bluuebunny/crossref_metadata_2025_split huggingface dataset:
Time Course of Fat Accumulation in Various Organs of Young OLETF Rats
Authors: Sakakura Kanta , Nishihira Misuzu , Iwamoto Yumi , Yamamoto Yui , Fujita Naoto , Urakawa Susumu 
Published: May 2022

PurposeExcessive fat accumulation induces chronic inflammation in the white adipose tissue (WAT), which leads and accelerates to obesity‐related health problems such as glucose intolerance, insulin resistance, and dyslipidemia. Determination of the time course of fat accumulation in obesity might allow identification of target tissues to treat the health problems in each stage. The purpose of the present study was to investigate the relationship between inflammatory changes and time course of fat accumulation in various organs.MethodsMale Otsuka Long‐Evans Tokushima fatty (OLETF) rats were used as spontaneous obesity animals. All rats were fed a standard diet containing 55.3% carbohydrate and water ad libitum. From 4‐ to 20‐week‐old, blood samples from vena cava and samples of the epididymal adipose tissue, retroperitoneal adipose tissue, mesenteric adipose tissue, and inguinal subcutaneous adipose tissue as WAT, brown adipose tissue (BAT) from interscapular region, liver, pancreas, and plantaris muscles were analyzed histologically every 2 weeks. Collected serum was used to detect triglyceride (TG) and free fatty acid (FFA) concentrations. Tissue samples were sliced and stained with hematoxylin and eosin or oil red O for histological observation.ResultsBody weight in OLETF rats increased linearly from 4‐ to 20‐week‐old. Although the wet weight and adipocyte diameter of the epididymal adipose tissue also increased throughout the experimental period, the values of inguinal subcutaneous adipose tissue increased by 10‐week‐old and were almost the same after 10‐week‐old. The liver TG levels increased from 4‐ to 8‐week‐old. However, hypertriglyceridemia, BAT whitening, and hepatic steatosis were not observed at this period. The serum TG levels increased after 10‐week‐old. Additionally, the number of whitened unilocular adipocytes with a diameter >10 μm in the BAT increased after 10‐week‐old and oil red O positive lipid droplets were usually observed in the liver at this period. Not only the serum TG levels but also the serum FFA levels increased after 18‐week‐old. Additionally, enlarged adipocytes with a diameter >130 μm and crown like structure indicating inflammatory change were often observed in the epididymal adipose tissue and retroperitoneal adipose tissue, not in mesenteric adipose tissue and inguinal subcutaneous adipose tissue, at this period. Fatty pancreas and intramuscular fat deposition were not observed throughout the experimental period.ConclusionAdipocyte hypertrophy in the visceral WAT and fatty liver could precede dyslipidemia in young OLETF rats and continuous obesity subsequently proceed to ectopic fat accumulation including BAT whitening and chronic inflammation in the epididymal adipose tissue and retroperitoneal adipose tissue with higher FFA levels. Hyperphagia mainly on carbohydrate could contribute to ectopic fat accumulation in the liver and BAT whitening, not to fatty pancreas and intramuscular fat deposition in young stage.
