bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nanog regulation of endothelial asymmetric cell division (851.20)
Authors: Baruah Jugajyoti , Wary Kishore 
Published: June 2021

Objective: To test whether inhibition of Glycogen Synthase Kinase‐3 (GSK‐3) enzyme or GSK‐3α/β‐ knockdown, induces Asymmetric Cell Division (ACD) in endothelial cells (ECs). Accordingly, we test the hypothesis that a subset of mature ECs retain the ability to divide unequally, a process called asymmetric cell division (ACD) and this event requires the activation of canonical Wnt signaling pathway.Methods and Results: Stimulation of cultured ECs with 6‐bromoindirubin‐3‐oxime (BIO) or shRNA mediated GSK‐3α/β‐knockdown stabilized β‐catenin and increased expression of NANOG, thereby increased proliferation as well as migration of these cells in culture. Accordingly, BIO stimulation and 5‐bromo‐2'‐deoxyuridine (BrdU) pulse labeling of ECs followed by microscopy showed increased ACD in a subset of BrdU‐positive cells. The immunostaining of BrdU positive ECs (stalk‐cells) showed expression of NOTCH‐1 (NICD) and DLL4 (tip‐cells). Immunohistochemistry and biochemical analyses indicated altered expression of NOTCH and NUMB in asymmetrically dividing cells. Western blot analyses confirmed increased expression of NOTCH‐1, DLL4, NUMB and JAM‐A, protein differentially expressed during ACD, in response to BIO stimulation. In a converse experiment, NANOG‐knockdown carried‐out in ECs after receiving BIO showed decreased ACD, while control knockdown had no effect. Interestingly, re‐expression of VEGFR‐2/Flk1 cDNA into NANOG‐depleted ECs partially restored the ability of these cells to divide asymmetrically.Conclusion: Thus, our data indicate that the activation of canonical Wnt signaling induces ACD in a subset of ECs, an event that may partly be responsible for mediating EC dedifferentiation and regeneration.Grant Funding Source: Supported by American Heart Association Grant to KKW (12GRNT12070159).
Measurement of Evans blue dye concentration in central and peripheral tissue following administration in vitro and in vivo: dose‐ and replicate‐dependent effects on absorbance, fluorescence, and extraction by trichloroacetic acid (851.3)
Authors: Wang Hwai‐Lee , Lai Ted 
Published: June 2021

The Evans blue dye (EBD) is the most commonly used inert tracer for tracking endogenous plasma protein extravasation into the brain. Even though several methods have been developed for measuring EBD concentration extravasated into central and peripheral tissues, the accuracy and precision of these methods remains poorly defined. First, we measured the absorbance and fluorescence of EBD solutions prepared in 96‐well plates using standard photo‐spectrometer. EBD absorbance increased in a dose‐dependent manner, and logarithm‐transformation revealed a sigmoidal curve with Hill coefficient of 2.086. The steepest slope of the curve lied between 5 µg/ml to 500 µg/ml, and hence this concentration range was the region at which measurements would be most precise. On the other hand, EBD fluorescence also increased in a concentration‐dependent manner, but with region of precision from 0.2 µg/ml to at least 5 µg/ml. Nevertheless, substantial photo‐bleaching of EBD fluorescence was found with increasing concentrations. Thus, our results recommends the use of fluorescent measurements to define EBD concentrations between 0.2 µg/ml to 5 µg/ml, and the use of absorbance measurements to define EBD concentrations between 5µg/ml to 500 µg/ml. Second, we determined the efficiency at which EBD can be retrieved from plasma proteins using the trichloroacetic acid (TCA) extraction technique. The degree of extraction was substantial even with 1:1 plasma‐to‐TCA ratio, and reached maximum with 1:2 ratio where increased ratios failed to further increase EBD extraction. Notably, contrary to earlier reports, we failed to completely extract EBD from plasma sample even with substantial amount of TCA. Third, we determined the sample‐to‐TCA ratio required to maximally extract EBD from central and peripheral tissue in rats receiving intravenous EBD. Taken together, the data presented here will be of great value in guiding future research using EBD as a tool to study central and peripheral vascular permeability.Grant Funding Source: Supported by the National Science Council of Taiwan
Differential role for STIM1 in the regulation of vascular function (851.7)
Authors: Kassan Modar , Zhang Wei , Ait Aissa Karima , Stolwijk Judith , Feske Stefan , Trebak Mohamed , Matrougui Khalid 
Published: June 2021

