bluuebunny/crossref_metadata_2025 huggingface dataset:
Stichopus japonicus acid mucopolysaccharide inhibits growth of human liver cancer cells by modulating mitochondrial apoptosis pathway (644.15)
Authors: Song Yang , Chen Chen , Jin Yang
Published: June 2021

Sea cucumber has traditionally been used as medicinal food for promoting health and warding off disease. Stichopus Japonicus Acid mucopolysaccharide (SJAMP) isolated from sea cucumbers have been shown to have anti‐tumor property. Several working mechanisms are proposed to explain SJAMP’s anti‐tumor effect. In this study we investigated whether SJAMP exerts its anti‐tumor effect via the mitochondrial apoptosis pathway in a cell‐based model. After incubating human liver cancer cell line Hep G2 cells with SJAMP at 0.25, 1, and 4 μg/mL for 24 h, we examined, using the indicated methods, cell morphology (transmission electron microscopy), mitochondrial membrane potential (flow cytometry), and expression of gene (real time RT‐PCR) and protein (immunohistochemistry) of apoptosis‐related molecules. Results showed that SJAMP treatment significantly reduced mitochondrial membrane potential, down‐regulated gene and protein expression of anti‐apoptotic Bcl‐2 and Survivin (P<0.05), and up‐regulated gene expression of pro‐apoptotic Bax, CytC, Caspase‐3, and Smac (P<0.05). These findings indicate that SJAMP may inhibit growth of Hep G2 cells by promoting mitochondria‐mediated apoptosis pathway.

Grant Funding Source: Supported by National Natural Science Foundation
Effect of hold time and formulation on nutrient retention in 2450 MHz microwave processed fruit purees (647.31)
Authors: Truong An , Truong An , Harris Gabriel , Simunovic Josip , Sandeep K.P.
Published: June 2021

Consumer demands for convenient, healthy foods have increased in recent years. Apples, peaches, and pineapples are high in bioactive compounds and may have anti‐cancer and anti‐diabetic effects. Common thermal processing techniques result in poor product quality and destruction of phytonutrients. Continuous flow microwave technology can process homogeneous and multiple‐phase foods and maintain high nutrient retention. This study aimed to quantify the effects of microwave processing on nutrient retention in fruit purees containing apple, peach, and pineapple. Different formulations of the fruits were pureed and filled in plastic, single serve cups and heated in a 2450 MHz household microwave oven at 100 ºC for 1, 2, and 3 minutes, a more rigorous process than pasteurization. Purees were extracted with acidified methanol and analyzed for total phenolics (TP) and antioxidant activity (AA) using Folin‐Ciocalteu and ORAC method, respectively. Vitamin C (VC) was extracted with 3% metaphosphoric acid, 8% acetic acid in water and analyzed on HPLC. There were no significant losses in any of the nutrients after microwave processing. With increased processing time, fruit cocktail processed for 3 min showed no significant losses in VC or AA and a significant increase in TP. These results demonstrate that microwave processing of fruit purees retains phytonutrients and may enhance their extraction from the fruit matrix.
Chemical composition, antioxidant capacity and shelf life stability of blueberry and cranberry flavored maple sap water beverages (647.5)
Authors: Hou Juncai , Walsh Helen , Liu Ning , Levin Elise , Wang Guorong , Guo Ming
Published: June 2021

Maple sap water is a by‐product of reverse osmosis filtration of maple sap during the production of maple syrup. The objective of this study was to develop blueberry and cranberry flavored beverages using maple sap water. The flavored sap water beverages were formulated using blueberry and cranberry concentrates, acidulant, and sugar and were pasteurized before bottling. Physicochemical characteristics, microbiological quality, ascorbic acid content, and antioxidant capacity of the beverages were analyzed during storage at 4 or 23 °C for 8 weeks. No growth of the total plate count, coliform, and yeast/mold in the beverages was seen initially, nor was it detected throughout the 8‐week storage either at 4 or 23 °C. The sugar content, pH, and titratable acidity for the flavored maple sap water beverages were significantly impacted by storage temperature, time and their interaction (P < 0.05). The storage temperature, time and their interaction significantly affected the area and peak of color absorption at 450 to 600 nm of flavored maple sap water beverages (P<0.01). The ascorbic acid content and total antioxidant capacity of flavored maple sap water beverages were significantly decreased during storage (P<0.01). Results showed that the maple sap water can be used for formulation of flavored functional beverage.
Anti‐melanogenic effect of the ethanol extract of Commelina communis L. in melanoma cells (647.50)
Authors: Kang Moon‐Kyung , Mun Yeun‐Ja , Woo Won‐Hong , Lee Young‐Eun
Published: June 2021

