Angiotensin II type 2 receptor signaling attenuates aortic aneurysm in mice through ERK antagonism.

What Is the Optimal Medical Therapy for Marfan Syndrome?

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the Fibrillin-1 gene. It is associated with formation of thoracic aortic aneurysms that can potentially be a life-threatening condition due to aortic rupture or dissection. Excessive non-canonical transforming growth factor beta signalling, mediated by activation of extracellular signal-regulated kinases 1/2 (ERK1/2), as well as inducible nitric oxide synthase (NOS2)-dependent nitric oxide production, have been identified to drive aortic pathology in MFS through induction of elastin fragmentation and smooth muscle cell apoptosis. Despite promising results in animal studies, specific pharmacological interventions approved for clinical use in patients with MFS-related aortic disease are rare. Nitro-oleic acid (NO2-OA) is an endogenously generated signalling modulator, which is available as an oral compound and has been shown to inhibit ERK1/2 activation and NOS2 expression in different disease models, thereby exerting promising therapeutic effects. In this study, we investigated whether NO2-OA decreases aortic dilation in MFS. Eight-week-old MFS (Fbn1C1041G/+) mice were treated with NO2-OA or vehicle for 4 weeks via subcutaneously implanted osmotic minipumps. Echocardiography indicated progressive ascending aortic dilation and wall stiffening in MFS mice, which was significantly attenuated by NO2-OA treatment. This protective effect was mediated by inhibition of aortic ERK1/2, Smad2 as well as nuclear factor kappa B overactivation and consequent attenuation of elastin fragmentation by matrix metalloproteinase 2, apoptosis, and collagen deposition. Critically, the therapeutic efficacy of NO2-OA in MFS was further emphasized by demonstrating its capability to reduce lethal aortic complications in Fbn1C1041G/+ mice challenged with Angiotensin II. NO2-OA distinctly attenuates progression of aortic dilation in MFS via modulation of well-established disease-mediating pathways, thereby meriting further investigation into its application as a therapeutic agent for the treatment of this condition.

Marfan syndrome (MFS) is a connective tissue disorder that causes complications throughout the body. However, the cardiovascular effects of MFS, specifically aortic aneurysms, are the leading cause of morbidity and mortality in patients. Both the transforming growth factor beta (TGF‐β) and angiotensin II type I receptor (AT1R) signaling pathways are known to contribute to the progression of MFS aneurysms. Recently, our laboratory reported that low‐intensity mild exercise could improve aortic function and structure in the mouse model of MFS. Losartan, an AT1R blocker, has been shown to slow down the progression of MFS aneurysms in both the mouse model and human patients. In this study, we have explored the potential of a combinational therapy of exercise and losartan in a well‐established mouse model of MFS associated aortic aneurysm in order to determine if there are additive protective and delaying effects on the progression of aortic aneurysm in the mouse model. Treatment consisted of 0.6 g/L (full dose) or 0.3 g/L (half dose) of losartan in drinking water combined with a 55% VO2 max exercise regimen (8 m/min, 30 min/day, 5 days/week). Mice were divided into experimental groups: control, MFS, MFS + exercise, MFS + 0.6 g/L losartan, MFS + 0.3 g/L losartan, MFS + exercise + 0.6 g/L losartan, and MFS + exercise + 0.3 g/L losartan. The biophysical properties of the aorta, such as the aortic diameter and pulse wave velocity (PWV), were determined by high resolution high frequency ultrasound imaging system (Vevo2100, FUJIFILM VisualSonics) in 3‐month‐old MFS and control mice. Aortic diameter measurements of the sinus of Valsalva were significantly higher in 3‐month‐old MFS mice as compared to control. However, losartan treatment, mild exercise, or combination of both had no effects on aortic root growth in MFS mice. In addition, measurements of the aortic annulus and sinotubular junction were not significantly different among experimental groups. MFS mice exhibited higher PWV as compared to control mice, indicating increased stiffness of aortic wall in these mice. Combination of mild aerobic exercise with full and half dose of losartan seems to have some effects on the aortic wall by decreasing the aortic diameter, although no statistical significant differences were observed among the treated and non‐treated groups. This study presents the effects of combination of mild exercise and losartan during the early stage of aneurysm progression in MFS mice. With the continuation of the longitudinal study at 6 and 9 months of age and as the aneurysm progresses, we will be able to continue our evaluation of aortic and cardiac function and structure in MFS mice subjected to exercise only, losartan only, or the combinational therapy. This study provides additional information on the most effective therapeutic approach to delay the progression of aneurysm in MFS.Support or Funding InformationThis study is supported by funding from The Marfan Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the fibrillin‐1 (FBN1) gene. This mutation manifests in a variety of phenotypic changes in the musculoskeletal, cardiovascular, and pulmonary systems, with a notable vascular effect leading to aortic aneurysm, dissection, and rupture. This results in a significant increase in morbidity and mortality in these patients. Studies