Activation of the Renin-Angiotensin-Aldosterone System Is Attenuated in Hypertensive Compared with Normotensive Pregnancy.

Hypertension during pregnancy increases the risk of adverse maternal and fetal outcomes, but the mechanisms of pregnancy hypertension are not precisely understood. Elevated plasma renin activity and aldosterone concentrations play an important role in the normal physiologic adaptation to pregnancy. These effectors are reduced in patients with pregnancy hypertension, creating an opportunity to define the features of the renin-angiotensin-aldosterone system (RAAS) that are characteristic of this disorder. In the current study, we used a novel LC-MS/MS-based methodology to develop comprehensive profiles of RAAS peptides and effectors over gestation in a cohort of 74 pregnant women followed prospectively for the development of gestational hypertension and pre-eclampsia (HYP, 27 patients) versus those remaining normotensive (NT, 47 patients). In NT pregnancy, the plasma renin activity surrogate, (PRA-S, calculated from the sum of Angiotensin I + Angiotensin II) and aldosterone concentrations significantly increased from the first to the third trimester, accompanied by a modest increase in the concentrations of angiotensin peptide metabolites. In contrast, in HYP pregnancies, PRA-S and angiotensin peptides were largely unchanged over gestation, and third-trimester aldosterone concentrations were significantly lower compared with those in NT pregnancies. The results indicated that the predominant features of pregnancies that develop HYP are stalled or waning activation of the RAAS in the second half of pregnancy (accompanied by unchanging levels of angiotensin peptides) and the attenuated secretion of aldosterone.

Combined angiotensin-converting enzyme and aminopeptidase inhibition for treatment of experimental ventilator-induced lung injury in mice.

Introduction: Ventilator-induced lung injury (VILI) may aggravate critical illness. Although angiotensin-converting enzyme (ACE) inhibition has beneficial effects in ventilator-induced lung injury, its clinical application is impeded by concomitant hypotension. We hypothesized that the aminopeptidase inhibitor ALT-00 may oppose the hypotension induced by an angiotensin-converting enzyme inhibitor, and that this combination would activate the alternative renin-angiotensin system (RAS) axis to counteract ventilator-induced lung injury. Methods: In separate experiments, C57BL/6 mice were mechanically ventilated with low (LVT, 6 mL/kg) and high tidal volumes (HVT, 30 mL/kg) for 4 h or remained unventilated (sham). High tidal volume-ventilated mice were treated with lisinopril (0.15 µg/kg/min) ± ALT-00 at 2.7, 10 or 100 µg/kg/min. Blood pressure was recorded at baseline and after 4 h. Lung histology was evaluated for ventilator-induced lung injury and the angiotensin (Ang) metabolite profile in plasma (equilibrium levels of Ang I, Ang II, Ang III, Ang IV, Ang 1-7, and Ang 1-5) was measured with liquid chromatography tandem mass spectrometry at the end of the experiment. Angiotensin concentration-based markers for renin, angiotensin-converting enzyme and alternative renin-angiotensin system activities were calculated. Results: High tidal volume-ventilated mice treated with lisinopril showed a significant drop in the mean arterial pressure at 4 h compared to baseline, which was prevented by adding ALT-00 at 10 and 100 µg/kg/min. Ang I, Ang II and Ang 1-7 plasma equilibrium levels were elevated in the high tidal volumes group versus the sham group. Lisinopril reduced Ang II and slightly increased Ang I and Ang 1-7 levels versus the untreated high tidal volumes group. Adding ALT-00 at 10 and 100 µg/kg/min increased Ang I and Ang 1-7 levels versus the high tidal volume group, and partly prevented the downregulation of Ang II levels caused by lisinopril. The histological lung injury score was higher in the high tidal volume group versus the sham and low tidal volume groups, and was attenuated by lisinopril ± ALT-00 at all dose levels. Conclusion: Combined angiotensin-converting enzyme plus aminopeptidase inhibition prevented systemic hypotension and maintained the protective effect of lisinopril. In this study, a combination of lisinopril and ALT-00 at 10 µg/kg/min appeared to be the optimal approach, which may represent a promising strategy to counteract ventilator-induced lung injury that merits further exploration.