Commelina communis Leded.., a annual plant, has been used as an antidiabetic, antioxidant and hypoglycemic agent in korea herbal medicine. We analyzed the anti‐melanogenic effects of ethanol extracts from Commelina communis Leded. (CCL) in B16F10 cells. CCL significantly reduced tyrosinase activity of the cells about 62.76±3.5% and 52.09±7.9% at concentrations of 12.5 μg/ml and 25 μg/ml, respectively. Also, melanin synthesis of cells was decreased with 66.11% and 52.30% by CCL, respectively. Tyrosinase gene and protein expressions were inhibited by the treatment with CCL. Moreover, CCL significantly reduced α‐melanocyte stimulating hormone (α‐MSH)‐induced melanin synthesis and tyrosinase activity, also decreased α‐MSH‐induced expression of melanogenic enzyme like as tyrosinase, tyrosinase‐related protein (TRP)‐1 and TRP‐2. This results show that the anti‐melanogenic activity of CCL is correlated with the suppression of tyrosinase gene and protein expressions in B16F10 cells.
Connective tissue growth factor (CTGF/CCN2) expression in quiescent hepatic stellate cells is inhibited by a Twist‐1‐miR‐214 axis (649.7)
Authors: Chen Li , Charrier Alyssa , Brigstock David
Published: June 2021

Connective tissue growth factor (CTGF/CCN2) drives hepatic fibrosis in vivo by stimulating fibrogenic pathways in hepatic stellate cells (HSC). Under normal conditions, CTGF/CCN2 expression is very low in quiescent HSC but CTGF/CCN2 expression is dramatically increased as HSC become activated during liver injury and adopt a fibrogenic phenotype. We have investigated the molecular basis for the suppressed CTGF/CCN2 levels in quiescent HSC. CTGF/CCN2 up‐regulation in fibrotic or steatotic livers, or in culture‐activated or ethanol‐treated primary mouse HSC was associated with a reciprocal down‐regulation of microRNA‐214 (miR‐214). By using protector or reporter assays to investigate the activity of the 3’‐untranslated region (UTR) of CTGF/CCN2 mRNA, induction of CTGF/CCN2 expression in HSC by fibrosis‐inducing stimuli was due to reduced expression of miR‐214 which otherwise inhibited CTGF/CCN2 expression by directly binding to the CTGF/CCN2 3’‐UTR. In primary mouse HSC, miR‐214 was co‐expressed with the transcription factor Twist‐1 which is a predicted target of an E box in the miR‐214 promoter. Expression of miR‐214 and Twist‐1 in HSC was dose‐dependently inhibited by ethanol or culture‐induced activation in a manner reciprocal to that of CTGF/CCN2. Transfection of primary D6 HSC (high endogenous Twist‐1 levels) with Twist‐1 siRNA reduced expression of miR‐214, but increased CTGF/CCN2 production. An opposite effect was shown by transfecting P6 HSC (low endogenous Twist‐1 levels) with Twist‐1 expression plasmids. Twist‐1 stimulated luciferase activity in HSC transfected with a wild‐type miR‐214 promoter but not with a mutant miR‐214 promoter lacking the E‐box site. We conclude that CTGF/CCN2 expression in HSC is dynamically regulated by an inhibitory Twist‐1‐miR214 axis. This regulatory mechanism may have utility for therapeutic intervention in pathways of CTGF/CCN2‐dependent fibrosis.

