bluuebunny/crossref_metadata_2025_split huggingface dataset:
GRK5‐mediated inhibitory phosphorylation is essential for inverse agonism at the cardiac mineralocorticoid receptor
Authors: Desimine Victoria Lynn , Parker Barbara Marie , Wertz Shelby Lynn , Duarte Yanelys , Maning Jennifer , McCrink Katie Anne , Pollard Celina Marie , Koch Walter J , Lymperopoulos Anastasios 
Published: June 2021

BackgroundIn the heart, aldosterone binds the mineralocorticoid receptor (MR) to exert damaging effects, e.g. fibrosis, apoptosis, oxidative stress, etc. GRK2 and GRK5 are the most abundant cardiac G protein‐coupled receptor (GPCR)‐kinases (GRKs). Both phosphorylate GPCRs but also non‐GPCR substrates. We recently showed that GRK5 blocks the cardio‐toxic MR‐dependent effects of aldosterone in the heart by directly phosphorylating the cardiac MR and inhibiting its transcriptional activity. MR antagonist (MRA) drugs are beneifical in human CHF by blocking the cardiac MR. Novel non‐steroidal MRAs, such as finerenone, may provide better cardio‐protection against aldosterone's cardio‐toxic actions than the classic steroidal MRAs, such as sprironolactone and eplerenone. Indeed, finerenone was very recently shown to be a more potent and efficacious inverse agonist at the MR than eplerenone in terms of cardiac fibrosis/adverse remodeling attenuation. Thus, in the present study, we sought to investigate potential differences between finerenone and eplerenone at engaging GRK5‐dependent cardiac MR phosphorylation and subsequent blockade.MethodsWe used the cardiomyocyte cell line H9c2 and neonatal rat ventricular myocytes (NRVMs). We measured MR phosphorylation via phospho‐serine immunoblotting & transcriptional activity via the luciferase reporter assay.ResultsGRK5 phosphorylates the MR in H9c2 cardiomyocytes in response to finerenone but not to eplerenone. Contrary to eplerenone, finerenone alone potently and efficiently suppresses MR transcriptional activity in H9c2 cardiac myocytes. GRK5‐dependent phosphorylation is necessary for the robust inverse agonism displayed by finerenone at the cardiac MR, since CRISPR‐mediated GRK5 gene deletion renders finerenone incapable of blocking cardiac MR transcriptional activity. Conversely, this non‐steroidal MRA fully suppresses cardiac MR transcriptional activity upon GRK5 overexpression in H9c2 cardiomyocytes. Eplerenone alone cannot suppress cardiac MR basal activity upon GRK5 overexpression or knockout. Finally, finerenone blocks aldosterone's effects more efficiently than eplerenone in control and GRK5‐overexpressing NRVMs.ConclusionsFinerenone appears a more efficacious cardiac MR blocker than eplerenone, partly via GRK5‐dependent cardiac MR inhibition, which eplerenone does not induce. This non‐canonical effect of GRK5 on the MR is essential for the efficient blockade of aldosterone's deleterious actions in the heart.Support or Funding Information1) American Heart Association 2) NSU President's Faculty Research & Development GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PARTICIPATION OF ESTRADIOL AND NITRIC OXIDE ON THE VASOCONSTRICTOR EFFECT OF BRADIKYNIN
Authors: Campos Maria Elena Hernández , Romero Karla Stephania Dominguez , Hernandez Ignacio Valencia 
Published: June 2021