have shown that there is a connection between MFS and intracranial aneurysms (IA). The prevalence of IA in patients with aortic disease is quadrupled compared to that in the general population. There is a modestly increased prevalence of ischemic stroke in hospitalized patients with MFS when compared with healthy controls. The reduced cerebral blood flow triggered by cardiac and peripheral vascular dysfunction could further make the brain more vulnerable to vascular dementia and Alzheimer’s pathology. Studies have found that 50% school‐age children with MFS had one or more neuropsychologic deficits. Despite the reported neurological complications in MFS patients, our understanding of cerebrovascular function and structure in MFS is very limited. Losartan, an AT1R blocker has shown positive effects on slowing the progression of aortic root aneurysm in the well‐established mouse model of MFS (Fbn1 +/‐ p. Cys1041Gly). Alternatively, the cardiovascular benefits of moderate exercise training have been well documented in the literature. In addition, studies have shown that aerobic exercise can improve cognitive function, decrease neuropsychiatric and neurodegenerative symptoms. In this study we aim to investigate the impact of MFS pathogenesis and Losartan treatment and mild aerobic exercise on other vessels such as the posterior cerebral artery, coronary and pulmonary arteries.At 4 weeks of age, male and female mice were divided into experimental groups: control (Ctrl), MFS, MFS + Losartan, MFS + exercise. MFS mice received 0.6 g/L of Losartan in drinking water or subjected to an exercise regimen of 8m/min, 30min/day, 5days/week. At 6 months of age, in vivo ultrasound imaging was performed to measure aortic pulse wave velocity (PWV), and peak blood flow of the coronary, pulmonary and posterior cerebral arteries. Blood pressure (BP) measurements were also taken using the tail‐cuff method.Our results showed that PWV, an index of aortic stiffness is increased in MFS mice compared to Ctrl, while Losartan and exercise reduced the PWV in MFS mice bringing it back to the values of healthy Ctrl. The posterior cerebral artery peak blood flow was significantly reduced in MFS and MFS + Losartan groups compared to Ctrl. No significant difference was observed in the MFS + exercise group compared to Ctrl. The coronary and pulmonary peak blood flow showed no significant change between MFS and control groups and MFS and intervention groups. There was also no statistical significance in the systolic and diastolic BP between the groups.This study provides an early insight into the disease progression of MFS as well as investigates the preliminary potential effects of Losartan and exercise on peak blood flow in coronary, pulmonary and cerebral arteries in the well‐established MFS mouse model.

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the fibrillin-1 (FBN1) gene. This mutation manifests in a variety of phenotypic changes with a notable cardiovascular effect leading to aortic aneurysm, dissection, and rupture. This results in a significant increase in morbidity and mortality in these patients. The angiotensin II type I receptor (AT1R) signalling pathway has previously been shown to contribute to the cardiovascular effects in the progression of this disease. Prior studies have demonstrated the protective effects of Losartan, an AT1R blocker, on slowing the progression of aortic root aneurysm in the well-established mouse model of MFS (Fbn1 +/- p. Cys1041Gly). In this study, we are further investigating the impact of MFS pathogenesis and Losartan treatment on other vessels such as the posterior cerebral artery and coronary artery. Mice (male + female) were divided into experimental groups: Control, MFS, MFS + 0.6g/L losartan. Drug therapy consisted of 0.6g/L of losartan in drinking water. Water intakes were recorded to ensure the mice were receiving the treatment. At 7 months of age, in vivo ultrasound imaging was performed to measure aortic diameters, aortic pulse wave velocity (PWV), and peak blood flow of the coronary and posterior cerebral arteries. In order to assess elastin fragmentation in the aortic wall, 5μm aortic cross-sections were subjected to Van Geison elastin staining. Blood pressure (BP) measurements were also obtained, using the tail-cuff method. Our results demonstrate that Losartan reduces aortic root diameters at the sinus of Valsalva and reduced the PWV, an index of aortic stiffness, in MFS mice at 7 months of age. Losartan also reduces elastin fragmentation in the aortic wall of MFS mice. The systolic coronary peak flow demonstrates a statistically significant decrease in MFS when compared to CTRL. Losartan treatment has no effect on the systolic coronary peak flow. There is no statistical significance in diastolic coronary peak flow between CTRL vs. MFS and MFS vs. MFS + Losartan. There is no statistical significance in comparison of the posterior cerebral artery peak flow between CTRL to MFS and MFS to MFS + Losartan. There is also no statistical significance in the systolic and diastolic BP between the groups. Our results show that Losartan has beneficial effects in delaying the progression of aortic aneurysm, reduces vessel wall stiffness, and reduces elastin fragmentation in the aortic root. This study also provides an early insight into the disease progression of MFS in the less investigated coronary and cerebral arteries, as well as investigates the preliminary potential effects of Losartan on peak flow in coronary and cerebral vessels in the well-established MFS mouse model.