The systemic renin-angiotensin system in COVID-19.

SARS-CoV-2 gains cell entry via angiotensin-converting enzyme (ACE) 2, a membrane-bound enzyme of the "alternative" (alt) renin-angiotensin system (RAS). ACE2 counteracts angiotensin II by converting it to potentially protective angiotensin 1-7. Using mass spectrometry, we assessed key metabolites of the classical RAS (angiotensins I-II) and alt-RAS (angiotensins 1-7 and 1-5) pathways as well as ACE and ACE2 concentrations in 159 patients hospitalized with COVID-19, stratified by disease severity (severe, n = 76; non-severe: n = 83). Plasma renin activity (PRA-S) was calculated as the sum of RAS metabolites. We estimated ACE activity using the angiotensin II:I ratio (ACE-S) and estimated systemic alt-RAS activation using the ratio of alt-RAS axis metabolites to PRA-S (ALT-S). We applied mixed linear models to assess how PRA-S and ACE/ACE2 concentrations affected ALT-S, ACE-S, and angiotensins II and 1-7. Median angiotensin I and II levels were higher with severe versus non-severe COVID-19 (angiotensin I: 86 versus 30 pmol/L, p < 0.01; angiotensin II: 114 versus 58 pmol/L, p < 0.05), demonstrating activation of classical RAS. The difference disappeared with analysis limited to patients not taking a RAS inhibitor (angiotensin I: 40 versus 31 pmol/L, p = 0.251; angiotensin II: 76 versus 99 pmol/L, p = 0.833). ALT-S in severe COVID-19 increased with time (days 1-6: 0.12; days 11-16: 0.22) and correlated with ACE2 concentration (r = 0.831). ACE-S was lower in severe versus non-severe COVID-19 (1.6 versus 2.6; p < 0.001), but ACE concentrations were similar between groups and correlated weakly with ACE-S (r = 0.232). ACE2 and ACE-S trajectories in severe COVID-19, however, did not differ between survivors and non-survivors. Overall RAS alteration in severe COVID-19 resembled severity of disease-matched patients with influenza. In mixed linear models, renin activity most strongly predicted angiotensin II and 1-7 levels. ACE2 also predicted angiotensin 1-7 levels and ALT-S. No single factor or the combined model, however, could fully explain ACE-S. ACE2 and ACE-S trajectories in severe COVID-19 did not differ between survivors and non-survivors. In conclusion, angiotensin II was elevated in severe COVID-19 but was markedly influenced by RAS inhibitors and driven by overall RAS activation. ACE-S was significantly lower with severe COVID-19 and did not correlate with ACE concentrations. A shift to the alt-RAS axis because of increased ACE2 could partially explain the relative reduction in angiotensin II levels.

Tidal Volume-Dependent Activation of the Renin-Angiotensin System in Experimental Ventilator-Induced Lung Injury.

OBJECTIVES: Ventilator-induced lung injury (VILI) is a major contributor to morbidity and mortality in critically ill patients. Mechanical damage to the lungs is potentially aggravated by the activation of the renin-angiotensin system (RAS). This article describes RAS activation profiles in VILI and discusses the effects of angiotensin (Ang) 1-7 supplementation or angiotensin-converting enzyme (ACE) inhibition with captopril as protective strategies. DESIGN: Animal study. SETTING: University research laboratory. SUBJECTS: C57BL/6 mice. INTERVENTIONS: Anesthetized mice ( n = 12-18 per group) were mechanically ventilated with low tidal volume (LV T , 6 mL/kg), high tidal volume (HV T , 15 mL/kg), or very high tidal volume (VHV T , 30 mL/kg) for 4 hours, or killed after 3 minutes (sham). Additional VHV T groups received infusions of 60 µg/kg/hr Ang 1-7 or a single dose of 100 mg/kg captopril. MEASUREMENTS AND MAIN RESULTS: VILI was characterized by increased bronchoalveolar lavage fluid levels of interleukin (IL)-6, keratinocyte-derived cytokine, and macrophage inflammatory protein-2 (MIP2). The Ang metabolites in plasma measured with liquid chromatography tandem mass spectrometry showed a strong activation of the classical (Ang I, Ang II) and alternative RAS (Ang 1-7, Ang 1-5), with highest concentrations found in the HV T group. Although the lung-tissue ACE messenger RNA expression was unchanged, its protein expression showed a dose-dependent increase under mechanical ventilation. The ACE2 messenger RNA expression decreased in all ventilated groups, whereas ACE2 protein levels remained unchanged. Both captopril and Ang 1-7 led to markedly increased Ang 1-7 plasma levels, decreased Ang II levels, and ACE activity (Ang II/Ang I ratio), and effectively prevented VILI. CONCLUSIONS: VILI is accompanied by a strong activation of the RAS. Based on circulating Ang metabolite levels and tissue expression of RAS enzymes, classical ACE-dependent and alternative RAS cascades were activated in the HV T group, whereas classical RAS activation prevailed with VHV T ventilation. Ang 1-7 or captopril protected from VILI primarily by modifying the systemic RAS profile.