Ovarian hormones privation in menopause can contribute to development of hypertension, which has high morbimortality in the world. Estrogens protect cardiovascular system by reducing angiotensin‐converting enzyme (ACE) activity, preventing bradykinin degradation and promoting a reduction in the vascular tone. These actions of estrogen are related with an increase in the synthesis of nitric oxide. However, bradykinin can produce, under certain conditions, vasoconstriction, a much less studied effect. Therefore, the objective of this work was to study the role of 17‐β estradiol (E2) on the vasoconstrictor effect of bradykinin. We used a group of 24 Wistar ovariectomized rats (OVX) and 6 Sham rats. After 14 days of recovery, a group of 6 OVX rats were administered wit E2 (5 μg/Kg/day s.c. for 5 days) and 6 OVX rats were administered with corn oil (vehicle). At the end of the treatment, rats were sacrificed and thoracic aorta extracted and cut in 5 mm length rings, subsets of aortas were obtained with and others without endothelium. Bradikynin concentration‐response curves were constructed, both in the presence and absence of L‐NAME. All procedures in animals complied with NOM‐062‐ZOO‐1999 and institutional animal care committee. The results showed that ovariectomy or treatment with estradiol did not modify blood pressure values. Treatment with estradiol, however, significantly reduced heart rate in OVX rats. Bradykinin produced a concentration‐dependent contractile effect that was of greater magnitude in rings without endothelium and was blocked by HOE140. Incubation with L‐NAME did not modify the contractile effect of bradykinin in the aortic rings from SHAM rats but inhibited this effect in the OVX treated rat with E2. These results indicate that in the presence of ovarian hormones, where estradiol seems to play an important role, bradykinin produces contraction independently of the endothelium and NO, this contraction seems to be mediated by the activation of B2 receptors.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ceramide‐Mediated Enhancement of Inflammatory Exosome Secretion from Podocytes during Hyperhomocysteinemia
Authors: Zhang Qinghua , Li Guangbi , Hong Jinni , Lohner Hannah , Li Pin‐Lan 
Published: June 2021

Exosome secretion has been shown increased during podocyte injury and associated glomerular diseases, and this increased exosome secretion may be important in mediating release of inflammasome products to activate glomerular inflammatory response leading to chronic glomerular disease. Given the crucial role of ceramide in the control of exosome biogenesis and release, the present study used a podocyte‐specific sphingomyelin phosphodiesterase 1 (Smpd1) transgenic mouse strain (Smpd1trg/Podocre mice) to test whether ceramide production enhanced by Smpd1 gene overexpression promotes inflammatory exosome release from podocyte leading to glomerular inflammation during Hcys. We demonstrated that in Smpd1trg/Podocre mice, hHcy (induced by the FF diet) produced significant podocyte inflammasome activation accompanied by large increases in urinary podocyte‐derived exosomes. By immunohistochemical detection, we found that the glomerular inflammatory responses was more remarkably activated by hHcy in Smpd1trg/Podocre mice than their littermates, as shown by increased staining of lymphocyte and T‐cell markers such as CD45 and CD43. Under electronic microscopy, we observed that hHcy induced more severe podocyte injury and exosome release around these podocytes in Smpd1trg/Podocre mice than their littermates. Using confocal microscopy, about 2.8 folds increase colocalization of multivesicle body marker (MVB) Vps16 and NLRP3 inflammasome product IL‐1β, but about 0.4 folds decrease interaction of Vps16 with lysosome (Lamp‐1) (a process determining MVB fate and exosome release) were found in the glomeruli of Smpd1trg/Podocre mice with hHcy compared to their littermates. In cultured podocytes, the nanoparticle tracking analysis confirmed that L‐Hcy treatment significantly increased microparticle release into supernatants especially from 50nm to 150nm and they were characterized as podocytes‐derived IL‐1β‐containing exosomes as shown by detection of synaptopodin, CD63 and IL‐1β via Western blot analysis. This L‐Hcy‐induced podocyte release of IL‐1β exosomes was mimicked by AC inhibitor carmofur, but attenuated by AC inducer, genistein. It was also found that L‐Hcy‐induced decreases in Vps16 and Lamp‐1 interaction could be simulated by carmofur and inhibited by genistein. All these results together suggest that increased ceramide in podocytes enhances release of inflammatory exosomes during hHcy by reduction of lysosome‐MVBs interaction and prolongation of MVBs to facilitate exosome secretion.Support or Funding Informationsupported by NIH grant DK54927, DK120491 and HL057244This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FACTORS ASSOCIATED TO THERAPEUTIC FAILURE OF LOSARTAN IN PATIENTS WITH HEART FAILURE. PHARMACOKINETIC MODEL
Authors: De León Díaz de León Edgar Alejandro , Almendra‐Pegueros Rafael Antonio , Gonzalez‐Correa Jose Antonio , Sanchez Teresa Tejerina , Moreno Ursula Fabiolo Medina , Moscoso Antonio Augusto Gordillo 
Published: June 2021

