.
Background
Angiotensin-(1–12) [Ang-(1–12)] functions as an endogenous substrate for the productions of Ang II and Ang-(1–7) by a non-renin dependent mechanism. This study evaluated whether Ang-(1–12) is incorporated by neonatal cardiac myocytes and the enzymatic pathways of 125I-Ang-(1–12) metabolism in the cardiac myocyte medium from WKY and SHR rats.
Methodology/Principal Findings
The degradation of 125I-Ang-(1–12) (1 nmol/L) in the cultured medium of these cardiac myocytes was evaluated in the presence and absence of inhibitors for angiotensin converting enzymes 1 and 2, neprilysin and chymase. In both strains uptake of 125I-Ang-(1–12) by myocytes occurred in a time-dependent fashion. Uptake of intact Ang-(1–12) was significantly greater in cardiac myocytes of SHR as compared to WKY. In the absence of renin angiotensin system (RAS) enzymes inhibitors the hydrolysis of labeled Ang-(1–12) and the subsequent generation of smaller Ang peptides from Ang-(1–12) was significantly greater in SHR compared to WKY controls. 125I-Ang-(1–12) degradation into smaller Ang peptides fragments was significantly inhibited (90% in WKY and 71% in SHR) in the presence of all RAS enzymes inhibitors. Further analysis of peptide fractions generated through the incubation of Ang-(1–12) in the myocyte medium demonstrated a predominant hydrolytic effect of angiotensin converting enzyme and neprilysin in WKY and an additional role for chymase in SHR.
Conclusions/Significance
These studies demonstrate that neonatal myocytes sequester angiotensin-(1–12) and revealed the enzymes involved in the conversion of the dodecapeptide substrate to biologically active angiotensin peptides.
doi:10.1371/journal.pone.0015759
PMCID: PMC3018413  PMID: 21249217
19.  Targeting the Degradation of Angiotensin II with Recombinant ACE2: Prevention of Angiotensin II-dependent Hypertension 
Hypertension  2009;55(1):90-98.
Angiotensin converting enzyme 2 (ACE2) cleaves angiotensin II (Ang II) to form Ang-(1-7). Here we examined whether soluble human recombinant ACE2 (rACE2) can efficiently lower Ang II and increase Ang-(1-7), and whether rACE2 can prevent hypertension caused by Ang II infusion as a result of systemic versus local mechanisms of ACE2 activity amplification.
rACE2 was infused via osmotic minipumps for three days in conscious mice or acutely in anesthetized mice. rACE2 caused a dose-dependent increase in serum ACE2 activity but had no effect on kidney or cardiac ACE2 activity. Following Ang II infusion (40pmol/min), rACE2 (1mg/kg/d) resulted in normalization of systolic blood pressure and plasma Ang II. In acute studies, rACE2 (1mg/kg) prevented the rapid hypertensive effect of Ang II (0.2mg/kg), and this was associated with both a decrease in Ang II and an increase in Ang-(1-7) in plasma. Moreover, during infusion of Ang II, the effect of rACE2 on blood pressure was unaffected by a specific Ang-(1-7) receptor blocker, A779 (0.2 mg/kg), and infusing supra-physiologic levels of Ang-(1-7) (0.2 mg/kg) had no effect on blood pressure.
We conclude that during Ang II infusion rACE2 effectively degrades Ang II and in the process normalizes blood pressure. The mechanism of rACE2 action results from an increase in systemic, not tissue, ACE2 activity and the lowering of plasma Ang II rather than the attendant increase in Ang-(1-7). Increasing ACE2 activity may provide a new therapeutic target in states of Ang II over-activity by enhancing its degradation, an approach that differs from the current focus on blocking Ang II formation and action.
doi:10.1161/HYPERTENSIONAHA.109.138420
PMCID: PMC2827767  PMID: 19948988
ACE2; Soluble; Recombinant; Angiotensin II; Angiotensin-(1-7)
20.  COSEHC global vascular risk management quality improvement program: rationale and design 
Background:
The Consortium for Southeastern Hypertension Control (COSEHC) promotes global risk factor management in patients with metabolic syndrome. The COSEHC Global Vascular Risk Management Study (GVRM) intends to quantify these efforts on long-term patient outcomes. The objectives of this study were to present baseline demographics of patients enrolled in the GVRM, calculate a modified COSEHC risk score using 11 variables (COSEHC-11), and compare it with the original COSEHC-17 and Framingham, Prospective Cardiovascular Münster (PROCAM), and Systemic Coronary Risk Evaluation (SCORE) risk scores.
Methods:
Deidentified electronic medical records of enrolled patients were used to calculate the risk scores. The ability of the COSEHC-11 score to predict the COSEHC-17 score was assessed by regression analysis. Raw risk scores were converted to probability estimates of fatal coronary heart disease (CHD) and compared with predicted risks from other algorithms.
