bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reciprocal Regulation of eNOS and Caveolin‐1 Function in Endothelial Cells
Authors: Chen Zhenlong , Minshall Rihcard 
Published: November 2023

Endothelial nitric oxide (eNOS)‐mediated NO production plays a critical role in the regulation of vascular tone, adhesivity, and maintenance of endothelial barrier function. Caveolin‐1 (Cav‐1), which is found highly expressed in endothelia, is an important regulator of vascular tone via modulation of eNOS activity. eNOS‐derived NO may also affect Cav‐1 functions directly and indirectly through nitrosation and phosphorylation‐dependent modifications, although the exact molecular mechanisms and effects on eNOS expression and activity in endothelial cells are not clear. Here, reciprocal regulation by the two proteins was investigated. First, we determined by semi‐quantitative Western blotting that the ratio Cav‐1 to eNOS expression in primary culture endothelial cells was approximately 200:1. Secondly, upon stimulation of ECs with Ca2+ ionophore A23187 to activate Ca2+‐dependent signaling, Cav‐1 nitrosation, phosphorylation, and monomerization occurred within minutes which could be blocked by L‐NAME (eNOS inhibitor) and PP2 (Src kinase inhibitor). Furthermore, oligmerization of Cav‐1 was enhanced when eNOS expression was reduced by siRNA. These data suggest that eNOS, through a NO‐Src signaling pathway, induces a conformational change to the functional oligomeric form of Cav‐1. Thirdly, Cav‐1 knockdown by siRNA reduced eNOS expression level, although NO production and eNOS phosphorylation per molecule were elevated 2‐fold. Finally, in HEK cells overexpressing eNOS, transfection of Cav‐1 resulted in a concentration‐dependent increase followed by a decrease in eNOS expression and activity. Thus, eNOS‐derived NO production promotes Cav‐1 nitrosylation, phosphorylation and monomerization, while lower levels of Cav‐1 expression stabilize eNOS expression and higher levels inhibit eNOS activity via phosphorylation‐dependent binding as part of a negative feedback regulatory mechanism.Support or Funding InformationFinancial Support: P01 HL60678 and Department of Anesthesiology
Endothelium‐Derived Microvesicles Alter Permeability of Human Brain Microvascular Endothelial Monolayers
Authors: Hunter Larry W. , Jayachandran Muthuvel , Miller Virginia M. 
Published: November 2023

The disruption of the cerebral microvascular endothelium and increases in capillary permeability are characteristic of many brain pathologies, including Alzheimer's disease, small vessel disease, ischemia/hypoxia, and hemorrhagic stroke. This study was designed to investigate whether microvesicles (MVs) released from activated human brain microvascular endothelial cells (HBMECs) alter permeability of endothelial monolayers. Male donor HBMECs were cultured to confluence in flasks, then treated for 24 hrs with medium alone (control) or with medium plus either tumor necrosis factor (TNFa, 20 ng/ml), thrombin (THR, 2 U/ml), angiotensin II (AngII, 10 nM), or hydrogen peroxide (H2O2, 0.8 mM). MVs released into the medium were isolated by differential centrifugation, labeled with annex in V plus antibodies against endothelial adhesion molecules which have modified expression during inflammation (PECAM‐1, ICAM‐1, E‐select in, integrin aV, or MCAM); and then analyzed by FACS. Total number of MVs released (MVs/ml) by each of the agonists (TNFa, 615.8±13.9 MV/μL; THR, 113.8±4.8 MV/μL; and H2O2, 152.8±23.5 MV/μL) were significantly greater (P<0.05) compared the number released from cells treated medium alone (82.2±5.4 MV/μL). Each agonist treatment released distinct types of MV identified by FACS. Permeability (% control) of the HBMEC monolayer assessed using FITC‐dextran transport across the monolayers increased after 90 minute exposure to all agonist treatments: 199.5%, 234.1%, 126.8%, and 588.3% for TNFa, THR, AngII, and H2O2, respectively. MV derived from the treated cells were labeled with PKH67 and applied to monolayers of (control) HBMECs cultures. Within 30 minutes of exposure punctate structures with PKH67 labeling were observed within the cytoplasm, and after 60 min, similar structures also associated with nuclei and some of which were co‐labeled with the lysosome marker lysotracker red. Co‐localization of labeled MVs with early endosomes, (labeled with anti‐EEA‐1 Ab/Alexa Fluor 647) was absent at these time points. Within these parameters, no differences in vesicle‐trafficking were observed between MVs generated from cells exposed to either medium, TNFa, or H2O2. These results suggest that treatment of HBMECs with agonists associated with inflammation and thrombosis increase release of distinct MVs which in turn increase paracellular permeability. Capillary endothelial permeability may be disturbed by populations of MVs released from proximal activated cells or MV derived from activated cells and carried in the blood.Support or Funding InformationSupported by the Aurora Fnd, NIH HL88988 and Mayo Clinic
How the activity of PKCα is increased and maintained in Heart Failure despite the up‐regulation of molecular braking mechanism?
Authors: Aslam Naveed , ZAHEER IRUM 
Published: November 2023