PIP2 and cholesterol interplay in inflammation and atherosclerosis
Authors: Gulshan Kailash , Iacano Amanda J. , Traughber Cynthia A. , Khan Mariam R. , Opoku Emmanuel , Smith Jonathan D. , Nunn Tina , Sangwan Naseer 
Published: May 2022

IntroductionCholesterol accumulation in arterial macrophages leads to foam cell formation, Nlrp3 inflammasome activation, and IL‐1b release. Phosphatidylinositol 4, 5‐bisphosphate (PIP2) plays a role in the cholesterol efflux pathway and IL‐1b release. The PIP2 phosphatase TMEM55b modulates cholesterol efflux but the mechanism of this effect is not clear. Miltefosine altered PIP2 localization and blunted Nlrp3 inflammasome in macrophages, but the mechanism of its anti‐atherosclerotic properties is not clear.ObjectivesTo determine the effect of cellular PIP2 levels on ABCA1 mediated cholesterol efflux. To determine the mechanism by which Miltefosine reduces atherosclerosis in mice.Methods and resultsThe correlation between cellular PIP2 levels and ABCA1 mediated cholesterol efflux was determined. PIP2 levels were markedly increased in cholesterol‐loaded cells vs. control macrophages (~ 22 % increase, N=3, mean±SD, *P=0.05). Expression of TMEM55b, a PIP2 phosphatase, in BHK cells resulted in ~ 70 % decrease in ABCA1 mediated cholesterol efflux (N=4, mean ± SD, P<0.0001). Miltefosine treated mice showed blunted in‐vivo Nlrp3 inflammasome assembly and had significantly lowered IL‐1b levels in peritoneal lavage (mean +SD; N=5, **p<0.001). Miltefosine‐fed mice showed significantly higher reverse cholesterol transport to plasma, liver, and feces. The hyperlipidemic apoE‐/‐ KO mice fed with Miltefosine (50 mg/kg/day) gained significantly less weight vs. controls and showed significantly decreased atherosclerotic lesions (≥50% smaller vs. controls; mean + SD, p<0.001 in females with N=10, and p<0.04 in males with N=12). Miltefosine altered gut microbiota in hyperlipidemic apoE‐/‐ KO mice.ConclusionPIP2 regulates ABCA1 mediated cellular cholesterol efflux. Miltefosine can dampen in‐vivo Nlrp3 inflammasome assembly, increase reverse cholesterol transport, reduce high fat diet‐induced weight gain, reduce atherosclerotic lesion area and alter gut microbiota.
Seizure Susceptibility Increases After Early‐Life Seizures in Zebrafish
Authors: Bhuiyan Kasulul , Ramon Alfonsina , Stegmuller Christopher , Lippman‐Bell Jocelyn 
Published: May 2022

Early‐life seizures (ELS) can lead to the development of epilepsy in humans (Elger et al., 2004). Dynamic animal models are essential to study the effects of ELS in the developing brain. Zebrafish have emerged as a valuable model for neuroscience research due to their rapid development, the potential for high‐throughput screening, and their genetic similarity with mammals (Stewart et al., 2014). While rodent ELS studies show spontaneous seizures later in life (Stafstrom & Sasaki‐Adams, 2003), the effects of ELS on later‐life seizures in zebrafish has yet to be explored.Based on human and rodent studies of ELS, we hypothesized that larval zebrafish would become more susceptible to seizures within two weeks after experiencing ELS. To test this, we induced ELS in larval zebrafish using the chemoconvulsant pentylenetetrazol (PTZ), a drug commonly used to induce seizures in zebrafish (Baraban et al., 2005). Zebrafish were exposed to 5mM PTZ for 40 minutes per day from 5‐7 days post‐fertilization (dpf). Clutchmate controls remained in a beaker inside an incubator for the duration of this time and were not exposed to PTZ. All fish were placed in a tank on an automated rack system at 8 dpf where they remained unperturbed until 21 dpf. At 21 dpf, zebrafish from both groups were pipetted into 12‐well plates where they were exposed to either 1, 2.5, or 5 mM PTZ for 20 minutes. For each condition and PTZ concentration, the number of stage two (whirlpool) seizures were measured.In this pilot study, we found in 0%, 36.4%, and 50% of control fish demonstrated stage two seizures when administered 1, 2.5, and 5 mM PTZ at 21 dpf, respectively (n=8,11, and 12). However, 12.5%, 66.7%, and 66.7% of 21 dpf zebrafish that had previously experienced ELS had stage two seizures at those same concentrations, respectively (n = 8, 12, and 12). The number of stage two seizures demonstrated per fish were similar between conditions for 1mM and 5mM PTZ, but at 2.5 mM PTZ, zebrafish that experienced prior ELS had three times as many stage two seizures as controls (mean = 1.6 ± 0.5 SE for ELS fish, and 0.5 ± 0.2 SE for controls).These observations provide evidence that larval zebrafish that experienced PTZ‐induced ELS as very young larvae (5‐7 dpf) may develop increased seizure susceptibility later in life. These results support the use of zebrafish in research aiming to identify the mechanisms by which ELS might lead to the development of epilepsy.