Dysregulation of ACE (Angiotensin-Converting Enzyme)-2 and Renin-Angiotensin Peptides in SARS-CoV-2 Mediated Mortality and End-Organ Injuries.

ACE (angiotensin-converting enzyme)-2 as the target for SARS-CoV-2 also negatively regulates the renin-angiotensin system. Pathological activation of ADAM17 (A disintegrin and metalloproteinase-17) may potentiate inflammation and diminish ACE2-mediated tissue protection through proteolytic shedding, contributing to SARS-CoV-2 pathogenesis. We aim to examine plasma soluble ACE2 and angiotensin profiles in relation to outcomes by enrolling consecutive patients admitted for COVID-19 with baseline blood collection at admission and repeated sampling at 7 days. The primary outcome was 90-day mortality, and secondary outcomes were the incidence of end-organ injuries. Overall, 242 patients were included, the median age was 63 (52-74) years, 155 (64.0%) were men, and 57 (23.6%) patients reached the primary end point. Baseline soluble ACE2 was elevated in COVID-19 but was not associated with disease severity or mortality. In contrast, an upward trajectory of soluble ACE2 at repeat sampling was independently associated with an elevated risk of mortality and incidence of acute myocardial injury and circulatory shock. Similarly, an increase in soluble tumor necrosis factor receptor levels was also associated with adverse outcomes. Plasma Ang I, Ang 1-7 (angiotensin 1-7) levels, and the Ang 1-7/Ang II (angiotensin II) ratio were elevated during SARS-CoV-2 infection related to downregulation of ACE activity at baseline. Moreover, patients having an upward trajectory of soluble ACE2 were characterized by an imbalance in the Ang 1-7/Ang II ratio. The observed dysregulation of ACE2 and angiotensin peptides with disease progression suggest a potential role of ADAM17 inhibition and enhancing the beneficial Ang 1-7/Mas axis to improve outcomes against SARS-CoV-2 infection.

Selective Inhibition of the C-Domain of ACE (Angiotensin-Converting Enzyme) Combined With Inhibition of NEP (Neprilysin): A Potential New Therapy for Hypertension.

[Figure: see text].

Aldosterone and cortisol synthesis regulation by angiotensin-(1-7) and angiotensin-converting enzyme 2 in the human adrenal cortex.