Heart failure (HF) disease is marked by dampened cardiac contractility. A milder target which could improve the contractile function without desensitization of β‐Adregenric system may help during HF condition and potentially improve survival. It seems that inhibition of PKCα activity might fit this criterion of a milder therapeutic target to boost contractility for HF patients. Observations show that PKCα activity is increased during HF disease. This seems to be bizarre as the increase in PKCα activity is also accompanied by up‐regulation of a molecular braking mechanism. Here, we explore the question that how PKCα activity can be increased and maintained during HF despite the presence of molecular brakes? More specifically, we investigate the possibility of a positive feedback loop through which PKCα might sustain its activity in heart disease condition. Through a computational approach we show that PKCα activity in cardiomyocytes is regulated through a local DAG signaling system. Our results also show that local DAG cascade is regulated through a two compartment signaling system. Our results imply that after large MI local homeostasis of DAG is disrupted and the loss of this balance leads to prolonged activation of PKCα, a key molecular target linked with LV remodeling and dysfunctional filling and ejection characteristics in mammalian heart. This study also proposes an explanation for how the DAG homeostasis is regulated during normal systolic and diastolic functions?Support or Funding InformationPrivately funded
Spilanthol from Acmella oleracea lowers Na+‐K+‐2Cl− ‐cotransporter activity and water channel aquaporin 2 membrane expression in mouse renal cells
Authors: Gerbino Andrea , Schena Giorgia , Milella Luigi , Miglionico Rocchina , Armentano Mariafrancesca , Barbosa Alan Franco , Procino Giuseppe , Svelto Maria , Carmosino Monica 
Published: November 2023

ObjectiveAcmella oleracea is well recognized in Brazilian traditional medicine as diuretic, although few scientific data have been published to support this effect. Aim of this study was to determine the molecular effect of Acmella oleracea extract and its main alkylamide spilanthol on two major processes involved in the urine concentrating mechanism: Na‐K‐2Cl symporter (NKCC2) activity in the thick ascending limb and water channel aquaporin 2 accumulation at the apical plasma membrane of collecting duct cells.MethodsPhosphorylation of NKCC2 was evaluated as index of its activation by Western blotting. Rate of aquaporin 2 apical expression was analysed by confocal laser microscopy. Spilanthol‐induced intracellular signalling events were dissected by video‐imaging experiments.ResultsExposure to spilanthol reduced the basal phosphorylation level of NKCC2 both in freshly isolated mouse kidney slices and in NKCC2‐expresing HEK293 cells. Spilanthol‐induced effect on NKCC2 activity was significantly larger than that exerted by the methanol plant extract. In addition, exposure to spilanthol strongly reduced both desmopressin and low Cl−‐dependent increase in NKCC2 phosphorylation in mouse kidney slices and NKCC2‐expressing HEK293 cells, respectively. Similarly, exposure to spilanthol reduced both desmopressin‐ and forskolin‐stimulated aquaporin 2 accumulation at the apical plasma membrane of collecting duct in mouse kidney slice and MCD4 cells. Finally, at cellular level, spilanthol rapidly reduced or reversed basal and agonist‐increased cAMP levels through a mechanism involving increases in intracellular [Ca2+].ConclusionsSpilanthol‐induced inhibition of cAMP production negatively modulates urine‐concentrating mechanisms thus holding great promise for its use as both diuretic and aquaretic.Support or Funding InformationThis work was supported by the Ricerca di interesse locale RIL to M. Carmosino, University of Basilicata and by Fondo per gli Investimenti della Ricerca di Base‐Rete Nazionale di Proteomica to M. Svelto (grant number RBRN07BMCT_009).
Postconditioning Reduces Early Reactive Oxygen Species Production in the Brain in a Porcine Model of Cardiac Arrest and Resuscitation
Authors: Matsuura Timothy R , Yannopoulos Demetris , Bartos Jason A , Olson Matthew D , Rees Jennifer N , Tsangaris Adamantios , Shekar Kadambari Chandra , McKnite Scott H , Aufderheide Tom P , Bienengraeber Martin P , Neumar Robert W , Dikalova Anna E , Dikalov Sergey I , Douglas Hunter F , Salzman Michele M , Riess Matthias L 
Published: November 2023