Grant Funding Source: NIH 5 R01 AA021276
A desmoplakin point mutation insensitive to PKC phosphorylation ameliorates pemphigus vulgaris autoantibody‐mediated loss of cell cohesion (650.4)
Authors: Dehner Carina , Waschke Jens , Spindler Volker
Published: June 2021

Desmoplakin (DP) serves to anchor intermediate filaments in desmosomal complexes. Recent data suggest that a specific DP point mutation (S2849G) exhibits increased keratin filament association and fosters Ca2+‐insensitivity of desmosomes in keratinocytes, presumably by rendering DP inaccessible for PKC phosphorylation. Previously, we have shown that depletion of the desmosomal adhesion molecule desmoglein 3 (Dsg3) induced by autoantibodies from patients with the blistering skin disease pemphigus vulgaris (PV‐IgG) is reduced in maturated desmosomes and dependent on PKC signaling. Thus, we investigated the role of DP‐S2849G for loss of cell cohesion mediated by PV‐IgG. In cell dissociation assays, expression of DP‐S2849G increased cell cohesion in two different human keratinocyte cell lines and ameliorated loss of cell adhesion induced by PV‐IgG or treatment with AK23, a pathogenic Dsg3 antibody derived from a pemphigus mouse model. Depletion of Dsg3 was inhibited by DP‐S2849G in the cytoskeletal (Triton‐X 100 insoluble) fraction and keratin filament retraction, a hallmark of PV, was efficiently blocked. In line with this, DP was phosphorylated upon incubation with PV‐IgG in a PKC‐dependent manner. Taken together, our data demonstrate the relevance of keratin filament anchorage to desmosomes for cell adhesion and suggest PKC‐mediated DP phosphorylation as a central mechanism in PV pathogenesis.
Acute mercury toxicity modulates cytochrome P450, soluble epoxide hydrolase and their associated arachidonic acid metabolites in C57BL/6 mouse heart (652.5)
Authors: El‐Kadi Ayman , Amara Issa , Elshenway Osama
Published: June 2021

Mercury exposure is associated with increased risk of cardiovascular disease and profound cardiotoxicity. However, the correlation between Hg2+‐mediated toxicity and alteration in cardiac cytochrome P450s (Cyp) and their dependent arachidonic acid metabolites has never been investigated. Therefore, we investigated the effect of acute mercury toxicity on the expression of Cyp‐epoxygenases and Cyp‐ω‐hydroxylases; and their associated arachidonic acid metabolites in mice hearts. In addition, we examined the expression and activity of soluble epoxide hydrolase (sEH) as a key player in arachidonic acid metabolism pathway. Mercury toxicity was induced by a single intraperitoneal injection (IP) of 2.5 mg/kg of mercuric chloride (HgCl2). Our results showed that mercury treatment caused a significant induction of the cardiac hypertrophy markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP); in addition to Cyp1a1, Cyp1b1, Cyp2b9, Cyp2b10, Cyp2b19, Cyp2c29, Cyp2c38, Cyp4a10, Cyp4a12, Cyp4a14, Cyp4f13, Cyp4f15, Cyp4f16 and Cyp4f18 gene expression. Moreover, Hg2+ significantly increased sEH protein expression and activity, in hearts of mercury‐treated mice, with a consequent decrease in 14,15‐, and 11,12‐epoxyeicosatrienoic acids (EETs) formation. Whereas the formation of 14,15‐, 11,12‐, 8,9‐dihydroxyeicosatrienoic acids (DHETs) was significantly increased. In conclusion, acute Hg2+ toxicity modulates the expression of several Cyp and sEH enzymes with a consequent decrease in the cardioprotective EETs which could represent a novel mechanism by which mercury causes progressive cardiotoxicity. Furthermore, inhibiting sEH might represent a novel therapeutic approach to prevent Hg2+‐induced hypertrophy.

