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1.  Hemodynamic and Hormonal Patterns of Untreated Essential Hypertension in Men and Women 
Knowledge of hemodynamic factors accounting for the development of hypertension should help to tailor therapeutic approaches and improves blood pressure control. Few data exist regarding sex differences of hemodynamic factors contributing to hypertension progression among untreated non-diabetic prehypertensive (PreHyp), Stage I and II subjects as define by the Joint National Commission-7 guidelines (JNC-7).
We utilized non-invasive impedance cardiography, applanation tonometry, and plasma measures of angiotensin II, angiotensin-(1-7), serum aldosterone, hs-C reactive protein, and cytokine biomarkers of inflammation to characterize the hemodynamic and hormonal profile of 100 untreated hypertensive subjects (39 females).
Despite there being no differences between females and males in terms of office blood pressure, heart rate, and body mass index, males demonstrated lower values of pulse pressure, systemic vascular resistance, brachial artery pulse wave velocity, and augmentation index. In each of the three hypertension categories, the increased blood pressure in males was associated with significant augmentations in stroke volume and cardiac output when compared to females. Sex related hemodynamic differences were associated in females with higher plasma levels of leptin, hs-C-reactive protein, plasma angiotensin II, and serum aldosterone and no differences in the serum concentrations of cytokinins. In women but not men, hs-C reactive protein correlated with plasma concentrations of TGF-β1 and body weight, in addition, plasma TGF-β1 correlated with levels of serum VCAM-1.
The impact of sex differences in the hemodynamic factors accounting for the elevation in arterial pressure in essential hypertensive subjects has been poorly characterized or this information is not available. We suggest that this gap in knowledge may adversely influence choices of drug-treatment since our study shows for the first time significant differences in the hemodynamic and hormonal mechanisms accounting for the increased blood pressure in women compared to men.
PMCID: PMC4280797  PMID: 24280597
angiotensin II; blood pressure; cardiac output; central aortic pressure; essential hypertension; inflammation; vascular disease
2.  Chymase Mediates Angiotensin-(1-12) Metabolism in Normal Human Hearts 
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.
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.
PMCID: PMC3605226  PMID: 23312967
ACE2; cardiac myocytes; angiotensin converting enzyme inhibitors; angiotensin II; heart disease; proangiotensin 12; angiotensin-(1-7)
3.  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.
PMCID: PMC3724686  PMID: 23901282
cardiovascular risk; coronary heart disease; dyslipidemia; electronic medical records; hypertension; metabolic syndrome
4.  Nebivolol Improves Insulin Sensitivity in the TGR(Ren2)27 Rat 
Metabolism: clinical and experimental  2011;60(12):1757-1766.
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.
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.
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.
PMCID: PMC3170670  PMID: 21640361
Insulin resistance; oxidative stress; skeletal muscle
5.  ACE2: Angiotensin II/Angiotensin-(1-7) balance in cardiorenal injury 
Our current recognition of the renin-angiotensin system is more convoluted than originally thought due to the discovery of multiple novel enzymes, peptides, and receptors inherent to this interactive biochemical cascade. Over the last decade angiotensin converting enzyme 2 (ACE2) has emerged as a key player in the pathophysiology of hypertension and cardiovascular and renal disease due to its pivotal role in metabolizing vasoconstrictive/hypertrophic/proliferative angiotensin II into favorable angiotensin-(1-7). This review addresses a considerable advancement in research on the role of tissue ACE2 in development and progression of hypertension and cardiorenal injury. We also summarize the results from recent clinical and experimental studies suggesting that serum or urine soluble ACE2 may serve as a novel biomarker or independent risk factor relevant for diagnosis and prognosis of cardiorenal disease. Recent proceedings on novel therapeutic approaches to enhance ACE2/angiotensin-(1-7) axis are also reviewed.
PMCID: PMC4286874  PMID: 24510672
6.  Salt Loading Exacerbates Diastolic Dysfunction and Cardiac Remodeling in Young Female Ren2 Rats 
Metabolism: clinical and experimental  2013;62(12):10.1016/j.metabol.2013.08.010.
Recent data would suggest pre-menopausal insulin resistant women are more prone to diastolic dysfunction than men, yet it is unclear why. We and others have reported that transgenic (mRen2)27 (Ren2) rats overexpressing the murine renin transgene are insulin resistant due to oxidative stress in insulin sensitive tissues. As increased salt intake promotes inflammation and oxidative stress, we hypothesized that excess dietary salt would promote diastolic dysfunction in transgenic females under conditions of excess tissue Ang II and circulating aldosterone levels.
For this purpose we evaluated cardiac function in young female Ren2 rats or age-matched Sprague-Dawley (SD) littermates exposed to a high (4%) salt or normal rat chow intake for three weeks.