OBJECTIVE: The branch of the renin--angiotensin system constituting angiotensin-(1-7) [Ang-(1-7)], the Ang II type 2 receptor, the Mas receptors and the Ang-(1-7)-forming enzyme ACE-2, by counteracting the Ang II type 1 receptor (AT1R)-mediated effects, are held to be cardiovascular protective in several conditions. However, whether Ang-(1-7) and ACE-2 are detectable in human adrenocortical tissues and whether they affect aldosterone and cortisol biosynthesis was unknown. METHODS: We measured angiotensin peptides with liquid chromatography tandem-mass spectrometry and ACE-2 mRNA with digital droplet (dd)PCR in human aldosterone-producing adenoma (APA) and APA-adjacent tissue obtained from patients with primary aldosteronism. We also investigated the effects of Ang-(1-7) and the ACE-2 activator diminazene aceturate (DIZE) on aldosterone synthase (CYP11B2) and 11ß-hydroxylase (CYP11B1) gene expression, in the absence or presence of the AT1R antagonist irbesartan, or of the MasR antagonist A779. RESULTS: APA and APA-adjacent adrenocortical tissues express ACE-2 mRNA and contain detectable amounts of Ang II and Ang-(2-8), but not of Ang I, Ang-(1-5), Ang (3-8) and Ang-(1-7). Under unstimulated and Ang II- stimulated conditions Ang-(1-7) did not blunt CYP11B1 and CYP11B2 mRNA. At supraphysiological concentrations (10-4 mol/l), Ang-(1-7) stimulated both CYP11B1 and CYP11B2 mRNA via the AT1R. The ACE-2 activator DIZE increased by 1.5-fold ACE-2 mRNA but did not blunt Ang II- upregulated CYP11B1 and CYP11B2 expression. CONCLUSION: These results do not support the hypothesis that the ACE-2/Ang-(1-7)/MasR axis play a protective role by counteracting enhanced aldosterone secretion in humans.

Dipeptidyl peptidase 3 modulates the renin-angiotensin system in mice.

Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent hydrolase involved in degrading oligopeptides with 4-12 amino acid residues. It has been associated with several pathophysiological processes, including blood pressure regulation, pain signaling, and cancer cell defense against oxidative stress. However, the physiological substrates and the cellular pathways that are potentially targeted by DPP3 to mediate these effects remain unknown. Here, we show that global DPP3 deficiency in mice (DPP3-/-) affects the renin-angiotensin system (RAS). LC-MS-based profiling of circulating angiotensin peptides revealed elevated levels of angiotensin II, III, IV, and 1-5 in DPP3-/- mice, whereas blood pressure, renin activity, and aldosterone levels remained unchanged. Activity assays using the purified enzyme confirmed that angiotensin peptides are substrates for DPP3. Aberrant angiotensin signaling was associated with substantially higher water intake and increased renal reactive oxygen species formation in the kidneys of DPP3-/- mice. The metabolic changes and altered angiotensin levels observed in male DPP3-/- mice were either absent or attenuated in female DPP3-/- mice, indicating sex-specific differences. Taken together, our observations suggest that DPP3 regulates the RAS pathway and water homeostasis by degrading circulating angiotensin peptides.

Critical Role of Neprilysin in Kidney Angiotensin Metabolism.

RATIONALE: Kidney homeostasis is critically determined by the coordinated activity of the renin-angiotensin system (RAS), including the balanced synthesis of its main effector peptides Ang (angiotensin) II and Ang (1-7). The condition of enzymatic overproduction of Ang II relative to Ang (1-7) is termed RAS dysregulation and leads to cellular signals, which promote hypertension and organ damage, and ultimately progressive kidney failure. ACE2 (angiotensin-converting enzyme 2) and NEP (neprilysin) induce the alternative, and potentially reno-protective axis by enhancing Ang (1-7) production. However, their individual contribution to baseline RAS balance and whether their activities change in chronic kidney disease (CKD) has not yet been elucidated. OBJECTIVE: To examine whether NEP-mediated Ang (1-7) generation exceeds Ang II formation in the healthy kidney compared with diseased kidney. METHODS AND RESULTS: In this exploratory study, we used liquid chromatography-tandem mass spectrometry to measure Ang II and Ang (1-7) synthesis rates of ACE, chymase and NEP, ACE2, PEP (prolyl-endopeptidase), PCP (prolyl-carboxypeptidase) in kidney biopsy homogenates in 11 healthy living kidney donors, and 12 patients with CKD. The spatial expression of RAS enzymes was determined by immunohistochemistry. Healthy kidneys showed higher NEP-mediated Ang (1-7) synthesis than Ang II formation, thus displaying a strong preference towards the reno-protective alternative RAS axis. In contrast, in CKD kidneys higher levels of Ang II were recorded, which originated from mast cell chymase activity. CONCLUSIONS: Ang (1-7) is the dominant RAS peptide in healthy human kidneys with NEP rather than ACE2 being essential for its generation. Severe RAS dysregulation is present in CKD dictated by high chymase-mediated Ang II formation. Kidney RAS enzyme analysis might lead to novel therapeutic approaches for CKD.