Elevated intracellular Ca(2+) triggers numerous signaling pathways including protein kinases such as the calmodulin-dependent kinases (CaMKs) and the extracellular signal-regulated kinases (ERKs). In the present study we examined Ca(2+)-dependent "cross-talk" between these two protein kinase families. Using a combination of pharmacological inhibitors and dominant-negative kinases (dnKinase), we identified a requirement for CaMKK acting through CaMKI in the stimulation of ERKs upon depolarization of the neuroblastoma cell line, NG108. Depolarization stimulated prolonged ERK and JNK activation that was blocked by the CaMKK inhibitor, STO-609; this inhibition of ERK activation by STO-609 was rescued by expression of a STO-609-insensitive mutant of CaMKK. However, activation of ERK by epidermal growth factor or carbachol were not suppressed by inhibition of CaMKK, indicating specificity for this "cross-talk." To identify the downstream target of CaMKK that mediated ERK activation upon depolarization, dnKinases were expressed. The dnCaMKI completely suppressed ERK2 activation whereas dnAKT/PKB or nuclear-targeted dnCaMKIV, other substrates for CaMKK, were not inhibitory. ERK activation upon depolarization or transfection with constitutively active (ca) CaMKI was blocked by dnRas. Additionally, depolarization of NG108 cells promoted neurite outgrowth, and this effect was blocked by inhibition of either CaMKK (STO-609) or ERK (UO126). Co-transfection with caCaMKK plus caCaMKI also stimulated neurite outgrowth that was blocked by inhibition of ERK (UO126). These data are the first to suggest that ERK activation and neurite outgrowth in response to depolarization are mediated by CaMKK activation of CaMKI.

Effectiveness of Combination of Losartan Potassium and Doxycycline versus Single-Drug Treatments in the Secondary Prevention of Thoracic Aortic Aneurysm in Marfan Syndrome

To examine the possible crosstalk and the roles of angiotensin (Ang) II type 1 (AT1) and type 2 (AT2) receptors in the control of apoptosis in fetal vascular smooth muscle cells (VSMCs). Fetal VSMCs were prepared from rat fetal aorta at embryonic day 20. Expression of Ang II receptors was measured by a radioligand binding assay. Apoptotic changes were assessed by caspase 3 activity and chromatin dye staining. Regulation of extracellular signal-regulated kinase (ERK) activity via Ang II receptors was analysed by determining phosphorylated ERK with Western blot. Ang II receptor-mediated activation of tyrosine phosphatase SHP-1 was assessed by protein tyrosine phosphatase assay. The expression of AT1 and AT2 receptors was approximately 70%: 30% per cell. Serum depletion induced apoptosis in fetal VSMCs and selective AT1 receptor stimulation attenuated the apoptotic changes, whereas selective AT2 receptor activation enhanced apoptosis. Ang II increased ERK phosphorylation, which was inhibited by addition of the AT1 receptor-specific antagonist CV11974, but enhanced by addition of the AT2 receptor-specific antagonist PD123319, suggesting that activation of AT2 receptor attenuated the AT1 receptor-mediated ERK phosphorylation. Moreover, we demonstrated that AT2 receptor stimulation activated SHP-1 in fetal VSMCs, whereas AT1 receptor stimulation did not. Transient transfection of a dominant-negative SHP-1 mutant into rat fetal VSMCs resulted in a significant decrease of the AT2 receptor-mediated inhibition of ERK phosphorylation and attenuated the proapoptotic effect of AT2 receptor. These results indicate that a crosstalk between AT1 and AT2 receptors regulates the survival of fetal VSMCs and substantiate SHP-1 as a key molecule in AT2 receptor signaling.