Role of Human Aldehyde Oxidase in the Generation of Reactive Oxygen Species during the Metabolism of Nicotine
Authors: Kundu Tapan K. , Hemann Craig , Leimkühler Silke , Zweier Jay L. 
Published: May 2022

Nicotine is the major addictive constituent of tobacco and alternative tobacco products including electronic cigarettes (e‐cigs). While e‐cig vaping has been proposed as safer than traditional tobacco smoking, recent studies has demonstrated that these products can also trigger oxidant stress and inflammation with resultant pulmonary and cardiovascular disease. E‐cig liquids contain high levels of nicotine typically of 12 to 24 mg/ml or more and with the use of high power delivery devices high, poorly controlled levels of nicotine can be delivered. When inhaling tobacco cigarette smoke or e‐cig vape, nicotine is rapidly absorbed through the lungs. The major pathway of nicotine metabolism in humans consists of the C‐oxidation of nicotine to produce cotinine involving a two‐step process: the first step is CYP2A6‐catalyzed 5′‐oxidation to nicotine‐Δ1′(5′)‐iminium ion (NI), which is in equilibrium with 5′‐hydroxynicotine; and the second step is aldehyde oxidase (AO) catalyzed oxidation of NI to cotinine.Using both cytochrome‐c reduction and EPR spin trapping techniques, we show for the first time that the oxidation of NI by human AO produces superoxide radical anion (O2• −) with high flux rates. Purified recombinant human AO (hAO) was prepared and characterized by SDS gel electrophoresis and optical absorption spectroscopy. hAO, 40 nM was incubated in the presence of NI concentrations from 1‒50 µM. The kinetic parameters, Km and Vmax values, for superoxide generation from NI were determined to be 2.7 ± 0.4 μM and 694 ± 24 nmol min−1 mg−1 of protein, respectively. From quantitation of the measured superoxide and the amount of NI consumed, we estimate that ~60% of the oxygen consumed in this process is converted to superoxide with ~40% hydrogen peroxide. Superoxide generation was almost completely quenched by either 0.5 μM raloxifene, a potent inhibitor of AO, or 0.1 μM Copper‐Zinc‐superoxide dismutase (SOD1) with no superoxide generation detected either in the absence of either AO or nicotine‐Δ1′(5′)‐iminium ion.Therefore, human AO efficiently catalyzes the oxidation of NI to cotinine with concomitant production of reactive oxygen species. Due to the high expression levels of AO in lung tissue as well as its rapid rate of NI metabolism with low Km, AO rapidly and efficiently converts NI to cotinine with the formation of superoxide and hydrogen peroxide. Thus, nicotine metabolism will be associated with the formation of superoxide, hydrogen peroxide and secondary reactive oxygen species that can in turn cause lung oxidative stress and inflammation.
Internalization of Angiotensin‐(1‐12) in Adult Retinal Pigment Epithelial‐19 Cells
Authors: Ahmad Sarfaraz , Wright Kendra N. , Ferrario Carlos M. , Cheng Heng‐Jie , Choudhary Mayur , Malek Goldis , Sappington Rebecca M. 