Results:
Of the 177,404 patients enrolled, 43,676 had data for all 11 variables. The COSEHC-11 score (mean ± standard deviation) of these 43,676 patients was 31.75 ± 11.66, implying a five-year fatal CHD risk of 1.4%. The COSEHC-11 score was highly predictive of the COSEHC-17 score (R2 = 0.93; P < 0.0001) and correlated well with the SCORE algorithm.
Conclusion:
The COSEHC-11 risk score is statistically similar to the COSEHC-17 risk score and should be a viable tool for evaluating its ability to predict five-year cardiovascular mortality in the coming years.
doi:10.2147/VHRM.S13746
PMCID: PMC3169675  PMID: 21931496
cardiovascular risk; electronic medical records; metabolic syndrome
21.  Reduced circulating levels of angiotensin‐(1–7) in systemic sclerosis: a new pathway in the dysregulation of endothelial‐dependent vascular tone control 
Annals of the Rheumatic Diseases  2007;66(10):1305-1310.
Objective
Systemic sclerosis (SSc) impairs endothelium‐dependent vasodilatation. Among angiotensin I (Ang I)‐derived compounds, vasoconstrictor angiotensin II (Ang II) and vasodilator angiotensin‐(1–7) (Ang‐(1–7)), cleaved from ACE and neutral endopeptidase (NEP) 24.11, respectively, play an important role in vascular tone regulation. Ang‐(1–7) may act independently or by activating other vasodilating molecules, such as nitric oxide (NO) or prostaglandin I2 (PGI2). Our aim was to assess, in patients with SSc, circulating levels of Ang I, Ang II and Ang‐(1–7), with their metabolising enzymes ACE and NEP, and levels of NO and PGI2, and to correlate them to the main characteristics of SSc.
Methods
Levels of Ang I, Ang II, Ang‐(1–7), NEP, ACE, NO and PGI2 were measured in 32 patients with SSc, who were also assessed for humoral and clinical characteristics, and 55 controls.
Results
Plasma Ang I, Ang II and Ang‐(1–7) levels were lower in patients with SSc than in controls (p<0.001in all cases). When Ang II and Ang‐(1–7) levels were expressed as a function of the available Ang I, lower Ang‐(1–7) levels in patients with SSc than in controls were confirmed (p<0.001), while no difference was found for Ang II levels. In patients with SSc, the Ang II/Ang‐(1–7) ratio indicated a prevalence of Ang II over Ang‐(1–7), while in controls Ang‐(1–7) was prevalent (p<0.001). Levels of ACE, NEP, NO and PGI2 were lower in patients with SSc than in controls (p<0.05 in all cases).
Conclusion
In patients with SSc, prevalence of the vasoconstricting Ang II over the vasodilator Ang‐(1–7) suggests a dysfunction of the angiotensin‐derived cascade that may contribute to dysregulation of vascular tone.
doi:10.1136/ard.2006.064493
PMCID: PMC1994289  PMID: 17360781
angiotensin (1–7); angiotensin converting enzyme; endothelium; neutral endopeptidase; systemic sclerosis
22.  Nebivolol Reduces Proteinuria and Renal NADPH Oxidase-Generated Reactive Oxygen Species in the Transgenic Ren2 Rat 
American Journal of Nephrology  2009;30(4):354-360.
Background/Aims
Renin-angiotensin-aldosterone system (RAAS) and sympathetic nervous system activation are crucial in the pathogenesis of hypertension, cardiovascular and renal disease. NADPH oxidase-mediated increases in reactive oxygen species (ROS) are an important mediator for RAAS-induced cardiovascular and renal injury. Increased levels of ROS can diminish the bioactivity of nitric oxide (NO), a critical modulator of RAAS effects on the kidney. Thereby, we hypothesized that in vivo nebivolol therapy in a rodent model of activated RAAS would attenuate glomerular damage and proteinuria through its actions to reduce NADPH oxidase activity/ROS and increase bioavailable NO.
Methods
We utilized the transgenic Ren2 rat which displays heightened tissue RAAS, hypertension, and proteinuria. Ren2 rats (6–9 weeks of age) and age-matched Sprague-Dawley littermates were treated with nebivolol 10 mg/kg/day (osmotic mini-pump) for 21 days.
Results
Ren2 rats exhibited increases in systolic blood pressure, proteinuria, kidney cortical tissue total NADPH oxidase activity and subunits (Rac1, p67phox, and p47phox), ROS and 3-nitrotyrosine, as well as reductions in podocyte protein markers; each of these parameters improved with nebivolol treatment along with increases in renal endothelial NO synthase expression.
Conclusions
Our data suggest that nebivolol improves proteinuria through reductions in renal RAAS-mediated increases in NADPH oxidase/ROS and increases in bioavailable NO.
doi:10.1159/000229305
PMCID: PMC2814025  PMID: 19609077
Nebivolol; Proteinuria; NADPH oxidase; Reactive oxygen species
23.  Mineralocorticoid Receptor Antagonism Attenuates Vascular Apoptosis and Injury via Rescuing Akt Activation 
Hypertension  2008;53(2):158-165.