Marfan Syndrome (MFS), a connective tissue disorder, resulting from mutations in the Fibrillin‐1 gene, is associated with several clinical manifestations with the most life‐threatening being aortic aneurysm, dissection, and rupture. The mechanism underlying MFS pathogenesis seems to result from crosstalk between the Angiotensin‐II (AngII) pathway and over activation of transforming growth factor‐beta (TGF‐b) signaling. Studies show that Losartan, an angiotensin II receptor type I (ATRI) blocker, can block progression of aortic aneurysm in mice, partially due to its inhibitory effects on TGF‐b signaling. It has been established that caveolin‐1 (Cav1), a coat protein of caveolae that is highly expressed in endothelial and smooth muscle, regulates AngII and TGF‐β signaling pathways, through its interactions with ATR1 and TGF‐β receptors. Interestingly, Cav1 knockout (Cav1KO) animal models illustrated increased elastin synthesis and nitric oxide (NO) production. Our previous study in the MFS mouse model reported reduced NO production in the aortic wall, proposing a potential link between Cav‐1 activation and aneurysm progression in MFS mice. Hence, in this study, we aimed to investigate the effects of genetic manipulation of Cav1 expression on the progression of aortic aneurysm in a well‐established mouse model of MFS‐associated aortic aneurysm, by generating MFS mice lacking Cav1 expression (MFS/Cav1KO).In vivo analysis of biophysical properties in 3‐ and 6‐ month old wild type, MFS, Cav1KO, and MFS/Cav1KO mice was performed using Vevo 2100 high‐resolution ultrasound imaging system (FUJIFILM VisualSonics). Aortic diameters at the aortic annulus, sinus of Valsalva, and sinotubular junction were significantly increased in MFS and MFS/CAV1KO mice as compared to control and Cav1KO groups at 3, and 6 months in systole and diastole. Pulse wave velocity (PWV), a reliable indicator of aortic wall stiffness, was significantly increased in MFS as compared to control and Cav1KO mice, with no significant changes observed between MFS and MFS/Cav1KO groups at 3 months of age. Interestingly, at 6 months of age, MFS/Cav1KO mice presented higher values for PWV as compared to MFS groups, indicating that deletion of Cav1 gene in MFS mice may have detrimental effects on aortic wall structure as MFS mice age.Cardiac parameters were also measured. Our data showed that at 3 months of age, a marked increase in heart rate, ejection fraction, and fractional shortening were detectable in MFS mice as compared to control subjects. Measurements of cardiac output and stroke volume revealed no significant differences among experimental groups. Interestingly, left ventricular mass was significantly greater in MFS/CAV1KO compared to wild type and MFS mice, suggesting that Cav1 plays a protective role in maintaining normal cardiac structure in MFS mice. At this point, our collected data suggest that Cav1 may have some protective effects on aortic and cardiac structure and function during the development of aortic aneurysm in the mouse model of MFS.Support or Funding InformationThis study was funded by The Marfan Foundation (M.E.), and a Midwestern University Graduate Fund (T.C.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Our lab studies molecular determinants of vessel wall homeostasis, with particular emphasis on molecules and pathways that contribute to aortic aneurysm. The study of Marfan syndrome (MFS), an autosomal dominant condition with a strong predisposition for aortic root aneurysm and dissection, has provided a tractable means to gain a foothold in the pathogenesis of this complex phenotype. Our initial studies demonstrated that MFS is caused by mutations in the extracellular matrix protein fibrillin-1. Early pathogenetic models that singularly invoked inherent weakness of the tissues as the driving force in disease progression boded poorly for the development of productive treatment strategies and failed to reconcile multiple disease manifestations, including bone overgrowth, myxomatous valve changes, and muscle and fat hypoplasia. Using mouse models of MFS, we demonstrated that fibrillin-1 is not simply a structural protein but rather regulates the local bioavailability and activation of the growth factor transforming growth factor (TGF) β . Many important manifestations of MFS, including emphysema, valve degeneration, myopathy, and aortic aneurysm, could be attenuated or prevented in fibrillin-1–deficient mice upon systemic administration of TGF β antagonists. Similar protection was achieved through the use of the angiotensin II type 1 receptor blocker (ARB) losartan, which lowers both blood pressure and TGF β signaling and is in widespread clinical use for the treatment of hypertension. These observations led to the first clinical trial for MFS that is based upon a refined mechanistic understanding of disease. We now have performed comprehensive interrogation of a modifier network in mouse models of MFS. These data reveal parsing of the angiotensin II signaling cascades in the pathogenesis and prevention of aortic aneurysm. Signaling through the type 2 (AT2) receptor is protective, whereas loss of AT2 signaling accelerates disease and attenuates the protection afforded by ARBs, suggesting that losartan provides protection, at least in part, through shunting of angiotensin II signaling through AT2. We also demonstrate a predominant role of noncanonical TGF β signaling in the pathogenesis of ascending aortic aneurysm and show that the mechanism of AT2-mediated protection derives from cross-talk with this pathway. Taken together, these data explain why ARBs markedly outperform angiotensin-converting enzyme inhibitors in our mouse model of MFS. They also define noncanonical TGF β and AT2 signaling as prognostic and therapeutic modifiers of vascular disease and provide rationale and incentive for additional clinical trials in MFS and related disorders.