Background: The aim of this study was to determine the role of stromal interacting molecule‐1 (STIM1) in the regulation of vascular function using tissue‐specific STIM1 knockout mice either in endothelial cells or in smooth muscle cells.Methods &amp; results: We used male C57/BL6 homozygous and heterozygous mice lacking STIM1 specifically in either smooth muscle cells (STIM1SMC‐/‐, STIM1SMC‐/+) or in endothelial cells (STIM1EC‐/‐, STIM1EC‐/+). Systolic blood pressure and glucose levels were similar in all mice, but body weight was significantly reduced in STIM1EC‐/‐ and STIM1SMC‐/‐ mice. The contraction of resistance and conductance arteries in response to phenylephrine was significantly reduced in STIM1SMC‐/‐ mice only. However, contraction to thromboxane and potassium chloride was similar in all groups. The endothelium‐dependent relaxation was significantly impaired in STIMEC‐/+ and drastically reduced in STIM1EC‐/‐ mice while the endothelium‐independent vasorelaxation was similar among all groups. Acute down regulation of STIM1 in resistance arteries reduced endothelium‐dependent relaxation to acetylcholine and the contractile response to phenylephrine, while the contractile response to thromboxane was not affected. NADPH oxidase activity was only increased in STIMEC‐/+ and STIMEC‐/‐ mice. Calcium Ca2+ entry in microvascular endothelial cells stimulated with thrombin and histamine, two vasoactive compounds that cause NO production, had the pharmacological features of store‐operated Ca2+ entry (SOCE) and was dependent on STIM1 expression.Conclusions: STIM1 plays opposing roles in vascular smooth muscle vs. endothelial cells in the regulation of vascular reactivity. We conclude that STIM1 is a critical factor in the basic mechanisms of vascular endothelium‐dependent relaxation and the contractility in response to sympathetic activity but not in the intrinsic contractility of smooth muscle.
A novel estimate of endothelial cell autophagic flux is associated with greater vascular endothelial function and reduced oxidative stress in healthy middle‐aged/older adults (855.3)
Authors: van Boekel Anna , Santos‐Parker Jessica , LaRocca Thomas , Seals Douglas , Kaplon Rachelle 
Published: June 2021

Using only indirect, static markers of the dynamic cellular quality control system, autophagy, we have reported impaired autophagy in biopsied vascular endothelial cells (EC) of humans with aging that is related to endothelial dysfunction, as indicated by reduced endothelium‐dependent dilation (EDD), and oxidative stress. Here, we estimated dynamic autophagic flux in EC from 10 healthy adults aged 51‐73 by calculating the within‐subject difference in the expression of microtubule‐associated protein light chain 3 (LC3 ratio) between ECs incubated in the absence vs. presence of chloroquine (50 μM 1.5 hrs) to inhibit autophagy. EDD was assessed by: 1) brachial artery flow‐mediated dilation (FMD); and 2) forearm blood flow (FBF) to incremental brachial artery infusions of acetylcholine. Oxidative stress‐suppression of EDD was determined by ∆FBF to co‐infusion of vitamin C (vitC). The LC3 ratio was positively related to FMD (r=0.76, P<0.05) and FBF (r=0.56, P<0.05), but not endothelium‐independent dilation to NO donors (both P>0.05). The LC3 ratio also was inversely related to ∆FBF with vitC (r=‐0.57, P<0.05). These findings provide novel evidence that greater EC autophagic flux is associated with improved endothelial function linked to reduced oxidative stress in healthy middle‐aged/older adults.Grant Funding Source: NIH AG044031 TR001082 AG013038
Regulation of the epithelial sodium channel by specific palmitoyltransferases (DHHC) (860.6)
Authors: Mukherjee Anindit , Poland Paul , Wang Zhijian , Kleyman Thomas , Hughey Rebecca 
Published: June 2021