Published: May 2022

IntroductionThe characterization, by this laboratory, of angiotensin‐(1‐12) [Ang‐(1‐12)] as a primary angiotensin II (Ang II)‐forming substrate may be an unrecognized source of Ang II‐mediated microvascular complication in hypertension‐mediated retinopathy. We found that the plasma Ang‐(1‐12) level was 12‐66‐fold higher when compared to the angiotensin I in normal human subjects. For the first time, we investigated the endogenous expression and incorporation of Ang‐(1‐12) in cultured adult retinal pigment epithelial‐19 (ARPE‐19) cells. We also investigated the internalization of Ang‐(1‐12) in the presence of a highly specific monoclonal antibody (mAb) that was developed by us against the C‐terminus of the human Ang‐(1‐12) sequence.MethodARPE‐19 cells (passage 28‐30) were grown to post‐confluence in 10% serum supplemented DMEM/F12 medium for 2‐3 days. An immunofluorescence (IF) staining was performed to detect the internalized Ang‐(1‐12) using the human Ang‐(1‐12) mAb (1:5000 dilution) and the Alexa Flour 488 secondary antibody (1:1000 dilution). Internalization was also confirmed in ARPE‐19 using radiolabeled Ang‐(1‐12) [125I‐Ang‐(1‐12), purity >99%] in the presence and absence of the mAb by HPLC. For this, the APRE‐19 cells were incubated (4 h) with Ang‐(1‐12) alone or Ang‐(1‐12) + mAb. Afterward, cells were washed and harvested in isotonic buffer. The lysed cells were centrifuged at 28,000 g and the membrane pellet and supernatants (cytosolic fractions) were counted on a gamma counter. The cytosolic fractions were further analyzed by HPLC using a C18 column to confirm the internalization of intact Ang‐(1‐12) sequence.ResultsOur IF staining shows that Ang‐(1‐12) is endogenously expressed in ARPE‐19 cells. Further, an increased intensity of staining was detected in ARPE‐19 cells exposed to Ang‐(1‐12) (100 nM). High Ang‐(1‐12) radiolabeled counts were detected in the cytosolic fractions (3.1% of the total loaded radiolabeled) and very low counts in the membrane pellet (0.2%). We found that the internalization of 125I‐Ang‐(1‐12) was significantly decreased (~40%, P<0.0001) in the presence of the human Ang‐(1‐12) mAb (Fig. 1). The detection of a large peak (88%) of 125I‐Ang‐(1‐12) in the cytosolic fraction in the HPLC chromatogram further confirms that the intact Ang‐(1‐12) sequence is internalized by the cells (Fig. 2).ConclusionsAng‐(1‐12) is internalized by ARPE‐19 cells from extracellular spaces of the retina and the internalization of Ang‐(1‐12) decreased following exposure to the human Ang‐(1‐12) mAb. Overall our data suggests the human Ang‐(1‐12) mAb may be used as a novel therapeutic agent to prevent circulating Ang‐(1‐12) internalization into the retinal cells and subsequently reduce the intracellular generation of the Ang II from the Ang‐(1‐12) substrate.
Gross Anatomy of Bifid Xiphoid Process
Authors: Zamani Shahrzad , Meawad Sara , Van Der Horn Tamryn , Dhiman Shiv 
Published: May 2022

IntroductionXiphoid process is the ossified extension of the lower sternum in the chest midline of human adults. Natural variance in this process results in the less common bifurcated morphology.ObjectiveThe primary objective of this study was to explore the distribution of the bifid variant. As a secondary objective, we aimed to compare bifurcated Xiphoid processes to other possible variations.MethodsA case study was designed to explore the distribution of bifid variants in a sample of cadavers (n=30) at a large medical education institution. The dependent variable was binarized (bifid or normal) and univariate analyses were performed based on the height, width, and presence of bifurcation measured in each cadaver.ResultsData is reported using textual and diagrammatic visuals. A significant difference was observed between the measurements of the bifid and the normal variants. Additionally, we provide a review of the implications of this bony landmark for mediastinal pressure and surgeries performed on the thoracic and abdominal areas.ConclusionThis case study demonstrates a significant variation of the Xiphoid process, aiding clinicians in performing more accurate imaging and diagnosis. Future research should consider the physiological effects and clinical significance of this process.