HomeHypertensionVol. 76, No. 5Plasma Angiotensin Peptide Profiling and ACE (Angiotensin-Converting Enzyme)-2 Activity in COVID-19 Patients Treated With Pharmacological Blockers of the Renin-Angiotensin System Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBPlasma Angiotensin Peptide Profiling and ACE (Angiotensin-Converting Enzyme)-2 Activity in COVID-19 Patients Treated With Pharmacological Blockers of the Renin-Angiotensin System Ulrich Kintscher, Anna Slagman, Oliver Domenig, Robert Röhle, Frank Konietschke, Marko Poglitsch and Martin Möckel Ulrich KintscherUlrich Kintscher Correspondence to Ulrich Kintscher, Charité–Universitätsmedizin Berlin, Institute of Pharmacology, Center for Cardiovascular Research, Hessische Strasse 3-4, 10115 Berlin, Germany. Email E-mail Address: [email protected] https://orcid.org/0000-0001-7386-0990 From the Charité–Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Germany (U.K., A.S., M.M., R.R., F.K.) Institute of Pharmacology, Center for Cardiovascular Research, Germany (U.K.) DZHK (German Centre for Cardiovascular Research), Partner Site Berlin (U.K.) , Anna SlagmanAnna Slagman From the Charité–Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Germany (U.K., A.S., M.M., R.R., F.K.) Department of Emergency and Acute Medicine, Campus Mitte and Campus Virchow Clinic (A.S., M.M.) , Oliver DomenigOliver Domenig Attoquant Diagnostics, Vienna, Austria (O.D., M.P.). , Robert RöhleRobert Röhle https://orcid.org/0000-0002-8130-6524 From the Charité–Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Germany (U.K., A.S., M.M., R.R., F.K.) Institute of Biometry and Clinical Epidemiology, Coordinating Center for Clinical Studies (R.R.) Berlin Institute of Health, Germany (R.R., F.K.) , Frank KonietschkeFrank Konietschke From the Charité–Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Germany (U.K., A.S., M.M., R.R., F.K.) Institute of Biometry and Clinical Epidemiology (F.K.) Berlin Institute of Health, Germany (R.R., F.K.) , Marko PoglitschMarko Poglitsch Attoquant Diagnostics, Vienna, Austria (O.D., M.P.). and Martin MöckelMartin Möckel https://orcid.org/0000-0002-7691-3709 From the Charité–Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Germany (U.K., A.S., M.M., R.R., F.K.) Department of Emergency and Acute Medicine, Campus Mitte and Campus Virchow Clinic (A.S., M.M.) Originally published27 Aug 2020https://doi.org/10.1161/HYPERTENSIONAHA.120.15841Hypertension. 2020;76:e34–e36Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: August 27, 2020: Ahead of Print Pharmacological blockade of the renin-angiotensin system (RAS) with ACE (angiotensin-converting enzyme) inhibitors or angiotensin type 1 receptor blockers (ARB) reduces morbidity and mortality in various cardiovascular diseases. One of the key RAS-modulating enzymes, ACE2, has recently gained increasing attention because it converts not only angiotensin (Ang) II to the alternative RAS metabolite Ang-(1–7) but also functions as the cellular entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2.1 At the beginning of the SARS-CoV-2 pandemic, some investigators suggested that because ACE inhibitor or ARB may lead to upregulation of ACE2 expression/activity, use of these agents in coronavirus disease 2019 (COVID-19) patients might be associated with worsened outcomes.1 Meanwhile, several observational studies have shown that neither the risk of COVID-19 nor its severity is negatively affected by ACE inhibitor or ARB.2,3 However, it remains unclear how RAS activity, particularly ACE2, is regulated in COVID-19 and how this is altered by ACE inhibitor/ARB therapy. In this study, we analyzed distinct RAS components in plasma from patients with COVID-19 ±ACE inhibitor/ARB therapy using liquid chromatography-mass spectrometry/mass spectrometry.The study was approved by the Charité-Universitäts-medizin, Berlin, Germany, Institutional Ethics Committee (EA2/204/19, Amendment 1) and registered in the German Registry for Clinical Studies (DRKS00019207). Surplus plasma samples were collected at the time of admission to the emergency room from 6 different patient groups (total, n=58 [women, 21]): SARS-CoV-2 negative control group (control, n=9 [4]), SARS-CoV-2 negative with ACE inhibitor (control-ACE inhibitor, n=10 [2]), SARS-CoV-2 negative with ARBs (control-ARB, n=8 [5]), COVID-19 without ACE inhibitor/ARB (COVID, n=12 [5]), COVID-19 with ACE inhibitor (COVID-ACE inhibitor, n=10 [2]), and COVID-19 with ARBs (COVID-ARB, n=9 [3]). Equilibrium levels of Ang-peptides (Ang