BackgroundDecompensated heart failure is a common cause of hospitalization. Therapeutic use of ARA‐II has demonstrated safety in this patients. Circulating drug concentrations can be affected by some factors (age, sex, body surface, formulation), and variations in levels of plasma can be observed, causing a therapeutic failure and therefore, greater hemodynamic instability.AimTo Identify factors that affect plasma drug concentration (Losartan patent vs generic) and its active metabolite (E‐3174) as well as possible factors that affect the therapeutic failure in patients with heart failure.MethodsA double‐blind randomized controlled clinical trial in a single dose (n = 60), comparing Losartan potassium (50 mg) patent Vs generic, This study was approved by the ethics committee, registration number 98‐15. Inclusion criteria: both sexes, stable patients with FEVI less than 40% and informed consent signed. To determine the pharmacokinetic profile, the method was validated to show compliance with international requirements ICHQ2 (R1). After oral administration of the drug, a 12‐hour follow‐up was performed. The data were analyzed by means of multiple linear regression and t‐test in RStudio.Results24/60 patients have been analyzed. Mean age 56.2 ± 13.8 years, time of evolution of HF of 1–3 years. The developed and validated analytical method proved to be sensitive, linear, accurate, precise and with fewer run‐time (5 min Vs 15 min) than the standard method. The area under the curve (AUC) of Losartan patent and generic was 3.28 ± 0.77 and 3.12 ± 0.90 (p = 0.64) respectively and for its metabolite E‐3174 patent and generic was 5.27 ± 0.89 and 4.51 ± 0.88 (p = 0.56) respectively.The maximum concentration (Cmax), was greater in the patent formulation, the metabolite E3174, was lower in male and patients with higher BMI, these data aren't a statistical difference.Conclusionsthe differences in AUC and Cmax between both formulations aren't statistically significant, however, when analyzing the metabolite E‐3174 we see that after 6 hours, the patent formulation shows an increase in the concentration that is not observed in the generic, which can affect the pharmacological effect in the patient. The therapeutic effect (blood pressure reduction) was better for patent formulation Vs generic, even without statistically significant. For these reasons is essential to complete the sample size to establish a more accurate conclusion.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chalcone derivative L6H21 reduces EtOH‐LPS‐induced liver injury through inhibition of NLRP3 inflammasome activation
Authors: Gu Zelin , McClain Craig , Feng Wenke 
Published: June 2021

Chronic alcohol intake increases circulating endotoxin levels causing excessive inflammation that aggravates the liver injury. L6H21, a derivative of chalcone, has been found to inhibit inflammation in cardiac diseases and non‐alcoholic fatty liver disease. However, use of L6H21 in alcoholic liver disease (ALD) to inhibit exotoxin‐associated inflammation has not been explored. In this study, we examined the effects of L6H21 on EtOH‐LPS‐induced hepatic inflammation, steatosis and liver injury, and investigated the underlying mechanisms. C57BL6 mice were treated with 5% ethanol for ten days, and LPS was applied on the last day. L6H21 was given with alcohol. EtOH‐LPS treatment significantly increased hepatic steatosis and serum ALT and AST levels, which were reduced by L6H21 treatment. L6H21 treatment significantly reduced hepatic protein levels of TLR4, p‐P65 and p‐IκB, and inhibited p65 translocation into the nucleus. In addition, L6H21 treatment markedly reduced hepatic protein levels of NLRP3, cleaved caspase‐1 and cleaved‐IL‐1β, which were increased by EtOH‐LPS exposure. Moreover, L6H21 treatment effectively inhibited caspase‐1 associate apoptosis. In vitro study using Raw264.7 cells confirmed the effects of L6H21 on macrophage activation. Taken together, our results demonstrate that L6H21 suppresses the LPS/TLR4/NF‐κB‐mediated inflammation and NLRP3 inflammasome activation in ethanol and LPS‐induced liver injury. L6H21 might be used an alternative strategy for ALD prevention/treatment.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Asparaginase Immune Complexes Induce FcγRIII‐Dependent Basophil Activation and Concentration‐Dependent Hypersensitivity Reactions
Authors: Rathod Sanjay , Ramsey Manda , Finkelman Fred D , Fernandez Christian A 
Published: June 2021