Grant Funding Source: Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant RGPIN 250139
Discovery of combinational targets for Alzheimer’s disease based on traditional Chinese medicinal prescription (654.10)
Authors: Zhou Wen‐Xia , Cheng Xiao‐Rui , Zhang Yong‐Xiang
Published: June 2021

The present one‐drug, one‐target paradigm for anti‐Alzheimer’s disease (AD) treatment appears to be clinically unsuccessful, the combinational targets which composed of several biomolecules based on bio‐network may be optimum for the discovery of anti‐AD drugs. This study aims to search the combinational targets for AD based on traditional Chinese medicine (TCM) using network pharmacology strategy. The nootropic effects of five TCM prescriptions, namely Liuwei‐Dihuang decoction (LW), Bawei‐Dihuang decoction (BW), Huanglian‐Jiedu decoction (HL), Danggui‐Shaoyao‐San (DSS) and Tiaoxin Recipe (TXF), were validated by cognitive behavior test in senescence accelerated mouse prone 8 (SAMP8), and the combinational therapeutic targets were investigated using cDNA microarray, RT‐PCR, bioinformatics and computational biology techniques. The results showed LW, BW, HL, DSS, and TXF significantly improve the cognitive behavior of SAMP8. There are 117 genes expression in hippocampus and 122 genes expression in cerebral cortex regulated by LW, BW, HL, DSS and TXF in SAMP8. These genes regulated by five prescriptions involve in the biomolecular network. The hub nodes included 9 molecules, that is STUB1, STRN4, RPS6KA1, ROCK1, MAP3K3, TICAM2, MAPT, UBE2D2 and PHB. Furthermore, RPS6KA1, SLC17A7 and FHIT are the common nodes effected by one of five prescriptions and may be potential therapeutic target candidates for AD.

Grant Funding Source: the National Science and Technology Major Project (2012ZX09301003‐002‐001, 2013ZX09508104)
Patch clamp recordings of fast‐acting ion channels in rat dorsal root ganglion cells (654.3)
Authors: Svensson Dalén Jennie , Sinclair Jon , Karlsson Anna , Pihl Johan , Karila Paul
Published: June 2021

Manual patch clamp recordings offer direct insight into ion channel properties through the characterization of ion channel activity. Due to the high quality data it is considered the gold standard for ion channel research. Still, the limited throughput means that maximizing data from each cell, especially sensitive primary cells, is important.

We have developed a patch clamp‐based assay for characterization fast‐acting ion channels, in this case exemplified by P2X, in primary dorsal root ganglion (DRG) neurons. This assay is based on the use of primary DRG neurons in culture as a cell model for chronic pain. These neurons retain their sensory functionality and remain responsive to thermal, mechanical and functional stimuli, and when supplemented with nerve growth factor (NGF) they can be used to mimic peripheral sensitization. The assay utilizes a microfluidic perfusion system, Dynaflow Resolve, to facilitate a stable recording situation and fast and programmable solution exchange. At the start of the experiment, compounds and buffer are loaded in the 16 wells of the Dynaflow Resolve chip. Micro‐channels connect each well to a recording chamber where the cells are added. To be able to provide fast solution exchange the DRG neuron is lifted using the patch pipette and positioned in front of the micro‐channel outlets. Then, the cell is scanned through the discrete flow zones formed outside the channels.

A comparison between conventional bath perfusion and the automated Dynaflow Resolve system showed that both systems have similar capacity when it comes to obtaining successful high resistance seals. However, for collecting full 5‐8 point dose‐responses from single cells, the success rate for the Dynaflow Resolve superseded the conventional system. This enables a dramatically increased productivity when performing more complex pharmacological characterizations.
Antibacterial activities of a novel silver(I) complex with furosemide (654.8)
Authors: Cóssia Silva Maria , Lazarini Silmara , De Carvalho Renata , Amaral André , Corbi Pedro , Lustri Wilton
Published: June 2021