I, Ang II, Ang-[1–7], and Ang-[1–5]) were measured using liquid chromatography-mass spectrometry/mass spectrometry technology (Attoquant Diagnostics).4 Ang-based markers for ACE (Ang II/Ang I) and plasma renin activity (Ang I+Ang II) were calculated from Ang-peptide levels. ACE2 activity was assayed by a classical kinetic approach applying its natural substrate (ex vivo spiked Ang II) and measuring the turnover to Ang-(1–7)±ACE2 inhibitor MLN-4760. The inhibitor-sensitive ACE2-specific turnover was converted to an ACE2 concentration using a calibration curve of recombinant human ACE2. Ang-peptide concentrations/ratios, ACE2 activity, and age between groups were compared using the Kruskal-Wallis test. In case of a significant result, the Dunn-Test for pairwise comparisons using Bonferroni correction was applied. A P of <0.05 was considered statistically significant, although results have to be considered exploratory.Patient CharacteristicsAge (years, mean±SD): control, 44.8±19.7; control-ACE inhibitor, 63.6±17.8; control-ARB, 73.1±11.4 (P=0.02 versus control); COVID, 50±15.1; COVID-ACE inhibitor, 61.4±20.9; COVID-ARB, 74.2±10.1 (P=0.02 versus COVID); COVID severity (n/group), as defined previously,3 severe (intensive care unit admission, mechanical ventilation, and death): COVID (2), COVID-ACE inhibitor (1), COVID-ARB (1); acute renal failure ([n/group] control-ARB [2], COVID [1], COVID-ARB [1]); diuretic use (n/group): control (0), control-ACE inhibitor (4), control-ARB (6), COVID (0), COVID-ACE inhibitor (1), and COVID-ARB (3). Coexisting conditions are outlined in the Figure (A).ResultsAng-peptide equilibrium concentrations did not significantly differ between the control and COVID groups without ACE inhibitor/ARB treatment (Figure [B], left). More importantly, Ang I+II, Ang II/Ang I, and ACE2 activity were not significantly different between both groups (Figure [C]). These data suggest that patients with COVID-19 are not those with increased RAS activity levels and that particularly COVID-19–induced alternative RAS activation, potentially mediated through circulating ACE2, is not a typical feature in our patient cohort.Download figureDownload PowerPointFigure. Patient characteristics, Ang (angiotensin) peptide profiles, and ACE (angiotensin-converting enzyme)-2 levels. A, The presence of cardiovascular disease (hypertension, coronary artery disease, and chronic heart failure) and type 2 diabetes mellitus depicted as percentage of patients in each group. B, Plasma Ang-peptide concentrations and renin-angiotensin system (RAS) enzymatic cascade are depicted as RAS Fingerprints. The concentration of indicated Ang metabolites is reflected by the size of the corresponding sphere. Blue arrows indicate enzymes that are known to carry out metabolic conversions between connected Ang metabolites. Numbers represent median concentrations (pmol/L) and interquartile ranges in parentheses. C, Ang-based markers for plasma renin activity: Ang I+Ang II and ACE: Ang II/Ang I were calculated from Ang-peptide levels. ACE2 activity was measured as described above. Data are shown as dot plots and median. Significant P values within each group (control and coronavirus disease 2019 [COVID]) are indicated. control: severe acute respiratory syndrome coronavirus 2 negative control group without ACE inhibitor/angiotensin type 1 receptor blocker (ARB) therapy; control+ACE inhibitor: severe acute respiratory syndrome coronavirus 2 negative control group with ACE inhibitor therapy; control+ARB: severe acute respiratory syndrome coronavirus 2 negative control group with ARB therapy; COVID: patients with COVID-19 without ACE inhibitor/ARB; COVID+ACE inhibitor: patients with COVID-19 with ACE inhibitor therapy; COVID+ARB: patients with COVID-19 with ARB therapy. ACEi indicates ACE inhibitor; and CTRL, control.Comparison of all groups, including ACE inhibitor/ARB treatment groups, revealed no significant differences of Ang I+II levels between the groups (Figure [C], upper left). Ang I+II is a reliable marker for plasma renin activity and did not change significantly, despite the use of ACE inhibitor/ARB, while median values were clearly increased in patients on ACE inhibitor/ARB. This is consistent with previous observations demonstrating a broad spectrum of intensity in compensatory renin secretion in patients treated with ACE inhibitor or ARB.4 As expected, patients in the control-ACE inhibitor and COVID-ACE inhibitor group showed increased Ang I and markedly suppressed Ang II levels (Figure [B]), resulting in a significant reduction of the Ang II/Ang I ratio (Figure [C], lower left). Ang-(1–5) levels did not significantly differ between groups, whereas Ang-(1–7) was significantly increased in the COVID-ACE inhibitor group versus COVID without ACE inhibitor/ARB (P=0.01) and versus COVID-ARB (P=0.045). ACE2 activity was significantly higher in patients with COVID-19 treated with ACE inhibitor compared with patients with COVID-19 without ACE inhibitor/ARB (Figure [C], right). ACE2 activity was also increased in the control-ACE inhibitor and control-ARB group but did not reach statistical significance (Figure [C], right). ARB treatment in COVID-19 did not significantly affect ACE2 activity (Figure [C], right).The main findings of this study are as follows: (1) patients with COVID-19 are not characterized by major changes in RAS activity in plasma including ACE2 activity, (2) ACE inhibitor therapy significantly suppressed Ang II/Ang I ratios, the Ang-based marker for ACE, in COVID-19 and in non–patients with COVID-19, and (3) plasma ACE2 activity is increased in patients with COVID-19 treated with ACE inhibitor. These data are consistent with previously published results in SARS-CoV-2–negative patients treated with ACE inhibitor or ARB demonstrating an Ang II/Ang I suppression and a more profound increase of Ang-(1–7) under ACE inhibitor compared with ARBs.4 The data published so far on plasma ACE2 activity and Ang-(1–7) levels in patients without COVID treated with ACE inhibitor or ARBs are controversial.1 Some studies showed an increase in circulating ACE2 activity and Ang-(1–7) levels that cannot be proven by other studies.1 In addition, increased ACE2 activity has been identified in multiple cardiovascular diseases such as hypertension, coronary artery disease, and chronic heart failure, which are usually treated with ACE inhibitor.1 Whether the ACE inhibitor treatment in our study plays a role in ACE2 upregulation or whether these changes are mediated by the increased presence of cardiovascular disease in this group requires further investigation. Furthermore, the clinical significance of the elevated ACE2 activity in patients with COVID-19 treated with ACE inhibitor is currently not completely understood. Whether plasma ACE2 level may be a reliable marker of the full-length membrane bound form1 and whether ACE2 serves as a marker for disease severity or endothelial regeneration in the lung5 need to be clarified in future studies. Some of the major limitations of this study include small sample sizes, lack of a power analysis, lack of any data on blood pressure when the plasma samples were obtained, and lack of any data on duration of illness. Finally, it should be emphasized that the majority of the study patients were not experiencing severe COVID-19. However, we provide for the first time a snapshot of distinct systemic RAS components in patients with COVID-19 under ACE inhibitor/ARB therapy that helps to understand the clinical data on a molecular pharmacological level.AcknowledgmentsWe thank Fabian Holert, Jana Eberst, and Beata Hoeft for the support with sample preparation/handling and clinical data collection.Sources of FundingThis study was supported by institutional funding from the Charité–Universitätsmedizin Berlin, Germany. U. Kintscher is supported by the DZHK (German Centre for Cardiovascular Research) and by the BMBF (German Ministry of Education and Research); BER 5.4 PR, the Deutsche Forschungsgemeinschaft (KI 712/10-1), the BMBF/BfR1328-564 m, and the Einstein Foundation/Foundation Charité (EVF-BIH-2018-440).DisclosuresO. Domenig and M. Poglitsch are employees of Attoquant Diagnostics, Vienna, Austria. U. Kintscher received research grants/speaker honoraria from Bayer. U. Kintscher received speaker honoraria from Berlin Chemie, Boehringer Ingelheim, Daiichi Sankyo, Novartis, Sanofi, and Servier and participated in advisory boards of Berlin Chemie, Boehringer Ingelheim, Novartis, and Sanofi. M. Möckel received research grants/speaker honoraria from Roche Diagnostics and BRAHMS ThermoFisher; M. Möckel received speaker honoraria from Boehringer Ingelheim, Daiichi Sankyo, Novartis, and Bristol Myers Squibb and participated in advisory boards of Daiichi Sankyo and Boehringer Ingelheim. The other authors report no conflicts.FootnotesCorrespondence to Ulrich Kintscher, Charité–Universitätsmedizin Berlin, Institute of Pharmacology, Center for Cardiovascular Research, Hessische Strasse 3-4, 10115 Berlin, Germany. Email ulrich.kintscher@charite.de