BackgroundAsparaginase (ASNase) is an important drug for treatment of pediatric acute lymphoblastic leukemia. However, antibody responses to ASNase increase the risk of leukemia relapse by mediating ASNase‐induced hypersensitivity and blocking ASNase's therapeutic effects. Previously, we have demonstrated two mechanism of ASNase hypersensitivity: one involving an anti‐ASNase IgG response, and another involving cell‐associated anti‐ASNase IgE antibodies. These studies suggested that formation of ASNase immune complexes (ICs) is sufficient to induce ASNase hypersensitivity.ObjectiveTo detect ASNase ICs in mice after the onset of ASNase hypersensitivity and determine whether FcγRIII engagement by ASNase ICs can lead to immune cell activation.MethodsMice were sensitized to native E. coli ASNase using aluminum hydroxide adjuvant. Six μm protein G‐coated polystyrene beads were used to detect plasma ASNase ICs by flow cytometry after the onset of ASNase‐induced hypersensitivity. Fluorochrome‐labeled ASNase was used to detect ASNase IC binding to immune cells. The basophil activation test (BAT) was used to evaluate ASNase IC effects on basophils.ResultsASNase ICs were detected after the onset of murine ASNase hypersensitivity reactions and ASNase IC concentration strongly correlated with reaction severity (r2= 0.796, P=0.0005). ASNase ICs formed ex vivo at high anti‐ASNase IgG1 antibody concentrations. ASNase IC binding to naïve and sensitized peripheral blood neutrophils, macrophages/monocytes, and basophils was FcγRIII‐dependent. Consistent with these results, the BAT demonstrated that ASNase IC activation of naïve and sensitized basophils was FcγRIII‐dependent.ConclusionASNase‐sensitized mice form ICs after ASNase administration, which activate murine basophils, and likely mast cells, via FcγRIII. This suggests that ASNase ICs can contribute to the onset and severity of ASNase hypersensitivity.Support or Funding InformationUniversity of Pittsburgh School of Pharmacy and NIH Grants RO1 CA216815 to CAFThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transmission electron microscopic analysis of mitochondria changes in pulmonary arterial hypertension
Authors: Rybka Vladyslava , Shults Nataliia 
Published: June 2021

Mitochondria are dynamic organelles, which play crucial roles in many cellular processes. Their severe damage results in alterations of other ultrastructural components and leads to loss of cellular function. In pulmonary hypertension (PH), increasing of the pulmonary vascular resistance puts the strain on the right ventricle, leading to the demand for ATP production by mitochondria to ensure cardiomyocyte function. The failure of cardiac pump increases the peripheral resistance by inducing the neurohormonal activation and influences the skeletal muscle structure and function, leading to the muscle weakness. In a rat PH model, our previous morphometric study reported changes in the mitochondrial types. Type I–II mitochondria are defined as mitochondria with the preserved ultrastructure and Type III–IV mitochondria are defined as mitochondria with the severe cristae damage that are not able to produce the required amount of ATP for energy‐depended processes in cardiomyocytes. Fischer rats had 100% mortality because of right heart failure in PH. In their RV cardiomyocytes, the types III–IV of mitochondria were predominant, along with a drastic decrease in coefficient of energy efficiency of mitochondria (CEEM), while in LV types II–III of mitochondria were present. Whereas the SD rats that do not die of PH, mostly had mitochondria Type I–II in RV and LV, along with slight decrease in CEEM in RV. In both strains of rats with PH, the treatment with a Bcl‐2 inhibitor, Navitoclax (ABT‐263), lead to severe damage of the mitochondria structure, causing rats to die because of hearth failure. The transmission electron microscopy (TEM) analysis of mitochondria in skeletal muscle cells in PH revealed the decrease in mitochondria volume and the damage to the mitochondria structure. In skeletal muscle cells, Type III mitochondria were present in rats, which correlates with the reduction in aerobic capacities. These results suggest that the ultrastructural defects in mitochondria in both cardiac and skeletal muscle in PH impact the development of the hearth failure and death, thus PH can be described as a systemic disease.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sigma‐1 receptor agonists elicit arteriolar dilation via Akt/eNOS activation, and also have endothelial barrier protective properties
Authors: Motawe Zeinab Y. , Katnik Christopher P. , Trujillo Andrea N. , Cuevas Javier , Breslin Jerome W. 
Published: June 2021