IntroductionPostconditioning during cardiopulmonary resuscitation (CPR) is an effective intervention to improve neurological function and survival in a porcine model of prolonged cardiac arrest (CA). We tested if ischemic (IPoC) and anesthetic postconditioning (APoC) decrease reactive oxygen species (ROS) production in the brain upon reperfusion with CPR.MethodsAfter 15 min of untreated ventricular fibrillation, 10 pigs were randomized to receive active compression/decompression CPR with IPoC (three 20‐sec pauses in CPR), APoC with 4 Vol% sevoflurane for 3 min, or without postconditioning (control) before organ harvest at 4 min. Cortical brain tissue was immediately frozen in liquid nitrogen and stored at −80°C until ROS measurements were performed by Electroparamagnetic Resonance (EPR). Data are mean±SD. Statistics: ANOVA with SNK‐post hoc testing, alpha = 0.05.ResultsEPR spectral changes showed that APoC (7.0±0.3 arbitrary units) more so than IPoC (7.9±0.3) reduced ROS compared to controls (9.1±0.2).ConclusionsBoth IPoC and APoC result in decreased ROS production in the brain during early CPR. Either strategy has been shown to improve mitochondrial coupling of oxidative phosphorylation and ATP synthesis and delay mPTP opening post CA. Together, our findings provide further evidence that preservation of mitochondrial function is a key element of improving neurological outcome after CA and CPR.Support or Funding InformationR01HL123227
Malignant Hyperthermia‐associated Mutation of RyR1 Induces Mitochondrial Damages and Cellular Oxidation in the Heart
Authors: O‐Uchi Jin , Mishra Jyotsna , Jhun Bong Sook , Hurst Stephen , Fu Deming , Gomez Ludovic , Sheu Shey‐Shing 
Published: November 2023

Human pedigrees carrying the ryanodine receptor type 1 (RyR1) mutations frequently show skeletal muscle (SM) disorders including malignant hyperthermia (MH). There are also case reports describing sudden cardiac death (SCD) in MH patients in the conscious condition without anesthesia. These observations cannot be explained as a secondary effect of their SM dysfunction; however, the molecular mechanism underlying cardiac phenotypes in MH is still unknown. We previously reported that a low level of RyR1 is expressed in the mitochondria (termed as mitochondrial RyR1: mRyR1), but not in the sarcoplasmic reticulum (SR) in the cardiomyocytes and serves as an important mitochondrial Ca2+ influx pathway in addition to the mitochondrial Ca2+ uniporter in cardiomyocytes. We also reported, using knock‐in mice carrying a MH‐related RyR1 mutation Y522S (YS) that YS hearts exhibit disrupted mitochondrial morphology as well as compromised mitochondrial functions with a high cellular oxidative state, which can account for cardiac phenotypes in MH. In addition, YS heart developed multiple ventricular extrasystoles by β‐adrenergic stimulation. Therefore, we hypothesize that YS‐RyR1s form “leaky channel” at mitochondria and induce mitochondrial Ca2+ overload, which alters the cellular Ca2+ handling in cardiomyocyte. Using isolated mitochondria or isolated cardiomyocytes under confocal microscope, we found that YS cardiomyocytes have higher basal mitochondrial Ca2+ concentration, depolarized mitochondrial membrane potential and slower cytosolic Ca2+ clearance as compared to WT. Moreover, pretreatment of RyR1 blocker dantrolene prevented these changes in YS cardiomyocytes and normalized their Ca2+ handling profiles similar to those in WT. In conclusion, these results indicate that chronic mitochondrial Ca2+ overload via leaky mutant mRyR1 damages cardiac mitochondrial functions/structures, reduces cytosolic Ca2+ buffering capacity and induces cellular oxidation, which may increase arrhythmogenic events in MH.Support or Funding InformationThis work was partly supported by American Heart Association (AHA) grant (14BGIA18830032 to J.O.‐U.), Medical Research Grant from W.W. Smith Charitable Trust (H1403 to J.O.‐U.) and NIH grants (2R01HL093671 and 1R01HL122124 to SSS).
Angiotensin II‐mediated Mitochondrial Ca2+ Uptake and Superoxide Generation Activate Proliferative Pathway in Neonatal Cardiac Fibroblasts
Authors: O‐Uchi Jin , Fu Deming , Mishra Jyotsna , Jhun Bong Sook , Hurst Stephen , Sheu Shey‐Shing 
Published: November 2023

