Sarfaraz Ahmad

Chymase-Dependent Generation of Angiotensin II from Angiotensin-(1-12) in Human Atrial Tissue

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).

An evolving story of angiotensin-II-forming pathways in rodents and humans.

Lessons learned from the characterization of the biological roles of Ang-(1-7) [angiotensin-(1-7)] in opposing the vasoconstrictor, proliferative and prothrombotic actions of AngII (angiotensin II) created an underpinning for a more comprehensive exploration of the multiple pathways by which the RAS (renin-angiotensin system) of blood and tissues regulates homoeostasis and its altered state in disease processes. The present review summarizes the progress that has been made in the novel exploration of intermediate shorter forms of angiotensinogen through the characterization of the expression and functions of the dodecapeptide Ang-(1-12) [angiotensin-(1-12)] in the cardiac production of AngII. The studies reveal significant differences in humans compared with rodents regarding the enzymatic pathway by which Ang-(1-12) undergoes metabolism. Highlights of the research include the demonstration of chymase-directed formation of AngII from Ang-(1-12) in human left atrial myocytes and left ventricular tissue, the presence of robust expression of Ang-(1-12) and chymase in the atrial appendage of subjects with resistant atrial fibrillation, and the preliminary observation of significantly higher Ang-(1-12) expression in human left atrial appendages.

Advances in the renin angiotensin system focus on angiotensin-converting enzyme 2 and angiotensin-(1-7).

The contribution of the renin angiotensin system to physiology and pathology is undergoing a rapid reconsideration of its mechanisms from emerging new concepts implicating angiotensin-converting enzyme 2 and angiotensin-(1-7) as new elements negatively influencing the vasoconstrictor, trophic, and pro-inflammatory actions of angiotensin II. This component of the system acts to oppose the vasoconstrictor and proliferative effects on angiotensin II through signaling mechanisms mediated by the mas receptor. In addition, a reduced expression of the vasodepressor axis composed by angiotensin-converting enzyme 2 and angiotensin-(1-7) may contribute to the expression of essential hypertension, the remodeling of heart and renal function associated with this disease, and even the physiology of pregnancy and the development of eclampsia.

ACE2: Angiotensin II/Angiotensin-(1–7) Balance in Cardiac and Renal 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 in 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 the considerable advancement in research on the role of tissue ACE2 in the development and progression of hypertension and cardiac and renal injury. We 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. We also review recent proceedings on novel therapeutic approaches to enhance ACE2/angiotensin-(1–7) axis.

Cardiac Kallikrein-Kinin System Is Upregulated in Chronic Volume Overload and Mediates an Inflammatory Induced Collagen Loss

Background The clinical problem of a “pure volume overload” as in isolated mitral or aortic regurgitation currently has no documented medical therapy that attenuates collagen loss and the resultant left ventricular (LV) dilatation and failure. Here, we identify a potential mechanism related to upregulation of the kallikrein-kinin system in the volume overload of aortocaval fistula (ACF) in the rat. Methodology/Principal Findings LV interstitial fluid (ISF) collection, hemodynamics, and echocardiography were performed in age-matched shams and 4 and 15 wk ACF rats. ACF rats had LV dilatation and a 2-fold increase in LV end-diastolic pressure, along with increases in LV ISF bradykinin, myocardial kallikrein and bradykinin type-2 receptor (BK2R) mRNA expression. Mast cell numbers were increased and interstitial collagen was decreased at 4 and 15 wk ACF, despite increases in LV ACE and chymase activities. Treatment with the kallikrein inhibitor aprotinin preserved interstitial collagen, prevented the increase in mast cells, and improved LV systolic function at 4 wk ACF. To establish a cause and effect between ISF bradykinin and mast cell-mediated collagen loss, direct LV interstitial bradykinin infusion in vivo for 24 hrs produced a 2-fold increase in mast cell numbers and a 30% decrease in interstitial collagen, which were prevented by BK2R antagonist. To further connect myocardial stretch with cellular kallikrein-kinin system upregulation, 24 hrs cyclic stretch of adult cardiomyocytes and fibroblasts produced increased kallikrein, BK2R mRNA expressions, bradykinin protein and gelatinase activity, which were all decreased by the kallikrein inhibitor-aprotinin. Conclusions/Significance A pure volume overload is associated with upregulation of the kallikrein-kinin system and ISF bradykinin, which mediates mast cell infiltration, extracellular matrix loss, and LV dysfunction–all of which are improved by kallikrein inhibition. The current investigation provides important new insights into future potential medical therapies for the volume overload of aortic and mitral regurgitation.

Uptake and Metabolism of the Novel Peptide Angiotensin-(1-12) by Neonatal Cardiac Myocytes

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.

Salt-Induced Renal Injury in Spontaneously Hypertensive Rats: Effects of Nebivolol

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.

Chymase Mediates Angiotensin-(1-12) Metabolism in Normal Human Hearts

Identification of angiotensin-(1-12) [Ang-(1-12)] in forming angiotensin II (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. Reverse phase HPLC characterized the products of (125)I-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 (125)I-Ang-(1-12) remained intact, whereas exclusion of chymostatin from the inhibitor cocktail led to significant conversion of Ang-(1-12) into Ang II. In addition, chymase-mediated hydrolysis of (125)I-Ang I was higher compared with Ang-(1-12). Negligible Ang-(1-12) hydrolysis occurred by ACE, ACE2, and neprilysin. A high chymase activity was detected for both (125)I-Ang-(1-12) and (125)I-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.

Hemodynamic and Hormonal Changes to Dual Renin–Angiotensin System Inhibition in Experimental Hypertension

We examined the antihypertensive effects of valsartan, aliskiren, or both drugs combined on circulating, cardiac, and renal components of the renin–angiotensin system in congenic mRen2.Lewis hypertensive rats assigned to: vehicle (n=9), valsartan (via drinking water, 30 mg/kg per day; n=10), aliskiren (SC by osmotic mini-pumps, 50 mg/kg per day; n=10), or valsartan (30 mg/kg per day) combined with aliskiren (50 mg/kg per day; n=10). Arterial pressure and heart rate were measured by telemetry before and during 2 weeks of treatment; trunk blood, heart, urine, and kidneys were collected for measures of renin–angiotensin system 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 angiotensin (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 whereas 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 end points 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 attributable to loss of tubuloglomerular feedback by Ang II.

Characterization of the cardiac renin angiotensin system in oophorectomized and estrogen-replete mRen2.Lewis rats.

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.