Sigma‐1 receptors (σ1) have previously gained considerable attention pertaining to neuroprotection through its actions on calcium homeostasis, and the mitochondria‐associated ER membrane domain. Our laboratory recently showed that the σ1 agonist afobazole causes an endothelial NO‐dependent decrease in contraction amplitude. However, little is known about the mechanisms and functions of σ1 receptor in endothelial cells. In the current study, we investigated how σ1 agonists impact brain arteriolar tone and the Akt/eNOS pathway. Parenchymal arterioles were isolated from brains of Sprague‐Dawley rats and mounted onto glass micropipettes in a 37 °C bath containing albumin‐physiological salt solution. The nonselective σ agonist afobazole was applied to the bath at concentrations of 50, 100, 150, 200, and 250 μM in the presence or absence of the σ1 antagonist BD1047. The selective σ1 agonist, PRE‐084 was used to stimulate human umbilical vein endothelial cells (HUVEC), in which phosphorylation of endothelial NO synthase (eNOS) or Akt phosphorylation on their activation sites (S1177 and S473, respectively) were evaluated by western blotting. NO production was measured by fluorescence microscopy in cells loaded with the indicator dye DAF‐FM. Trans‐endothelial electrical resistance (TER) was used to determine the ability of PRE‐084 to attenuate disruption of endothelial barrier function caused by the the mitochondrial function inhibitors CCCP and antimycin‐A, or the inflammatory cytokine IL‐1β. The results show that afobazole significantly dilated rat brain arterioles, which was inhibited by the σ1 antagonist BD‐1047. Selective activation of σ1 with PRE‐084 significantly increased eNOS phosphorylation on S1177 and this was diminished in the presence of BD‐1047. Akt phosphorylation on S473 following PRE‐084 treatment was also enhanced. In addition, afobazole and PRE‐084 enhanced NO production in endothelial cells. PRE‐084 significantly attenuated decreases in TER caused by IL‐1β or CCCP, but not antimycin‐A, suggesting a potential barrier‐protective role for σ1 receptor, with some limitations that may be related to the source of mitochondrial dysfunction during inflammation. Collectively, our results suggest that σ1 receptor activation can cause vasodilation via the Akt/eNOS pathway in endothelial cells, and that σ1 may enhance endothelial barrier function under inflammatory conditions.Support or Funding InformationSupported by NIH grant R01GM120774.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prolonged Administration of the KCa Channel Activator SKA‐31 Improves Cardiac Function and Blood Pressure in Type 2 Diabetic Goto‐Kakizaki Rats
Authors: Mishra Ramesh C , Belke Darrell , John Cini Mathew , Wulff Heike , Braun Andrew P 
Published: June 2021

Endothelial dysfunction represents an independent risk factor contributing to the onset and progression of cardiovascular complications (e.g. hypertension, cardiomyopathy, stroke) in patients with Type 2 Diabetes (T2D). It is further predicted that reduction/reversal of endothelial dysfunction will mitigate T2D‐associated cardiovascular deficits. Small‐ and intermediate‐conductance, Ca2+‐activated K+ channels (KCa2.3 and KCa3.1, respectively) are prominently expressed in the vascular endothelium, and pharmacologic activators of these channels (e.g. SKA‐31) induce robust vasodilation upon acute application to isolated resistance arteries and intact animals. However, the effects of prolonged, in vivo administration of a KCa activator on the cardiovascular system have not been examined to date. In our current study, we have hypothesized that daily administration of SKA‐31 (10 and 30 mg/kg, I.P. injection) to male, non‐obese Type 2 Diabetic Goto‐Kakizaki rats (T2D GK, 14 weeks of age) for 12 weeks would oppose the development of diabetes‐related cardiac dysfunction. Echocardiographic analyses revealed that left ventricular (LV) ejection fraction, stroke volume and fractional shortening all significantly declined over time in vehicle‐treated rats, but remained at pre‐trial levels in animals administered either 10 or 30 mg/kg SKA‐31 for 12 weeks (n = 5–6/group). Chronic blood pressure measurements over the same period via radiotelemetry further revealed that SKA‐31 treatment lowered mean arterial pressure, compared with vehicle treated animals, with effects observed on both systolic and diastolic pressures (n = 5–6/group). To determine if the observed improvements in cardiac performance may be due to direct changes in the responsiveness of the coronary vasculature, isolated hearts from vehicle and SKA‐31 treated T2D GK rats were mounted in a Langendorff perfusion system under constant pressure (n ≥ 3/group). We observed that endothelium‐dependent, agonist‐evoked increases in coronary flow were similar in hearts from vehicle and SKA‐31 treated animals, as was baseline LV developed pressure. Histological staining of isolated hearts revealed differing levels of LV collagen content in the 3 treatment groups. In summary, these results indicate that prolonged SKA‐31 administration in T2D GK rats can improve cardiac function, which likely occur via both direct and indirect effects.Support or Funding InformationThis study was supported by research funding to APB from the Canadian Institutes of Health Research (MOP‐142467) and the Natural Sciences and Engineering Research Council of Canada (RGPIN‐2017‐04116), and to HW from the National Institutes of Health (R21 NS101876).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.