Cardiac fibroblasts (CFs) are the most prevalent cell type in the heart in addition to cardiomyocytes and play key roles in regulating myocardial physiological function and pathophysiological remodeling. Clinical observations and basic research data strongly suggest that CFs can respond to various stimuli including, angiotensin II (AT‐II), the levels of which are increased in the remodeling heart and participates in remodeling of the failing heart by AT‐II‐mediated pathological CF proliferation. It has been shown that CF proliferation may occur via enhanced production of reactive oxygen species (ROS), but the detailed signal transduction remains unclear. We previously reported that the enhancement of mitochondrial Ca2+ uptake by mitochondrial Ca2+ uniporter (MCU) induces mitochondrial superoxide (mtSO) generation in cardiomyocytes. Therefore, we hypothesize that Ang‐II stimulation enhances mitochondrial Ca2+‐ induced mtSO generation in CFs, which can activate ROS‐dependent proliferation signaling in CFs. First, using a mitochondria‐targeted Ca2+ biosensor, we found that AT‐II (≥1 μM) stimulation induces significant mitochondrial Ca2+ uptake in response to the Ca2+ release from the endoplasmic reticulum in neonatal rat CFs (NCF). In addition, AT‐II stimulation increases the mtSO levels detected by a mtSO indicator MitoSOX Red. These effects were significantly blocked by the pretreatment of an AT‐receptor antagonist losartan. Next, we confirmed that AT‐II application activates proliferative pathway, including ERK1/2, p38 and JNK1/2 in time‐dependent manner, which was abolished by losartan pretreatment. Lastly, pretreatment of a mitochondria‐targeted antioxidant, Mito‐tempo also significantly inhibited AT‐II‐mediated activation of the proliferative pathway without changing the AT‐II‐induced the mitochondrial Ca2+ uptake profile. Our results indicate that mtSO generation induced by mitochondrial Ca2+ accumulation works as a stimulator of the Ang II–induced proliferative pathway in cardiac fibroblasts.Support or Funding InformationThis work was partly supported by American Heart Association (AHA) grant (14BGIA18830032 to J.O.‐U.), Medical Research Grant from W.W. Smith Charitable Trust (H1403 to J.O.‐U.) and NIH grants (2R01HL093671 and 1R01HL122124 to SSS).
High fat‐diet induced obesity does not necessarily promote vascular remodeling/fibrosis in rats
Authors: Fernandes Roxanne , Carter‐Taylor Evan , Galligan James , Xu Hui 
Published: November 2023

Inflammation and hyperglycemia are responsible for obesity associated vascular remodeling/fibrosis. Our lab has previously observed that high fat (HF) diet‐induced obesity and hyperglycemia promote vascular remodeling/fibrosis, but HF diet does not necessarily promote hypertension in male mice. However, it is unclear if vascular remodeling/fibrosis and inflammation are unique changes in all HF diet‐induced obese rodent models. In current studies, we compared the blood pressure, vascular and tissue histological structures of heart, mesentery, and kidney in male and female Sprague Dawley rats fed with HF (60 kcal% fat) or control (10 kcal% fat) diet up to 20 or 48 weeks. Vascular responsibilities to norepinephrine and wall thickness/distensibility were evaluated in pressurized mesenteric arteries (MA) and veins (MV) in vitro. The plasma levels of TNA‐α, monocyte chemoattractant protein‐1 (MCP‐1), insulin, glucose and leptin were also evaluated in these rats. HF fed female (20 weeks) and male (48 weeks) rats were obese and hyperleptinemia, but normotensive and with unchanged vascular contractility. These rats did not exhibit vascular remodeling/fibrosis, hyperglycemia, and systemic inflammation, when compared with control diet fed rats. After 48 weeks of exposure to HF diet, female rats were obese, hyperleptinemia, hypertensive and with reduced vascular reactivity to norepinephrine, however, these rats did not exhibit vascular remodeling/fibrosis, hyperglycemia, or systemic inflammation either. Our findings suggested that obesity is not the determinant in the development of vascular fibrosis/remodeling. HF diet induced hyperglycemia and tissue inflammation may be the predominant mediators in obesity associated vascular remodeling/fibrosis.Support or Funding InformationSupported by Undergraduate Research in Biomedical Sciences Program (NIH grant HL103156, for Dr. Ewart to support Carter‐Taylor) and NIH grant 2P01HL070687 (For Drs Galligan and Xu)
Hydrogen Sulfide Suppresses Diabetic Myocardial ER Stress: An Uncanonical Way
Authors: Veeranki Sudhakar , Metreveli Naira , Tyagi Suresh C 
Published: November 2023