The epithelial sodium channel (ENaC) has a key role in the maintenance of extracellular fluid volume and control of blood pressure. ENaC is a member of the ENaC/degenerin family and is assembled with three structurally similar subunits (alpha, beta and gamma). We have shown that palmitoylation of specific cytoplasmic Cys residues in the beta and gamma subunits activate mouse ENaC by increasing channel open probability. In assessing channels that lack palmitoylation sites on the beta and/or gamma subunit, our data indicate that gamma subunit palmitoylation has a dominant role in activating ENaC. There is a family of 23 palmitolytransferases (referred to as DHHCs) in mice that catalyze the transfer of palmitate to cytoplasmic and transmembrane Cys in proteins. To directly assess the physiological significance of ENaC palmitoylation, we co‐expressed ENaC individually with all 23 palmitoyltransferases to identify specific palmitoyltransferases that can activate ENaC in Xenopus oocytes. We observed that five DHHCs significantly increased (1.8‐2.5 fold p < 0.05 ‐ 0.001) wild type channel activity. Co‐expression of these activating DHHCs with ENaC lacking Cys for either beta or gamma subunit palmitoylation revealed one DHHC with specificity for the gamma subunit. Using RT‐PCR we found that all five of the activating DHHC are present in mpkCCDc14 cells that express endogenous ENaC providing a model system to study regulation of ENaC palmitoylation. Results of our studies provide new insights regarding a regulatory pathway that has a large effect on the modulation of channel open probability.Grant Funding Source: supported by NIH 5R01DK065161‐09
Regulation of SERCA2a by chronic ethanol: role of Akt (864.8)
Authors: Jeffress Miara , Umoh Nsini , Haddad Georges 
Published: June 2021

Alcoholic cardiomyopathy is one of the leading causes of non‐ischemic dilated cardiomyopathy accounting for 36% of cases. Almost 50% of alcoholics develop this disease. Previously our lab has demonstrated the up‐regulation of intracellular Ca2+ (IC) in dilated cardiomyopathy by Akt. Here we investigate the role of Akt in regulating Ca2+ cycling proteins and contractility in chronic ethanol exposed cardiocytes. Parallel measures of IC and sarcomere/cellular contractions were assessed (Ionoptix) with the expression level (western blot) of Ca2+ handling proteins (sarcoplasmic reticulum calcium ATPase(SERCA2a), phospholamban(PLN), Na+‐Ca2+ exchanger (NCX) and L‐type Ca2+ channels(LTCC)) from control, low (LA: 0.02%), and high (HA: 0.45%) alcoholic rat hearts (4 months on Lieber DiCarli liquid diet), with and without Ad.EGFP and Ad.Akt transfection. Our results show an inotropically damaging increase in IC with HA but not with LA. In parallel, NCX protein expression is diminished in HA whereas that of LTCCs is increased. In addition, SERCA2a levels are increased in LA and decreased in HA. Upon Ad.Akt transfection SERCA2a levels increased in HA. Protein levels of phosphorylated PLN also increased in LA and decreased in HA hearts. This suggests that chronic ethanol elevates IC to harmful levels through cytoplasmic trapping via NCX which is exacerbated by reduced SERCA2a through PLN in an Akt‐dependent manner.Grant Funding Source: Supported by NIH/NIAAA/1R15AAA019816‐01A1 and 2G12 RR003048
Ebselen prevents the carotid body chemosensory potentiation and reverses the hypertension induced by intermittent hypoxia (873.1)
Authors: Moya Esteban , Arias Paulina , Carlos Varela , Oyarce Maria , Iturriaga Rodrigo 
Published: June 2021