Ovariectomy‐Induced Arterial Stiffening is Associated with Downregulation of Tissue Resident Macrophage Markers
Authors: Kilanowski‐Doroh Isabella , Ogola Benard , McNally Alexandra , D'Souza Shaina , Rojo Michelle , McLachlan James , Machado Heather , Lindsey Sarah 
Published: May 2022

Tissue resident macrophages play a major role in the regulation of the extracellular matrix (ECM). The ECM provides an essential scaffold and mechanical stability for blood vessels, but remodeling due to acute injury or chronic inflammatory conditions like hypertension promotes vascular stiffening. We have previously shown that pulse wave velocity (PWV), a measure of arterial stiffness, is higher in male and ovariectomized (OVX) female mice versus intact female mice. RNA‐sequencing of aortas from intact and OVX mice revealed changes in several genes involved in fibrosis as well as macrophage function. Therefore, we hypothesized that estrogen protects from arterial stiffening through maintenance of “protective” macrophages and limiting pro‐fibrotic ECM components. Based on the RNAseq results, we assessed the impact of estrogen on aortic expression of four fibrosis‐associated genes: decorin (DCN), endothelin‐1 (Edn1), type III collagen α1 (Col3a1), and connective tissue growth factor (CTGF) as well as four cell surface macrophage markers: colony stimulating factor 1 receptor (CSF1R), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), AXL receptor tyrosine kinase (Axl), and growth arrest specific 6 (Gas6).MethodsFemale C57BL/6 mice were ovariectomized (OVX) at 8 weeks or remained intact. PWV was obtained as a measure of arterial stiffness using a Vevo 1100 high resolution ultrasound. Mice were euthanized between 20‐23 weeks of age for tissue collection. RNA was isolated from aortas using a Qiagen RNeasy Mini Kit and gene expression was quantified using droplet digital polymerase chain reaction (ddPCR). Statistical analysis was performed using an unpaired t‐test.ResultsAbdominal PWV was increased in OVX mice compared with intact mice (p<0.0001). Surprisingly, OVX induced a significant decrease (p=0.045) in DCN expression, while a downward trend was found for CTGF (p=0.10) and Col3a1 (p=0.08) but not Edn1 (p=0.59). OVX also decreased the expression of macrophage markers CSF1R (p=0.022) and LYVE1 (p=0.047), with a downward trend for Gas6 (p=0.096) but not Axl (0.161).ConclusionIncreased PWV in OVX mice was not accompanied by upregulation of pro‐fibrotic genes but was associated with decreases in macrophage markers CSF1R and LYVE1 in the aorta. Therefore, estrogen loss may promote vascular stiffening through changes in the amounts or functions of specific subsets of macrophages in the vasculature. Our data support previous studies establishing a relationship between CSF1R, LYVE1, and arterial homeostasis. Future studies will examine the impact of estrogen on aortic tissue resident macrophages using flow cytometry and the downstream signaling that promotes arterial stiffening.
Biophysical Properties of Endothelial Mechanotransduction Mediated by Glycocalyx
Authors: Rivera Gonzalez Xaymara , Yazdanparast Tafti Sajedehalsadat , Dragovich Matthew , Zhang Xiaohui 
Published: May 2022

Endothelial surface glycocalyx (ESG) is a carbohydrate‐rich layer found on the endothelial cell (EC) surface lining all blood vessels' inner lumen. Composed of membrane glycoproteins, glycosaminoglycans (GAGs), such as hyaluronan and heparan sulfate (HS), and proteoglycans, the ESG forms a bulky, gel‐like layer serving critical functions in blood flow mechanotransduction, endothelial permeability maintenance, leukocyte adhesion, and inflammation control. ESG is known for sensing shear stress of blood flow and transducing the mechanical signal into nitric oxide (NO) production. NO is an essential signaling molecule to regulate vascular tone. To date, the molecular pathways of ESG‐mediated mechanotransduction have not been completely clear.We utilize a custom‐built AFM with fluorescence imaging capabilities to vertically stretch the ESG, which applies a pulling force to specific ESG components on the surface of a single live mouse brain endothelial cell (bEnd.3) and quantify the cell's NO production in real‐time. NO production is quantified by the fluorescent dye DAF‐FM. AFM probe is coated with monoclonal