In the hippocampus, extracellular signal-regulated kinase (ERK) and the non-receptor protein proline-rich tyrosine kinase 2 (PYK2) are activated by depolarization and involved in synaptic plasticity. Both are also activated under pathological conditions following ischemia, convulsions, or electroconvulsive shock. Although in non-neuronal cells PYK2 activates ERK through the recruitment of Src-family kinases (SFKs), the link between these pathways in the hippocampus is not known. We addressed this question using K(+)-depolarized rat hippocampal slices. Depolarization increased the phosphorylation of PYK2, SFKs, and ERK. These effects resulted from Ca(2+) influx through voltage-gated Ca(2+) channels and were diminished by GF109203X, a protein kinase C inhibitor. Inhibition of SFKs with PP2 decreased PYK2 tyrosine phosphorylation dramatically, but not its autophosphorylation on Tyr-402. Moreover, PYK2 autophosphorylation and total tyrosine phosphorylation were profoundly altered in fyn-/- mice, revealing an important functional relationship between Fyn and PYK2 in the hippocampus. In contrast, ERK activation was unaltered by PP2, Fyn knock-out, or LY294002, a phosphatidyl-inositol-3-kinase inhibitor. ERK activation was prevented by MEK inhibitors that had no effect on PYK2. Immunofluorescence of hippocampal slices showed that PYK2 and ERK were activated in distinct cellular compartments in somatodendritic regions and nerve terminals, respectively, with virtually no overlap. Activation of ERK was critical for the rephosphorylation of a synaptic vesicle protein, synapsin I, following depolarization, underlining its functional importance in nerve terminals. Thus, in hippocampal slices, in contrast to cell lines, depolarization-induced activation of non-receptor tyrosine kinases and ERK occurs independently in distinct cellular compartments in which they appear to have different functional roles.

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the fibrillin-1 gene. Patients with MFS are at risk of aortic aneurysm formation and dissection. Usually, blood pressure-lowering drugs are used to reduce aortic events; however, this is not sufficient for most patients. In the aorta of smooth muscle cell-specific sirtuin-1-deficient mice, spontaneous aneurysm formation and senescence are observed. Resveratrol is known to enhance sirtuin-1 activity and to reduce senescence, which prompted us to investigate the effectiveness of resveratrol in inhibition of aortic dilatation in the Fbn1(C1039G/+) MFS mouse model. Aortic senescence strongly correlates with aortic root dilatation rate in MFS mice. However, although resveratrol inhibits aortic dilatation, it only shows a trend toward reduced aortic senescence. Resveratrol enhances nuclear localization of sirtuin-1 in the vessel wall and, in contrast to losartan, does not affect leukocyte infiltration nor activation of SMAD2 and extracellular signal-regulated kinases 1/2 (ERK1/2). Interestingly, specific sirtuin-1 activation (SRT1720) or inhibition (sirtinol) in MFS mice does not affect aortic root dilatation rate, although senescence is changed. Resveratrol reduces aortic elastin breaks and decreases micro-RNA-29b expression coinciding with enhanced antiapoptotic Bcl-2 expression and decreased number of terminal apoptotic cells. In cultured smooth muscle cells, the resveratrol effect on micro-RNA-29b downregulation is endothelial cell and nuclear factor κB-dependent. Resveratrol inhibits aortic root dilatation in MFS mice by promoting elastin integrity and smooth muscle cell survival, involving downregulation of the aneurysm-related micro-RNA-29b in the aorta. On the basis of these data, resveratrol holds promise as a novel intervention strategy for patients with MFS.

Seven-transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β-arrestins, whose recruitment to the activated receptor is regulated by G protein-coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal-regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT(1A)R) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)-based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well-established function in the desensitization of G-protein activation, GRK2 exerts a strong negative effect on β-arrestin-dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2-dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT(1A)R, and HEK293 cells expressing other 7TMRs.

Angiotensin II induces fibronectin expression in human peritoneal mesothelial cells via ERK1/2 and p38 MAPK