Emerging evidence indicates that mineralocorticoid receptor (MR) blockade reduces the risk of cardiovascular events beyond those predicted by its blood pressure (BP)-lowering actions; however, the underlying mechanisms remain unclear. To investigate whether protection elicited by MR blockade is through attenuation of vascular apoptosis and injury, independently of BPlowering, we administered a low dose of the MR antagonist spironolactone or vehicle for 21 days to hypertensive transgenic Ren2 rats with elevated plasma aldosterone levels. Although Ren2 rats developed higher systolic BPs compared to Sprague-Dawley (SD) littermates, low dose spironolactone treatment did not reduce systolic BP compared with untreated Ren2 rats. Ren2 rats exhibited vascular injury as evidenced by increased apoptosis, hemidesmosome-like structure loss, mitochondrial abnormalities, and lipid accumulation compared with SD, and these abnormalities were attenuated by MR antagonism. Protein kinase B (Akt) activation is critical to vascular homeostasis via regulation of cell survival and expression of apoptotic genes. Akt serine473 phosphorylation was impaired in Ren2 aortas, and restored with MR antagonism. In vivo MR antagonist treatment promoted anti-apoptotic effects by increasing phosphorylation of BAD serine136 and expression of Bcl-2 and Bcl-xL, decreasing cytochrome c release and BAD expression, and suppressing caspase-3 activation. Furthermore, MR antagonism substantially reduced the elevated NADPH oxidase activity and lipid peroxidation, expression of angiotensin II, angiotensin type 1 receptor and MR, in Ren2 vasculature. These results demonstrate that MR antagonism protects the vasculature from aldosterone-induced vascular apoptosis and structural injury via rescuing Akt activation, independent of BP effects.
doi:10.1161/HYPERTENSIONAHA.108.121954
PMCID: PMC2692525  PMID: 19114643
Aldosterone; Oxidative Stress; Akt Activation; Vascular Apoptosis and Injury
24.  New angiotensins 
Accumulation of a large body of evidence during the past two decades testifies to the complexity of the renin–angiotensin system (RAS). The incorporation of novel enzymatic pathways, resulting peptides, and their corresponding receptors into the biochemical cascade of the RAS provides a better understanding of its role in the regulation of cardiovascular and renal function. Hence, in recent years, it became apparent that the balance between the two opposing effector peptides, angiotensin II and angiotensin-(1–7), may have a pivotal role in determining different cardiovascular pathophysiologies. Furthermore, our recent studies provide evidence for the functional relevance of a newly discovered rat peptide, containing two additional amino acid residues compared to angiotensin I, first defined as proangiotensin-12 [angiotensin-(1–12)]. This review focuses on angiotensin-(1–7) and its important contribution to cardiovascular function and growth, while introducing angiotensin-(1–12) as a potential novel angiotensin precursor.
doi:10.1007/s00109-008-0340-4
PMCID: PMC2713173  PMID: 18437333
(3–6): Renin–angiotensin system; Novel angiotensins; Angiotensin-(1–7); Angiotensin-(1–12); Hypertension; Cardiovascular growth
25.  Activation of Local Chorionic Villi Angiotensin II Levels But Not Angiotensin (1–7) in Preeclampsia 
Hypertension  2008;51(4):1066-1072.
The chorionic villi in the placenta are responsible for the regulation of fetal oxygen and nutrient transport. Although the peripheral renin-angiotensin system is activated during normal pregnancy, the regulation of the local chorionic villi renin-angiotensin system remains unknown. Therefore, placental chorionic villous tissue was collected from nulliparous third-trimester normotensive or preeclamptic subjects and was analyzed for angiotensin peptide content, angiotensinogen, renin, angiotensin-converting enzyme (ACE), ACE2, neprilysin, angiotensin II type 1 (AT1), angiotensin II type 2, Mas receptor mRNAs, and angiotensin receptor density and subtype. Angiotensin II in chorionic villi was significantly higher in preeclamptic subjects, whereas angiotensin (1–7) was not different. Angiotensinogen and AT1 receptor gene expression was significantly higher in preeclamptic subjects. No differences were observed in renin, ACE, ACE2, or neprilysin gene expression. Mas receptor mRNA in preeclamptic subjects was decreased. The AT1 receptor was the predominant receptor subtype in normal and preeclamptic chorionic villi. There was no difference in the density of the AT1, angiotensin II type 2, and angiotensin (1–7) receptors. These results indicate that enhanced chorionic villous expression of angiotensin II may result from increased angiotensinogen. Elevated angiotensin II, acting through the AT1 receptor, may favor vasoconstriction in placental chorionic villi and contribute to impaired fetal blood flow and decreased fetal nutrition observed during preeclampsia.
doi:10.1161/HYPERTENSIONAHA.107.103861
PMCID: PMC2705753  PMID: 18259034
preeclampsia; renin angiotensin system; pregnancy; placenta; angiotensin receptors; Mas receptor; angiotensin (1–7)

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