The use of metal complexes in medicine dates back to the 16th century. Nowadays one of the best examples of a metal‐based drug used in medicine is silver‐sulfadiazine (SSD). This compound is used topically as a cream in the treatment of burns and wounds preventing the appearance of skin bacterial infections. Due to the fact that several metal complexes are extensively applied in the treatment of various diseases the knowledge and understanding of their chemical and pharmacological properties are of high significance. The present research aims to establish conditions for the synthesis and evaluation of the antibacterial activities of a novel silver complex with furosemide (Ag‐fur). Elemental analysis of C, H and N showed a 1:1 metal/ligand composition for the complex, with the molecular formula AgC12H10ClN2O5S. Although insoluble in water, the Ag‐fur complex is soluble in dimethyl sulfoxide. The antibacterial activities of the complex were primarily evaluated by antibiogram assays using the disc diffusion method. The Ag‐fur complex has shown to possess significant antibacterial activity against Staphylococcus aureus (Gram‐positive), Escherichia coli and Pseudomonas aeruginosa (Gram negative) when compared to AgNO3 and to the standard antibiotic ceftriaxone.

Grant Funding Source: Supported by: Conselho Nacional de Desenvolvimento Científico e Tecnológico ‐ CNPq
Thalassia testudinum extract modulates hepatic cytochome P450 system in Wistar rats (657.16)
Authors: Rodeiro Guerra Idania , Hernandez Sandra Luz , Hernandez Ivones , Padron Saul Alejandro , Herrera Jose Alfredo , Olguin Sitlali , Camacho Rafael , Ronquillo Maria Dolores , Fernandez Miguel David , Menendez Roberto , Espinosa‐Aguirre Javier
Published: June 2021

We disclose here the effects over several P450 isoforms of one extract from Thalassia testudinum marine plant with cytoprotective and neuroprotective properties. Male Wistar rats were administered orally with 20, 200 and 400 mg/kg of the extract for 10 days. The activities of 7‐ethoxy, 7‐methoxy, 7‐penthoxy and 7‐benzoxyl resorufin‐O‐deethylases, 4‐nitrophenol hydroxylation and erythromycin N‐demethylation were used to asses the function of CYP1A1, CYP1A2, CYP2B1, CYP1B2, 2E1 and 3A in liver microsomes. Protein expressions of cythocromes were determined by western blot and mRNA levels by RT‐PCRq. Microsoms were used as metabolic fraction for the Ames test. Other animals, after receive the same doses of the extract, were administered orally with theophylline (10 mg/kg), blood samples were taken in order to determine the theophylline concentration in plasma. Thalassia testudinum extract (200 mg/kg) was able to induce CYP1A1 (1.5‐fold). Meanwhile, after 400 mg/kg the activity did increase, but no significant differences were found. The lowest dose (20 mg/kg) induced increments of CYP1A2 and 2B1 activities, but no significant differences were seen. A reduction (near to 20%) of CYP2B2, 2E1 and 3A activities was also found. The increase of the CYP1A1 activity was in agreement with the elevation of the protein, but mRNA levels were no modified. The extract increased the mutagenicity of the benzo(a)pyrene at the highest doses (1.8 and 2.3‐fold), confirming its influence on the CYP1A1 activity. Pharmacokinetic profile of theophylline changed in treated rats: Cmax and clearance of theophylline were significantly increased. The elimination rate constant increased twice and the half‐life time was 2‐fold reduced. Thalassia testudinum acts as inducer of the CYP1A1/2 enzymes under these experimentals, suggesting potential herb‐drug interaction when patients use the extract concomitantly or in advance of drugs which are substrates of P450s, especially CYP1A1/2.
Changes in extrasynaptic GABAA receptors in the periaqueductal gray with morphine tolerance (659.7)
Authors: Tonsfeldt Karen , Li Minghua , Suchland Katherine , Ingram Susan
Published: June 2021