Retinoic acid receptor α as a novel contributor to adrenal cortex structure and function through interactions with Wnt and Vegfa signalling.

Primary aldosteronism (PA) is the most frequent form of secondary arterial hypertension. Mutations in different genes increase aldosterone production in PA, but additional mechanisms may contribute to increased cell proliferation and aldosterone producing adenoma (APA) development. We performed transcriptome analysis in APA and identified retinoic acid receptor alpha (RARα) signaling as a central molecular network involved in nodule formation. To understand how RARα modulates adrenal structure and function, we explored the adrenal phenotype of male and female Rarα knockout mice. Inactivation of Rarα in mice led to significant structural disorganization of the adrenal cortex in both sexes, with increased adrenal cortex size in female mice and increased cell proliferation in males. Abnormalities of vessel architecture and extracellular matrix were due to decreased Vegfa expression and modifications in extracellular matrix components. On the molecular level, Rarα inactivation leads to inhibition of non-canonical Wnt signaling, without affecting the canonical Wnt pathway nor PKA signaling. Our study suggests that Rarα contributes to the maintenance of normal adrenal cortex structure and cell proliferation, by modulating Wnt signaling. Dysregulation of this interaction may contribute to abnormal cell proliferation, creating a propitious environment for the emergence of specific driver mutations in PA.

Apelin protects against abdominal aortic aneurysm and the therapeutic role of neutral endopeptidase resistant apelin analogs.

Abdominal aortic aneurysm (AAA) remains the second most frequent vascular disease with high mortality but has no approved medical therapy. We investigated the direct role of apelin (APLN) in AAA and identified a unique approach to enhance APLN action as a therapeutic intervention for this disease. Loss of APLN potentiated angiotensin II (Ang II)-induced AAA formation, aortic rupture, and reduced survival. Formation of AAA was driven by increased smooth muscle cell (SMC) apoptosis and oxidative stress in Apln-/y aorta and in APLN-deficient cultured murine and human aortic SMCs. Ang II-induced myogenic response and hypertension were greater in Apln-/y mice, however, an equivalent hypertension induced by phenylephrine, an α-adrenergic agonist, did not cause AAA or rupture in Apln-/y mice. We further identified Ang converting enzyme 2 (ACE2), the major negative regulator of the renin-Ang system (RAS), as an important target of APLN action in the vasculature. Using a combination of genetic, pharmacological, and modeling approaches, we identified neutral endopeptidase (NEP) that is up-regulated in human AAA tissue as a major enzyme that metabolizes and inactivates APLN-17 peptide. We designed and synthesized a potent APLN-17 analog, APLN-NMeLeu9-A2, that is resistant to NEP cleavage. This stable APLN analog ameliorated Ang II-mediated adverse aortic remodeling and AAA formation in an established model of AAA, high-fat diet (HFD) in Ldlr-/- mice. Our findings define a critical role of APLN in AAA formation through induction of ACE2 and protection of vascular SMCs, whereas stable APLN analogs provide an effective therapy for vascular diseases.

Aldosterone Stimulates Its Biosynthesis Via a Novel GPER-Mediated Mechanism.

CONTEXT: The G protein-coupled estrogen receptor (GPER) mediates an aldosterone secretagogue effect of 17ß-estradiol in human HAC15 adrenocortical cells after estrogen receptor ß blockade. Because GPER mediates mineralocorticoid receptor-independent aldosterone effects in other cell types, we hypothesized that aldosterone could modulate its own synthesis via GPER activation. METHODS: HAC15 cells were exposed to aldosterone in the presence or absence of canrenone, a mineralocorticoid receptor antagonist, and/or of the selective GPER antagonist G36. Aldosterone synthase (CYP11B2) mRNA and protein levels changes were the study end points. Similar experiments were repeated in strips obtained ex vivo from aldosterone-producing adenoma (APA) and in GPER-silenced HAC15 cells. RESULTS: Aldosterone markedly increased CYP11B2 mRNA and protein expression (vs untreated samples, P < 0.001) in both models by acting via GPER, because these effects were abolished by G36 (P < 0.01) and not by canrenone. GPER-silencing (P < 0.01) abolished the aldosterone-induced increase of CYP11B2, thus proving that aldosterone acts via GPER to augment the step-limiting mitochondrial enzyme (CYP11B2) of its synthesis. Angiotensin II potentiated the GPER-mediated effect of aldosterone on CYP11B2. Coimmunoprecipitation studies provided evidence for GPER-angiotensin type-1 receptor heterodimerization. CONCLUSION: We propose that this autocrine-paracrine mechanism could enhance aldosterone biosynthesis under conditions of immediate physiological need in which the renin-angiotensin-aldosterone system is stimulated as, for example, hypovolemia. Moreover, as APA overexpresses GPER this mechanism could contribute to the aldosterone excess that occurs in primary aldosteronism in a seemingly autonomous fashion from angiotensin II.