Oxidative stress is associated with hypertension and enhanced carotid body (CB) chemosensory responses induced by chronic intermittent hypoxia (CIH), but the underlying mechanism is not fully understood. We tested if ONOO‐ is involved in the CB enhanced chemosensivity. Accordingly, we studied the effects of Ebselen, a specific ONOO‐ scavenger, on 3‐nitrotirosine immunoreactivity (3‐NT‐ir) in the CB, the chemosensory discharge and arterial blood pressure (BP) in rats exposed to CIH. Male Sprague‐Dawley rats were exposed to CIH (5% O2, 12 times/h, 8 h/day), BP was measured with telemetry and Ebselen was administrated using subcutaneous osmotic pumps (10 mg/Kg day). At the end of the experiments, CB chemosensory discharge was recording, and rats were perfused with parafolmaldehyde 4% for 3‐NT‐ir. CIH increased 3‐NT‐ir in the CB, enhanced the chemosensory responses to hypoxia, elevated BP during normoxia and potentiated the BP responses to hypoxia. Ebselen prevented the CIH‐induced CB chemosensory potentiation, and reversed the elevated BP and the potentiated pressor responses to hypoxia. Our results showed that 3‐NT formation caused by CIH, contributes to the CB chemosensory potentiation, and that ONOO‐ is involved in the CIH‐induced hypertension.Grant Funding Source: Supported by FONDECYT 1100405
Cytokine‐mediated regulation of catecholamine biosynthesis in adrenal chromaffin cells (876.5)
Authors: Byrne Collin , Khurana Sandhya , Peltsch Heather , Kumar Aseem , Tai T.C. 
Published: June 2021

Catecholamines, neurohormones with extensive functions in the sympathetic regulation of blood pressure, have a well‐established role in cardiovascular disease and hypertension. Epidemiological data has shown that, in addition to elevations in the catecholamine adrenaline, hypertensive patients demonstrate altered plasma cytokine profiles, suggesting that hypertension entails a complex interplay of the neuroendocrine and immune systems. From preliminary work, we have found that spontaneously hypertensive rats (SHR) exhibit higher plasma levels of the cytokine CCL2, and decreased levels of interleukin (IL)‐1β and interferon (IFN)‐δ, compared to their normotensive counterparts. To delineate the relationship between cytokine‐mediated signalling and catecholamine synthesis, we tested a panel of cytokines for their ability to affect the expression of tyrosine hydroxylase (TH), dopamine beta hydroxylase (DBH) and phenylethanolamine N‐methyltransferase (PNMT), key enzymes for catecholamine biosynthesis. Adrenal medulla derived PC12 cells were treated with the cytokines IL‐1β, IL‐2, IL‐6, IL‐10, tumor necrosis factor‐α, IFN‐α, transforming growth factor‐β1, or CCL2, and transcriptional changes were analyzed using RT‐PCR. Alterations in the transcripts of TH, DBH and PNMT upon cytokine treatments were observed illustrating cytokine‐mediated regulation of catecholamine biosynthesis.Grant Funding Source: Supported by NSERC and CIHR
Inhibition of thiazide‐sensitive sodium chloride cotransporter phosphorylation provokes a global compensatory response (892.17)
Authors: Grimm P , Wall Susan , Delpire Eric , Wade James , Welling Paul 
Published: June 2021