Compared to SD littermates, at 10 weeks of age, female Ren2 rats fed normal chow showed elevations in left ventricular (LV) systolic pressures, LV and cardiomyocyte hypertrophy, and displayed reductions in LV initial filling rate accompanied by increases in 3-nitrotyrosine content as a marker of oxidant stress. Following 3 weeks of a salt diet, female Ren2 rats exhibited no further changes in LV systolic pressure, insulin resistance, or markers of hypertrophy but exaggerated increases in type 1 collagen, 3-nitrotryosine content, and diastolic dysfunction. These findings occurred in parallel with ultrastructural findings of pericapillary fibrosis, increased LV remodeling, and mitochondrial biogenesis.
These data suggest that a diet high in salt in hypertensive female Ren2 rats promotes greater oxidative stress, maladaptive LV remodeling, fibrosis, and associated diastolic dysfunction without further changes in LV systolic pressure or hypertrophy.
PMCID: PMC3833978  PMID: 24075738
angiotensin II; TG (mRen2) 27 rat; Diastolic Dysfunction; Cardiac Remodeling; Oxidative Stress; Perivascular Fibrosis
7.  Clinical utility of fixed-dose combinations in hypertension: evidence for the potential of nebivolol/valsartan 
Despite significant advances in pharmacologic approaches to treat hypertension during the last decades, hypertension- and hypertension-related organ damage are still a high health and economic burden because a large proportion of patients with hypertension do not achieve optimal blood pressure control. There is now general agreement that combination therapy with two or more antihypertensive drugs is required for targeted blood pressure accomplishment and reduction of global cardiovascular risk. The goals of combination therapies are to reduce long-term cardiovascular events by targeting different mechanism underlying hypertension and target organ disease, to block the counterregulatory pathways activated by monotherapies, to improve tolerability and decrease the adverse effects of up-titrated single agents, and to increase persistence and adherence with antihypertensive therapy. Multiple clinical trials provide evidence that fixed-dose combinations in a single pill offer several advantages when compared with loose-dose combinations. This review discusses the advances in hypertension control and associated cardiovascular disease as they relate to the prospect of combination therapy targeting a third-generation beta (β) 1-adrenergic receptor (nebivolol) and an angiotensin II receptor blocker (valsartan) in fixed-dose single-pill formulations.
PMCID: PMC4251532  PMID: 25473311
blood pressure control; hypertension; β1-adrenergic receptor; renin angiotensin system; fixed-dose combination therapy; nebivolol; valsartan
8.  Statin Treatment in Hypercholesterolemic Men Does Not Attenuate Angiotensin II-Induced Venoconstriction 
PLoS ONE  2014;9(9):e103909.
Experimental studies suggested that statins attenuate vascular AT1 receptor responsiveness. Moreover, the augmented excessive pressor response to systemic angiotensin II infusions in hypercholesterolemic patients was normalized with statin treatment. In 12 hypercholesterolemic patients, we tested the hypothesis that statin treatment attenuates angiotensin II-mediated vasoconstriction in hand veins assessed by a linear variable differential transducer. Subjects ingested daily doses of either atorvastatin (40 mg) or positive control irbesartan (150 mg) for 30 days in a randomized and cross-over fashion. Ang II–induced venoconstriction at minute 4 averaged 59%±10% before and 28%±9% after irbesartan (mean ± SEM; P<0.05) compared to 65%±11% before and 73%±11% after 30 days of atorvastatin treatment. Plasma angiotensin levels increased significantly after irbesartan treatment (Ang II: 17±22 before vs 52±40 pg/mL after [p = 0.048]; Ang-(1–7): 18±10 before vs 37±14 pg/mL after [p = 0.002]) compared to atorvastatin treatment (Ang II: 9±4 vs 11±10 pg/mL [p = 0.40]; Ang-(1–7): 24±9 vs 32±8 pg/mL [p = 0.023]). Our study suggests that statin treatment does not elicit major changes in angiotensin II-mediated venoconstriction or in circulating angiotensin II levels whereas angiotensin-(1–7) levels increased modestly. The discrepancy between local vascular and systemic angiotensin II responses might suggest that statin treatment interferes with blood pressure buffering reflexes.
Trial Registration NCT00154024
PMCID: PMC4179232  PMID: 25264877
9.  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.
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.
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.
PMCID: PMC2853260  PMID: 19958778
hypertension; renin-angiotensin-aldosterone system (RAAS) inhibition; angiotensin II; angiotensin-(1–7); combination therapy; aliskiren; prorenin/renin receptor
11.  COSEHC global vascular risk management quality improvement program: rationale and design 
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.
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.
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.
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.
PMCID: PMC3169675  PMID: 21931496
cardiovascular risk; electronic medical records; metabolic syndrome
12.  Attenuation of hypertension-mediated glomerulosclerosis in conjunction with increased angiotensin (1–7) 
Controversy exists as to whether angiotensin (1–7) (Ang (1–7)) acts as a protective hormone against renal injury.