Aldosterone LC-MS/MS Assay-Specific Threshold Values in Screening and Confirmatory Testing for Primary Aldosteronism.

Context: Current threshold values for primary aldosteronism (PA) diagnostic testing are based on measuring aldosterone (PAC) using immunoassays. Quantification of PAC by liquid chromatography-tandem mass spectrometry (LC-MS/MS) yields lower values. Objective: To compare aldosterone measurement by radioimmunoassay (RIA) with LC-MS/MS and evaluate performances of proposed LC-MS/MS-specific cutoffs for PA screening and confirmatory testing. Patients and Intervention: Forty-one patients underwent aldosterone/renin ratio (ARR) testing to screen for, and fludrocortisone suppression testing (FST) to confirm or exclude, PA. Renin (DRC) was measured by chemiluminescent immunoassay. Results: Median serum PACLC-MS/MS was 27.8% lower (P < 0.05) than plasma PACRIA in 164 pairs of FST samples. A positive correlation (Spearman coefficient, 0.894, P < 0.01; Pearson r coefficient, 0.861, P < 0.01) was observed between the two assays. Thirty-seven patients showed consistent FST diagnoses (29 positive, 8 negative), whereas four showed inconsistent FSTs by the two assays. Good agreement (κ coefficient, 0.736; P < 0.01) was observed between the current FST diagnostic PACRIA cutoff of 165 pmol/L and the proposed PACLC-MS/MS cutoff of 133 pmol/L. Among 37 patients with consistent FST results, no differences were observed in sensitivity (89.7% vs 93.1%) or specificity (87.5% vs 87.5%) for PA screening between the current ARR cutoff of 70 pmol/mU (PACRIA/DRC) and the proposed cutoff of 55 pmol/mU (PACLC-MS/MS/DRC). Conclusions: Adjustment of the current cutoffs for PA diagnostic testing is necessary if PAC is measured by LC-MS/MS. Our preliminary results suggest that the proposed LC-MS/MS cutoffs for ARR and FST perform as well as current RIA cutoffs.

Can Screening and Confirmatory Testing in the Management of Patients with Primary Aldosteronism be Improved?

Widespread application of the plasma aldosterone/renin ratio (ARR) as a screening test has led to the recognition that primary aldosteronism (PA) is the most common specifically treatable and potentially curable form of hypertension, accounting for 5-10% of patients. Maximal detection requires accurate diagnostic approaches and awareness and control of factors that confound results, including most antihypertensives, posture, time of day, dietary salt, and plasma potassium. Recent studies have revealed potential for false positives in patients on beta-adrenoceptor blockers, and, when direct renin concentration (but not plasma renin activity) is used to measure renin, in women during the luteal phase of the menstrual cycle or receiving estrogen-containing contraceptives or hormonal replacement therapy. In addition to verapamil slow release, hydralazine and prazosin, moxonidine has minimal effects on the ARR and can be used to control hypertension during work-up. Fludrocortisone suppression testing, while probably the most reliable means of definitively confirming or excluding PA, is time consuming and expensive, requiring a five day inpatient stay. A novel approach, upright (seated) saline infusion suppression testing (SST), has shown excellent reliability with much greater sensitivity than conventional recumbent SST in a recent pilot study, and requires only a day visit. Accurate measurement of aldosterone is essential for each step of PA workup: introduction of new, highly reliable high-throughput mass spectrometric methods into clinical practice has represented a major advance. In response to concerns raised about accuracy of renin assays, new mass spectrometric methods for measuring angiotensin II are currently being assessed in the clinical setting.