We hypothesize an adaptive transport process preserves salt balance when activation of the thiazide‐sensitive sodium‐chloride cotransporter (NCC) is impaired, as in Gitelman Syndrome. Here, we describe such a compensatory mechanisms taking place in SPAK kinase null mice, which have a Gitelman‐like phenotype owing to the loss of NCC phospho‐activation. Global transcriptional profiling by microarray, together with real‐time PCR, western blot analysis, immunolocalization and metabolic assays were statistically quantified to reveal that an intercalated cell (IC) reprogramming pathway synergizes with a α‐keto‐glutarate (α‐KG) paracrine signaling system to augment sodium chloride reabsorption in distal nephron. Acid secreting α‐IC are replaced with chloride reabsorbing pendrin positive IC (PPIC). Coordinate transcriptional activation of a multi‐gene transport system in PPIC, involving solute carriers (Slc26a; Slc4a8; Slc4a9), carbonic anhydrase 2 and 15, and V‐type H+‐ATPase subunits, creates a new electroneutral salt reabsorption pathway. In the proximal tubule, glutamate and α‐KG carriers (Slc3a1, Slc13a2, Slc38a3) increase while α‐KG metabolizing enzymes (Odc1 and Got2) decrease, causing [α‐KG] to increase in the tubular fluid. The GPCR (Oxgr1) for α‐KG increases on the PPIC apical surface, allowing paracrine delivery of α‐KG to stimulate the transport pathway. This study identifies the induction of an integrated compensatory NaCl reabsorption mechanism in PPIC, which may provide new molecular targets for diuretics.Grant Funding Source: Supported by RO1DK093501
Transcriptional regulation of renal organic anion transporter 1 by B‐cell CLL/lymphoma 6 (892.33)
Authors: Henjakovic Maja , Wegner Waja , Burckhardt Birgitta , Burckhardt Gerhard 
Published: June 2021

The human organic anion transporters 1 (OAT1) is crucial for the excretion of organic anions in renal proximal tubular cells and was classified as clinically relevant transporter in the kidneys. Our previous study indicated that renal male‐predominant expression of rat Oat1 and Oat3 appears to be regulated by transcription factor B‐cell CLL/lymphoma 6 (BCL6). The aim of this study was to characterize the effect of BCL6 on human OAT1 promoter.Luciferase assays were carried out on Opossum kidney (OK) cells transiently transfected with promoter constructs of OAT1 and expression vectors for BCL6. Transcription factor (TF) activation profiling plate array was used for monitoring the activation of multiple transcription factors in nuclear extracts of pcDNA3‐BCL6 and pcDNA3 transfected OK cells. The protein expression of several transcription factors was investigated using fluorescence microscopy and Western blot.BCL6 enhanced the promoter activity of OAT1, independently of predicted BCL6‐binding sites. Promoter sequence ‐63 to +1 bp of OAT1, which contained HNF1a binding sites, is the minimal regions required for BCL6 dependent activation. BCL6 enhanced the protein expression of HNF1α, a known OAT1 activator. HNF1α dependent activation of OAT1 promoter was increased by BCL6.BCL6 constitutes a promising candidate gene for the regulation of human OAT1 transcription, possibly via activation of HNF1α.
Role of mitochondrial fission and fusion in cardiomyocyte contractility (893.10)
Authors: Givvimani Srikanth , Pushpakumar Sathnur , Metreveli Naira , Narayanan Nithya , Kundu Sourav , Veeranki Sudhakar , Chaturvedi Pankaj , Tyagi Suresh 
Published: June 2021

Mitochondria constitute 30% of myocardial cell volume and are continuously engaged in two dynamic processes called fusion and fission which are regulated by Drp‐1(Dynamin related protein) and mitofusin protein‐2 (Mfn‐2) respectively. Previously, we showed that inhibition of Drp‐1 ameliorates cardiovascular dysfunction following pressure overload in an aortic banding model and myocardial infarction studies. As dynamic organelles, mitochondria are capable of changing their morphology in response to stress conditions. However, whether such changes can alter their function and in turn the cellular function is not known. Further, a direct role of mitochondrial fission and fusion on cardiomyocyte contractility has not yet been studied. In the present study, we hypothesized that modulating mitochondrial fission and fusion processes will alter cardiomyocyte contractility. To verify this, we used freshly isolated ventricular myocytes from wild type mouse hearts and transfected them with siRNA for DRP‐1 and Mfn‐2. Myocyte contractility studies were performed by IonOptix using a myopacer. Intracellular calcium and potassium measurements were done using flow cytometry. Immunocytochemistry was done to evaluate live cell mitochondria and its membrane potential sensitivity. Our results suggested that inhibition of mitofusion‐2 by siRNA significantly decreased the myocyte contractility suggesting the importance of fusion dynamics of mitochondria.Grant Funding Source: HL108621, HL 074185
Analysis of OSR1 kinase‐peptide binding substrate interaction through computational protein docking software (893.37)
Authors: Austin Thomas , Nannemann David , Meiler Jens , Delpire Eric 
Published: June 2021