Mu‐opioid receptors (MOPrs) reduce GABA‐mediated inhibition of periaqueductal gray (PAG) output neurons to activate the descending pain modulatory system and induce antinociception. The role of extrasynaptic GABAA receptors and tonic inhibition of PAG neurons in antinociception has not been explored. Rats were treated twice a day with saline or morphine (5 mg/kg, sc.) for 3 days. Superfusion of the GABAA antagonist, bicuculline (10 µM), inhibited miniature inhibitory postsynaptic currents and blocked a resting current, indicating the presence of both synaptic and extrasynaptic GABAA‐mediated currents in the PAG. Bicuculline reduced the holding current in saline rats 18 ± 4 pA (n = 10), with less effect in morphine rats (2 ± 1 pA, n = 6, p < 0.05). The bicuculline currents reversed at the expected chloride potential in chloride (‐24 ± 3 mV) and gluconate (‐84 ± 2 mV) intracellular solutions, suggesting the current is mediated by GABAA receptors. Tonic currents are often mediated by extrasynaptic GABAA receptors containing the delta‐subunit. THIP (10 µM), a GABAA delta subunit receptor agonist, induced currents in naïve and morphine treated slices (84 ± 22 pA, n= 8; 77 ± 24 pA, n = 6). The THIP induced currents were reduced by prior administration of met‐enkephalin (10 µM) (30 ± 7 pA, n = 5) compared to control (74 ± 16 pA, n = 7 , p < 0.05). These results suggest GABAA delta receptors may be modulated by activation of MOPrs in the PAG.

Grant Funding Source: DA027625
Significance WHO criteria in assessing pneumonia in children in Kazakhstan (660.7)
Authors: Gatauova Madina , Abduldayeva Aigul
Published: June 2021

To identify children under 5 years of pneumonia or lack thereof according to WHO criteria and the need for hospitalization. Retrospectively analyzed 852 medical records of children referred from in‐patient clinic with a diagnosis of “community‐acquired pneumonia” for the period 2012. In a retrospective analysis of 852 case histories diagnosed with” community acquired pneumonia” 60.92% of the respiratory pathology revealed the following.Febrile temperature with a duration >3 days was observed only in 182 children 21.36%.•shortness of breath and cyanosis :>50 breaths per minute at the age of 2‐12 months range 2‐6 months in 194 children, from 6 months to 12 months. In 92 children 10.8%. Dyspnea >40 breaths per minute in 26 children from one year to five years.Coughing was observed in 15.8% of affected children. Physical examination: retraction of the lower part of the chest, crackles on auscultation were observed in 54% of cases.Chest X‐ray was carried out in 78.3% of all children admitted. Infiltrative shadows were observed only in 151 children 17.7%. In 8.5% children ‐ polysegmental pneumonia, lobar pneumonia ‐ 0.35%, focal‐drain from 65 7.6%, draining 8.5%. Based on the above, according to the WHO criteria for hospitalization only needed 232 on the number of children admitted 27.2% with severe and very pneumonia,72.7% of children do not require hospitalization ‐ they have to be treated at home.
Expression dynamics of pro‐ and anti‐apoptotic proteins regulated by cAMP (663.19)
Authors: Wandrey Narine , Wilderman Andrea , Insel Paul
Published: June 2021

The second messenger cyclic AMP (cAMP) promotes apoptosis in many cell types, including certain lymphoid cells, by poorly understood mechanisms. Murine S49 T‐lymphoma cells are a model system to study cAMP‐mediated apoptosis and have a distinct advantage for such studies: a clonal variant (kin‐) that lacks protein kinase A (PKA) activity and cAMP‐induced apoptosis, thereby facilitating analysis of PKA‐regulated apoptotic components. We have compared protein levels and subcellular localization of pro‐ and anti‐apoptotic Bcl family proteins in wild‐type (WT) and kin‐ S49 cells treated with the cell‐permeable analog CPT‐cAMP (CPT). We found that WT, but not kin‐, cells treated with CPT time‐dependently increase their level of the S, L and EL isoforms of the pro‐apoptotic protein Bim. We also examined PKA‐promoted phosphorylation of Bad, which is anti‐apoptotic when phosphorylated. We detected no phosphorylation of Bad at Ser155 (a PKA target site) in kin‐ cells during 72 hr treatment with CPT. Unexpectedly, WT cells showed strong Ser155 phosphorylation of Bad (and an increased ratio of p‐Ser155 Bad/total Bad) in response to 24‐72 h CPT treatment. Thus, cAMP acts via PKA to enhance both pro‐ and anti‐apoptotic proteins. We speculate that the late increase in pBAD in WT S49 cells may be an attempt by the cells to reverse their ultimate apoptotic response to cAMP/PKA activation.