Clinical Relevance and Role of Neuronal AT1 Receptors in ADAM17-Mediated ACE2 Shedding in Neurogenic Hypertension.

RATIONALE: Neurogenic hypertension is characterized by an increase in sympathetic activity and often resistance to drug treatments. We previously reported that it is also associated with a reduction of angiotensin-converting enzyme type 2 (ACE2) and an increase in a disintegrin and metalloprotease 17 (ADAM17) activity in experimental hypertension. In addition, while multiple cells within the central nervous system have been involved in the development of neurogenic hypertension, the contribution of ADAM17 has not been investigated. OBJECTIVE: To assess the clinical relevance of this ADAM17-mediated ACE2 shedding in hypertensive patients and further identify the cell types and signaling pathways involved in this process. METHODS AND RESULTS: Using a mass spectrometry-based assay, we identified ACE2 as the main enzyme converting angiotensin II into angiotensin-(1-7) in human cerebrospinal fluid. We also observed an increase in ACE2 activity in the cerebrospinal fluid of hypertensive patients, which was correlated with systolic blood pressure. Moreover, the increased level of tumor necrosis factor-α in those cerebrospinal fluid samples confirmed that ADAM17 was upregulated in the brain of hypertensive patients. To further assess the interaction between brain renin-angiotensin system and ADAM17, we generated mice lacking angiotensin II type 1 receptors specifically on neurons. Our data reveal that despite expression on astrocytes and other cells types in the brain, ADAM17 upregulation during deoxycorticosterone acetate-salt hypertension occurs selectively on neurons, and neuronal angiotensin II type 1 receptors are indispensable to this process. Mechanistically, reactive oxygen species and extracellular signal-regulated kinase were found to mediate ADAM17 activation. CONCLUSIONS: Our data demonstrate that angiotensin II type 1 receptors promote ADAM17-mediated ACE2 shedding in the brain of hypertensive patients, leading to a loss in compensatory activity during neurogenic hypertension.

Effects of angiotensin-converting-enzyme inhibitor therapy on the regulation of the plasma and cardiac tissue renin-angiotensin system in heart transplant patients.

BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitors (ACEis) are beneficial in patients with heart failure, yet their role after heart transplantation (HTx) remains ambiguous. Particularly, the effects of ACEis on plasma and cardiac metabolites of the "classical" and "alternative" renin-angiotensin system (RAS) in HTx patients are unknown. METHODS: This cross-sectional study used a novel mass spectrometry-based approach to analyze plasma and tissue RAS regulation in homogenates of heart biopsy specimens from 10 stable HTx patients without RAS blockade and in 15 patients with ACEi therapy. Angiotensin (Ang) levels in plasma and Ang formation rates in biopsy tissue homogenates were measured. RESULTS: Plasma Ang II formation is exclusively ACE dependent, whereas cardiac Ang II formation is primarily chymase dependent in HTx patients. ACEi therapy substantially increased plasma Ang-(1-7), the key effector of the alternative RAS, leaving plasma Ang II largely intact. Importantly, neprilysin and prolyl-carboxypeptidase but not angiotensin converting enzyme 2 are essential for cardiac tissue Ang-(1-7) formation. CONCLUSION: ACE is the key enzyme for the generation of plasma Ang II, whereas chymase is responsible for cardiac tissue production of Ang II. Furthermore, our findings reveal that neprilysin and prolyl-carboxypeptidase are the essential cardiac enzymes for the alternative RAS after HTx. These novel insights into the versatile regulation of the RAS in HTx patients might affect future therapeutic avenues, such as chymase and neprilysin inhibition, beyond classical Ang II blockade.

Neprilysin is a Mediator of Alternative Renin-Angiotensin-System Activation in the Murine and Human Kidney.