Diabetes critically inhibits cardiac endogenous production of hydrogen sulfide (H2S), a cardioprotective gaseous signaling molecule, by suppressing expression of CBS, CSE and 3MST (known H2S producers endogenously) in the mouse models of type 1 diabetes (Akita or C57BL/6‐Ins2Akita/J). Diabetes induced ER stress has been previously identified as an initiator of cardiomyocyte apoptosis and cardiomyopathy. However, the mechanisms of ER stress induction are not clearly illustrated. Furthermore, H2S has been implicated as an inducer or suppressor of ER stress in a tissue type specific manner and molecular mechanisms of H2S mediated ER stress regulation are not entirely known. Here, we show that chronic H2S supplementation resulted in enhanced cardiac function concurrent with suppression of ER stress with significant reversal of elevated CHOP levels in the diabetic hearts. Interestingly, there were no significant changes in the sXBP1 levels in the diabetic hearts with or without H2S treatment suggesting differential regulation of different arms (PERK, ATF6 and IRE1) of ER stress pathway. Importantly, the ER stress suppression is accompanied by suppression of GRP78 (AKA Bip) mRNA levels with a dramatic and paradoxical elevation in its protein levels. Differential translational regulation and/or protein degradation are implicated in regulation of GRP78 protein levels which further highlighted that H2S supplementation not only suppresses ER stress, but also raises the threshold for ER stress induction through elevation of GRP78 protein levels. Remarkably, the H2S mediated diabetic ER suppression was also accompanied with reversal of elevated TLR4 receptors, a well‐known inducer of ER stress upon stimulation. TLR4 mutant diabetic mice (Akita background) are currently studied to unravel relative contribution of TLR4 dependent and independent mechanisms for H2S mediated reversal of ER stress during diabetic cardiomyopathy. In conclusion, H2S mediated raise in threshold for ER stress induction involve GRP78 protein preservation and TLR4 level suppression in the diabetic hearts.Support or Funding InformationNIH grants : HL108621, HL074185 and DK104653.
Physiological and transcriptomic data have equal variability in male and female rats
Authors: Dayton Alex , Skelton Meredith , Kaldunski Mary , Greene Andrew S , Cowley Allen W 
Published: November 2023

There are well known physiological differences between male and female animals that have been studied in multiple organ systems. Yet, the ratio of male to female animal studies in physiology is estimated at 3.7:1; a sex bias that may impact the interpretation and translation of the results to human conditions.It is well established that many phenotypes are significantly affected by estrogen and other circulating hormones. It is reasonable to suggest that the varying levels of these hormones during the estrous cycle would confound the study of these phenotypes in female rats by increasing the variance in female experimental groups. If the variance were greater in female rats than in males, it would require more experimental subjects (N) to detect an equivalent difference in phenotype. Since this would necessitate a change in experimental design to study female rats, it is important to determine whether this proposed increase in variance was supported by available data.We analyzed the phenotypic data available on the Rat Genome Database, a large, publically available dataset of physiological measurements of cardiovascular, renal, biochemical, vascular and lung phenotypes made in male and female rats. In total, this dataset comprised ~40 phenotypes measured in 6 genetically distinct strains of rat. We found no evidence that the variance or coefficient of variation was consistently greater in female rats than in male rats.We then wanted to see whether estrous stage impacted the blood pressure response to salt in the SS rat. In a smaller discrete study of the blood pressure response to high salt diet in a group of 16 female SS rats, we determined the phase of estrous by daily vaginal smears and cytology. At the end of the study, the number of estrous cycles determined for each animal over the three weeks of study (2 weeks of 8% salt). Six rats continued to cycle during the study with 3–5 cycles confirmed. The remaining eight rats had two or fewer cycles confirmed. When the blood pressure response to the high salt diet was analyzed, there was no difference between the cycling and non‐cycling female SS rats.To assess the impact of the estrous cycle on transcriptomic data, we analyzed the dataset of Y. Yu et al. (Nature Communications, 2014), in which genomic RNA‐seq measurements were made in several organs from both male and female Fischer 344 rats. This dataset consisted of RNA‐seq measurements made in 11 organs. Each RNA‐seq dataset contained measurements for ~20000 individual transcripts. We found no evidence that the variance or coefficient of variation of expression of individual genes was consistently greater in female rats than in male rats.Together, these analyses suggest that the commonly held view that the estrous cycle necessitates changes in experimental design due to increased variance is not supported by experimental data. We conclude that it is appropriate to use same experimental design for both sexes.