Grant Funding Source: Supported by: ASPET 2013 Summer Undergraduate Research Fellowship
Hydrogen peroxide induced homeostatic regulation in endothelial cells (667.10)
Authors: Patel Hemang , Chen Juan , Kavdia Mahendra
Published: June 2021

Transient and consistent increase in hydrogen peroxide (H₂O₂) levels is associated with several vascular pathologies. H₂O₂ has been evaluated for cytotoxic and vasoactive response to vasculature. However, its ability to regulate endothelial cell‐homeostasis remains unclear. To understand the consistent and transient H₂O₂ effects, we investigated the effect of continuous and transient H₂O₂ treatments, respectively on endothelial cells superoxide (O₂‾) production and gene expression. Following both H₂O₂ treatments, we measured gene expression levels of key ROS neutralizing enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase 1 (GPX1) and peroxiredoxin 1 (PRDX1), and cell signaling proteins such as ASK1, ATF2 and NFE2L2. We also measured O₂‾ production using dihydroethidium (DHE). Gene expression results indicated a significant down‐regulation in ASK1, CAT, TXNRD1 under continuous H₂O₂ exposure. Transient H₂O₂ exposure followed by remodeling period showed significant up‐regulation in GPX1 and TXNRD1 whereas genes expression of ASK1, SOD1 and PRDX1 returned to homeostasis. Interplay among functional and gene expression changes during continuous and transient H₂O₂ exposure suggested alternative molecular pathway regulation leading to redox homeostasis in endothelial cells.

Grant Funding Source: NIH R01 HL084337
Endothelial inflammation and monocyte adhesion following increased vascular wall shear stress in an ex vivo femoral artey model (670.10)
Authors: KadamBaldwin Aparna , Gersch Robert , Rosengart Todd , Frame Mary
Published: June 2021

Monocyte recruitment studies are primarily in vitro studies, or require the sacrifice of animals at multiple time points to analyze accumulation. With this ex vivo femoral artery model we can observe monocyte behavior in a whole vessel with all the extracellular matrix and structure intact. This study uses a previously characterized ex vivo femoral artery system. Ex vivo femoral arteries were subject to varying wall shear stresses (WSS)_physiological WSS (10.18Pa), 1.4x higher, 2x higher, and physiological + TNFα as appositive control. Following the induction of endothelial inflammation by WSS, fluorescently labeled monocytes were introduced into the perfusate or the vessels were flayed open an incubated with static monocytes. The subsequent adhesion of the monocytes to the endothelial wall was captured by video and microscopy. As hypothesized, TNFα positive control showed a significant increase in expression of all cytokines and adhesion molecules (p<0.05). Further, pro‐inflammatory cytokines MCP‐1, TNFα, and adhesion molecules P‐selectin, and VCAM‐1 were significantly upregulated with increases in WSS. It was further hypothesized that there would not be an upregulation of growth factors from an increase in shear stress alone. However, here, we see that the selected growth factors, FGF2, TGFβ, VEGF, Egr‐1 do in fact all increase in expression with the increases in WSS. The perfusion of monocytes through the femoral artery can be seen on video, however, the flow rate required to induce the experimental WSS was too high to allow for monocyte adhesion. The static monocyte incubation on arteries showed that higher WSS means more adhesion molecules and therefore more monocyte adhesion. We can conclude that monocyte adhesion is positively correlated with increases in WSS.
GLP‐1 affects human endothelial cell signaling by reducing VEGF‐A induced Ca2+ signaling and activating protein kinases (670.4)
Authors: Rotboel Cecilie , Tritsaris Katerina , Hansen Anker , Dissing Steen
Published: June 2021