Cardiovascular and renal pathologies are frequently associated with an activated renin-angiotensin-system (RAS) and increased levels of its main effector and vasoconstrictor hormone angiotensin II (Ang II). Angiotensin-converting-enzyme-2 (ACE2) has been described as a crucial enzymatic player in shifting the RAS towards its so-called alternative vasodilative and reno-protective axis by enzymatically converting Ang II to angiotensin-(1-7) (Ang-(1-7)). Yet, the relative contribution of ACE2 to Ang-(1-7) formation in vivo has not been elucidated. Mass spectrometry based quantification of angiotensin metabolites in the kidney and plasma of ACE2 KO mice surprisingly revealed an increase in Ang-(1-7), suggesting additional pathways to be responsible for alternative RAS activation in vivo. Following assessment of angiotensin metabolism in kidney homogenates, we identified neprilysin (NEP) to be a major source of renal Ang-(1-7) in mice and humans. These findings were supported by MALDI imaging, showing NEP mediated Ang-(1-7) formation in whole kidney cryo-sections in mice. Finally, pharmacologic inhibition of NEP resulted in strongly decreased Ang-(1-7) levels in murine kidneys. This unexpected new role of NEP may have implications for the combination therapy with NEP-inhibitors and angiotensin-receptor-blockade, which has been shown being a promising therapeutic approach for heart failure therapy.

Optimum AT1 receptor-neprilysin inhibition has superior cardioprotective effects compared with AT1 receptor blockade alone in hypertensive rats.

Neprilysin inhibitors prevent the breakdown of bradykinin and natriuretic peptides, promoting vasodilation and natriuresis. However, they also increase angiotensin II and endothelin-1. Here we studied the effects of a low and a high dose of the neprilysin inhibitor thiorphan on top of AT1 receptor blockade with irbesartan versus vehicle in TGR(mREN2)27 rats with high renin hypertension. Mean arterial blood pressure was unaffected by vehicle or thiorphan alone. Irbesartan lowered blood pressure, but after 7 days pressure started to increase again. Low- but not high-dose thiorphan prevented this rise. Only during exposure to low-dose thiorphan plus irbesartan did heart weight/body weight ratio, cardiac atrial natriuretic peptide expression, and myocyte size decrease significantly. Circulating endothelin-1 was not affected by low-dose thiorphan with or without irbesartan, but increased after treatment with high-dose thiorphan plus irbesartan. This endothelin-1 rise was accompanied by an increase in renal sodium-hydrogen exchanger 3 protein abundance, and an upregulation of constrictor vascular endothelin type B receptors. Consequently, the endothelin type B receptor antagonist BQ788 no longer enhanced endothelin-1-induced vasoconstriction (indicative of endothelin type B receptor-mediated vasodilation), but prevented it. Thus, optimal neprilysin inhibitor dosing reveals additional cardioprotective effects on top of AT1 receptor blockade in renin-dependent hypertension.

Pharmacodynamic effects of C-domain-specific ACE inhibitors on the renin-angiotensin system in myocardial infarcted rats.

INTRODUCTION: The renin-angiotensin system (RAS) is a dynamic network that plays a critical role in blood pressure regulation and fluid and electrolyte homeostasis. Modulators of the RAS, such as angiotensin-converting enzyme (ACE) inhibitors, are widely used to treat hypertension, heart failure and myocardial infarction. METHODS: The effect of ACE inhibitors (lisinopril and C-domain-selective LisW-S) on the constituent peptides of the RAS following myocardial infarction was examined in rats. Ten angiotensin peptides were analysed using a sensitive LC-MS/MS-based assay to examine both the circulating and equilibrium levels of these peptides. RESULTS: Administration of lisinopril or LisW-S caused a significant decrease in Ang 1-8/Ang 1-10 ratios as determined by circulating and equilibrium peptide level analysis. Furthermore, Ang 1-7 levels were elevated by both ACE inhibitors, but only lisinopril decreased the Ang 1-5/Ang 1-7 ratio. This indicates LisW-S C-domain specificity as Ang 1-5 is generated by hydrolysis of Ang 1-7 by the N-domain. Further corroboration of LisW-S C-domain specificity is that only lisinopril increased the circulating levels of the N-domain ACE substrate Ac-SDKP. CONCLUSION: LisW-S is able to effectively block ACE in vivo by C-domain-selective inhibition. The LC-MS/MS-based assay allows the evaluation of the pharmacologic impact of RAS inhibitors in different pathophysiological conditions.