antibodies against HS or glypican‐1 (a major core protein for HS) to exert pulling forces onto the HS or glypican‐1 on bEnd.3. To ensure that DAF intensity is a signal of NO production, we treated the cells with L‐NAME (an inhibitor of endothelial nitric oxide synthase). The DAF intensity was greatly reduced, which confirms that the DAF intensity is representing NO production.The AFM anti‐glypican‐1 coated probe showed an increase in DAF intensity, while the anti‐HS probe is shown to have a slight rise in DAF intensity. Since HS may attach to other proteins besides glypican, the data suggest that glypican‐1 is a more critical mechanotransducer. Our prior study indicated cation‐permeable, transient receptor potential (TRP) ion channels mediate stretch‐induced NO production. Probes were coated in glypican‐1 while the cells were exposed to Amiloride or SKF96365, blockers of TRPP2 and TRPC1 channels, respectively. After the Amiloride‐treated cells were mechanically stimulated, there was a reduction of NO production. However, the amount of NO output was still significant over the basal levels observed in cells. When the SKF96365‐treated cells were mechanically stimulated, production was inhibited entirely, thus, demonstrating TRPC1 channels play a vital role in allowing the Ca2+ into the cell to begin NO production cascade.These findings confirm the critical roles of HS, glypican‐1, and TRP channels on the rapid NO production resulting from mechanical signaling and have important implications for ESG‐related cardiovascular diseases such as diabetes, strokes, cardiac arrest, and atherosclerosis. It has been shown that intracellular Ca2+regulates eNOS activity. In future studies, we will measure the Ca2+intake mediated by TRP channels and delineate the interplay among mechanical stretch, TRP channel activation, Ca2+intake and NO production.
Electronic Cigarette Exposure Induces Vascular Endothelial Dysfunction with NADPH Oxidase Activation and eNOS Uncoupling
Authors: Ewees Mohamed G. , El‐Mahdy Mohamed A. , Eid Mahmoud S. , Mahgoup Elsayed M. , Khaleel Sahar A. , Zweier Jay L. 
Published: May 2022

BackgroundWe recently reported a mouse model of chronic electronic cigarette (e‐cig) exposure‐induced cardiovascular pathology, where long‐term exposure to e‐cig vape (ECV) induces cardiac abnormalities, impairment of endothelial function, and systemic hypertension. Using this model, we performed studies to delineate the underlying mechanisms of ECV‐induced vascular endothelial dysfunction (VED), which is a central trigger of cardiovascular disease.MethodsC57/BL6 male mice were exposed 2 hours/day, 5 days/week to ECV generated from e‐cig liquid containing 0, 6, or 24 mg/ml nicotine for 16 and 60 weeks. Blood pressure was measured by tail cuff and endothelial‐dependent and independent relaxation was measured on aortic segments using wire myography. Western blotting, immunofluorescence and ELISA were used to analyze the pathways involved in the mechanism of ECV‐induced VED.ResultsTime‐dependent elevation in blood pressure and systemic vascular resistance were observed, along with an impairment of acetylcholine‐induced aortic relaxation in ECV‐exposed mice, compared to air‐exposed control. Decreased intravascular nitric oxide (NO) levels and increased superoxide generation with elevated 3‐nitrotyrosine levels in the aorta of ECV‐exposed mice were observed, indicating that ECV‐induced superoxide reacts with NO to generate cytotoxic peroxynitrite. Exposure increased NADPH oxidase expression, supporting its role in ECV‐induced superoxide generation. Downregulation of endothelial nitric oxide synthase (eNOS) expression and Akt‐dependent eNOS phosphorylation occurred in the aorta of ECV‐exposed mice, indicating that exposure inhibited de novo NO synthesis. Following ECV exposure, the critical NOS cofactor tetrahydrobiopterin was decreased, with a concomitant loss of its salvage enzyme, dihydrofolate reductase. NADPH oxidase and NOS inhibitors abrogated ECV‐induced superoxide generation in the aorta of ECV exposed mice.ConclusionOur data demonstrate that ECV exposure activates NADPH oxidase and uncouples eNOS, causing a vicious cycle of superoxide generation and vascular oxidant stress that triggers VED and hypertension with predisposition to other cardiovascular disease.