We compared the degree of improvement of hypertensive nephropathy following 8 weeks’ treatment with either the angiotensin II receptor type 1 antagonist olmesartan medoxomil or the cardioselective beta blocker atenolol in 8-week-old spontaneously hypertensive rats (SHRs).
Both treatment regimens reduced mean blood pressure in a similar fashion, while bradycardia was present only in atenolol-treated SHRs. The heart weight:body weight ratio fell more in SHRs medicated with olmesartan versus those receiving atenolol. These changes were associated with increases in plasma Ang II in SHRs given the angiotensin II receptor blocker. At the end of treatment, plasma Ang (1–7) was higher in the olmesartan than atenolol or vehicle groups. The glomerular sclerosis (GS) index was lowered by olmesartan and atenolol compared with the vehicle group. While both olmesartan and atenolol attenuated renal perivascular collagen deposition (PVCD), the greatest effect was observed in SHRs receiving olmesartan. Elevations in plasma Ang (1–7) correlated negatively with reductions in GS or PVCD index, respectively.
While control of blood pressure remains a critical factor in the prevention of hypertensive nephropathy, Ang (1–7) may play a substantial role in preventing the structural changes in glomerulus through its effect on regulations of blood pressure and renal function.
PMCID: PMC4157897  PMID: 22089474
angiotensin (1–7); angiotensin II; angiotensin-converting enzyme 2; angiotensin receptor blockers; angiotensin receptors; atenolol; blood pressure; glomerulosclerosis
13.  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.
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.
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.
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).
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.
PMCID: PMC1994289  PMID: 17360781
angiotensin (1–7); angiotensin converting enzyme; endothelium; neutral endopeptidase; systemic sclerosis
14.  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
15.  Predominance of AT1 Blockade Over Mas–Mediated Angiotensin-(1–7) Mechanisms in the Regulation of Blood Pressure and Renin–Angiotensin System in mRen2.Lewis Rats 
American Journal of Hypertension  2013;26(5):583-590.
We investigated whether the antihypertensive actions of the angiotensin II (Ang II) receptor (AT1-R) blocker, olmesartan medoxomil, may in part be mediated by increased Ang-(1–7) in the absence of significant changes in plasma Ang II.
mRen2.Lewis congenic hypertensive rats were administered either a vehicle (n = 14) or olmesartan (0.5mg/kg/day; n = 14) by osmotic minipumps. Two weeks later, rats from both groups were further randomized to receive either the mas receptor antagonist A-779 (0.5mg/kg/day; n = 7 per group) or its vehicle (n = 7 per group) for the next 4 weeks. Blood pressure was monitored by telemetry, and circulating and tissue components of the renin–angiotensin system (RAS) were measured at the completion of the experiments.
Antihypertensive effects of olmesartan were associated with an increase in plasma renin concentration, plasma Ang I, Ang II, and Ang-(1–7), whereas serum aldosterone levels and kidney Ang II content were reduced. Preserved Ang-(1–7) content in kidneys was associated with increases of ACE2 protein but not activity and no changes on serum and kidney ACE activity. There was no change in cardiac peptide levels after olmesartan treatment. The antihypertensive effects of olmesartan were not altered by concomitant administration of the Ang-(1–7) receptor antagonist except for a mild further increase in plasma renin concentration.
Our study highlights the independent regulation of RAS among plasma, heart, and kidney tissue in response to AT1-R blockade. Ang-(1–7) through the mas receptor does not mediate long-term effects of olmesartan besides counterbalancing renin release in response to AT1-R blockade.
PMCID: PMC3657483  PMID: 23459599
angiotensin II; angiotensin-(1–7); AT1 receptor; blood pressure; heart; hypertension; kidney; mas receptor; olmesartan.
16.  Chymase Mediates Injury and Mitochondrial Damage in Cardiomyocytes during Acute Ischemia/Reperfusion in the Dog 
PLoS ONE  2014;9(4):e94732.