The oxidative‐stress‐responsive kinase 1 (OSR1) and the STE20/SPS1‐related proline/alanine‐rich kinase (SPAK) are key enzymes in a signaling cascade regulating the activity of Na+‐K+‐2Cl‐ cotransporters (NKCC1‐2) and Na+‐Cl‐ cotransporter (NCC). Both kinases have a conserved carboxyl‐terminal (CCT) domain, which recognizes a unique peptide (Arg‐Phe‐Xaa‐Val) motif present in OSR1‐ and SPAK‐activating kinases [with‐no‐lysine kinase 1 (WNK1) and WNK4] as well as their substrates (NKCC1, NKCC2, and NCC). In this study, various modalities of the Rosetta Software Suite including flexible peptide docking and protein design were utilized to predict several constructed hexapeptides that would favorably bind to the CCT domain after individual residue mutation or whole hexapeptide design. These constructs are all derived from a crystal structure of the CCT domain of OSR1 in complex with a hexapeptide (Gly‐Arg‐Phe‐Gln‐Val‐Thr) derived from WNK4 (2v3s). Among the favorable point mutations include an arginine to histidine substitution at position 2 and a phenylalanine to tyrosine substitution at position 3 of the hexapeptide. In addition, the designed hexapeptides FRFEVT (Phe‐Arg‐Phe‐Glu‐Val‐Thr) and TRFDVT (Thr‐Arg‐Phe‐Asp‐Val‐Thr) were both predicted to bind to the CCT domain. The results of these computations were confirmed through the use of yeast two‐hybrid screening and frequencies of computationally derived motifs were calculated in known proteins sequence by searching the NCBI mouse protein database. Our study show that the Rosetta Molecular Sotware Suite can very precisely model the binding of the GRFQVT peptide to the CCT/PF2 domain of OSR1 and our in silico mutagenesis study shows that the nature of the residues required for binding might be more flexible than previously recognized.Grant Funding Source: RO1 DK0935010, RO1 GM074771
Effect of nicotine exposure on cellular and molecular parameters of vitamin B1 (thiamin) uptake by pancreatic acinar cells (896.3)
Authors: Srinivasan Padmanabhan , Subramanian Veedamali , Kapadia Sameer , Said Hamid 
Published: June 2021

Pancreatic acinar cells take in thiamin by a specific carrier‐mediated process that involve both THTR‐1 and THTR‐2 (encoded by SLC19A2 and SLC19A3, respectively). In this study we examined the effect of chronic exposure of mouse pancreatic acinar in vivo (wild‐type and transgenic mice carrying the SLC19A2 and SLC19A3 promoters) and human pancreatic acinar cells in vitro to the cigarette smoke component nicotine on physiological and molecular parameters of the thiamin uptake process. Chronic exposure of mice to nicotine (0.77 mM nicotine in drinking water for 4 wks) leads to a significant decrease in thiamin uptake, expression of THTR‐1 and ‐2 protein, mRNA levels and activity of the SLC19A2 and SLC19A3 promoters expressed in transgenic mice. A decrease in the level of expression of protein and mRNA of the thiamin‐metabolizing enzymes TPKase and thiamin pyrophosphatase (TPPase) was observed. Similarly chronic exposure of human pancreatic acinar cells to nicotine (0.5 µM, 48 h) leads to a significant inhibition in thiamin uptake, as well as in the level of expression of THTR‐1 and ‐2 protein and mRNA. These results demonstrate, for the first time, that chronic exposure of pancreatic acinar cells to nicotine negatively impacts the physiological and molecular parameters of thiamin uptake and that this effect is exerted, at least in part, at the level of transcription of the SLC19A2 and SLC19A3 genes.Grant Funding Source: Supported by NIH grants AA 018071 and DK‐56061