Cardiac ischemia and reperfusion (I/R) injury occurs because the acute increase in oxidative/inflammatory stress during reperfusion culminates in the death of cardiomyocytes. Currently, there is no drug utilized clinically that attenuates I/R injury in patients. Previous studies have demonstrated degranulation of mast cell contents into the interstitium after I/R. Using a dog model of I/R, we tested the role of chymase, a mast cell protease, in cardiomyocyte injury using a specific oral chymase inhibitor (CI). 15 adult mongrel dogs had left anterior descending artery occlusion for 60 min and reperfusion for 100 minutes. 9 dogs received vehicle and 6 were pretreated with a specific CI. In vivo cardiac microdialysis demonstrated a 3-fold increase in interstitial fluid chymase activity in I/R region that was significantly decreased by CI. CI pretreatment significantly attenuated loss of laminin, focal adhesion complex disruption, and release of troponin I into the circulation. Microarray analysis identified an I/R induced 17-fold increase in nuclear receptor subfamily 4A1 (NR4A1) and significantly decreased by CI. NR4A1 normally resides in the nucleus but can induce cell death on migration to the cytoplasm. I/R caused significant increase in NR4A1 protein expression and cytoplasmic translocation, and mitochondrial degradation, which were decreased by CI. Immunohistochemistry also revealed a high concentration of chymase within cardiomyocytes after I/R. In vitro, chymase added to culture HL-1 cardiomyocytes entered the cytoplasm and nucleus in a dynamin-dependent fashion, and promoted cytoplasmic translocation of NR4A1 protein. shRNA knockdown of NR4A1 on pre-treatment of HL-1 cells with CI significantly decreased chymase-induced cell death and mitochondrial damage. These results suggest that the beneficial effects of an orally active CI during I/R are mediated in the cardiac interstitium as well as within the cardiomyocyte due to a heretofore-unrecognized chymase entry into cardiomyocytes.
PMCID: PMC3986229  PMID: 24733352
17.  Salt Loading Promotes Kidney Injury via Fibrosis in Young Female Ren2 Rats 
Cardiorenal Medicine  2014;4(1):43-52.
It is increasingly recognized that there is sexual dimorphism in kidney disease progression; however, this disparity is lost in the presence of diabetes where women progress at a similar rate to men. The renin-angiotensin-aldosterone system (RAAS) is known to regulate diabetes-induced kidney injury, and recent literature would suggest that gender differences exist in RAAS-dependent responses in the kidney. In this regard, these gender differences may be overcome by excessive salt intake. Thereby, we hypothesized that salt would promote proteinuria in transgenic female rats under conditions of excess tissue angiotensin (Ang) II and circulating aldosterone.
Materials and Methods
We utilized young female transgenic (mRen2)27 (Ren2) rats and Sprague-Dawley (SD) littermates and fed a high-salt diet (4%) over 3 weeks.
Compared to SD and Ren2 controls, female Ren2 rats fed a high-salt diet displayed increases in proteinuria, periarterial and interstitial fibrosis as well as ultrastructural evidence of basement membrane thickening, loss of mitochondrial elongation, mitochondrial fragmentation and attenuation of basilar canalicular infoldings. These findings occurred temporally with increases in transforming growth factor-β but not indices of oxidant stress.
Our current data suggest that a diet high in salt promotes progressive kidney injury as measured by proteinuria and fibrosis associated with transforming growth factor-β under conditions of excess tissue Ang II and circulating aldosterone.
PMCID: PMC4025048  PMID: 24847333
Angiotensin II; Transgenic (mRen2)27 rat; Proteinuria; Fibrosis; Reactive oxygen species

18.  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.
PMCID: PMC3576205  PMID: 23232645
angiotensin-(1–12); aliskiren; arterial remodeling; direct renin inhibitors; urinary protein; valsartan
19.  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.
PMCID: PMC3808369  PMID: 24204720
20.  Restoration of the Blood Pressure Circadian Rhythm By Direct Renin Inhibition and Blockade of Angiotensin II Receptors in mRen2.Lewis hypertensive rats 
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.
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.
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.
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.
PMCID: PMC3798054  PMID: 22222314
angiotensin II; aliskiren; blood pressure; circadian rhythm; direct renin inhibitors; hypertension; valsartan
21.  Low glial angiotensinogen improves body habitus, diastolic function, and exercise tolerance in aging male rats 
Cardiovascular endocrinology  2012;1(3):49-58.
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.
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.
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.
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.
PMCID: PMC3685869  PMID: 23795309
brain; cardiac aging; diastolic dysfunction; renin–angiotensin system; SERCA2; tissue Doppler
22.  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.
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)).
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.
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.
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.
PMCID: PMC3739444  PMID: 20300067
angiotensin-converting enzyme 2; angiotensin II; angiotensin-(1-7); blood pressure; cardiac hypertrophy; hypertension
23.  Decreased cardiac Ang-(1–7) is associated with salt-induced cardiac remodeling and dysfunction 
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).
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).
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.
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).
PMCID: PMC3733447  PMID: 19946038
ACE2; angiotensin II; angiotensin-(1–7); hypertension; salt
24.  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.
PMCID: PMC3348429  PMID: 22465166
Aldosterone; Combination; Renin inhibition; AT1R blockade; Podocyte; β-NAG; Oxidative Stress
25.  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.
PMCID: PMC2265080  PMID: 17692319
Hypercholesterolemia; LDL; monocyte; CD34; angiotensin; hematopoietic stem cell; hematopoiesis

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