GLP‐1 is a gut hormone and has until now mainly been in focus for its role in response to food intake. Here we have explored its potential role in capillary function by studying signaling in human dermal endothelial cells (HDMEC). We found that HDMEC express the GLP‐1R by use of RT‐PCR. GLP‐1 signaling in HDMEC causes partial inhibition and activation of pathways. By use of Ca2+ imaging we found that preincubation with GLP‐1 caused a reduction in VEGF‐A induced Ca2+ signaling due to a reduction in phosphorylation of PLCγ but also found that GLP‐1 causes phosphorylation of Src, Akt, ERK 1/2 and p38. GLP‐1 is an activator of Gs/βγ and we attempted to mimic the βγ‐subunit activation by treating HDMEC with pertussis toxin (PTX), which predominantly inhibits Gi. PTX also reduced VEGF‐induced Ca2+ release and in addition we found an enhanced Src phosphorylation. Our data are consistent with the GLP‐1 signaling being evoked through an enhanced Gs/βγ signaling since Gi inhibition elicits a similar response by favoring the Gs/βγ signaling pathway. The effects on VEGF‐induced Ca2+ transients implicate that GLP‐1 signaling could affect endothelial cell structure, metabolism and proliferation.
Regulation of HDL cholesterol metabolism and the impact of long‐term feeding of dietary jojoba oil and cholesterol on the development of atherosclerosis in New Zealand white rabbits: a foundational study (671.14)
Authors: Karam Tarek
Published: June 2021

Jojoba oil has been shown to maintain high‐density lipoprotein (HDL) concentration in hypercholesterolemic New Zealand White (NZW) rabbits, suggesting that jojoba oil regulates HDL metabolism and has a protective effect against atherosclerosis, one of the leading causes of death worldwide. We thus hypothesized that jojoba oil decreases the development of atherosclerosis in NZW rabbits. As a preliminary study, five NZW rabbits were fed either a normal chow diet (N), or a normal diet supplemented with either 3% jojoba seed oil (J), 1% cholesterol (C), or 1% cholesterol + 3% jojoba seed oil (CJ) for 7 weeks; each diet group contained one rabbit, except the C‐fed group which contained two. During the study, serum lipoprotein cholesterol concentrations were monitored via enzymatic assays and gel electrophoresis. Liver function was examined via serum albumin measurements. Rabbit aortas were extracted and assessed for the development of atherosclerotic lesions. Results indicated that NZW rabbits responded to the experimental diets as shown in previous studies. N and J‐fed rabbits showed no change in their lipoprotein profiles. C‐ and CJ‐fed rabbits had a decrease in HDL concentration; however, the decrease was less in the CJ rabbits than the C rabbits. Serum albumin measurement indicated proper liver function. Unfortunately, none of the rabbits developed atherosclerotic lesions and the effects on dietary jojoba oil on atherosclerosis development could not be assessed in this study. Despite the negative results, this preliminary study allowed us to better understand what must be done in order to study the original question of interest. In future studies, we plan to ensure atherosclerotic lesion development by conducting a dose response study.
Involvement of p38 MAP kinase in alcohol‐induced endothelial hyperpermeability (672.5)
Authors: Doggett Travis , Breslin Jerome
Published: June 2021

Recently we have demonstrated using a rat model of alcohol binge drinking that alcohol intoxication increases microvascular leakage in the gut. To investigate the endothelial mechanisms involved, we utilized a cultured endothelial cell (EC) model, utilizing human umbilical vein EC, human dermal microvascular EC, or human cardiac microvascular EC. We hypothesized that alcohol‐induced endothelial hyperpermeability is mediated by p38 MAP kinase signaling. Transendothelial electrical resistance (TER) of EC monolayers served as an index of barrier function. The pharmacological inhibitor SB203580 was used to test the role of p38 MAPK. In addition, localization of the intercellular junction protein VE‐cadherin was determined by immunofluorescence labeling. The results show that alcohol at concentrations greater than 20 mM significantly decrease TER and disrupt VE‐Cadherin continuity. Addition of 6 µM of SB203580, 30 min before addition of alcohol significantly inhibited the maximum decrease in TER (‐14.9% ± 1.9 vs. ‐36.8% ± 1.8, p<0.001). These data suggest that alcohol‐induced increase in endothelial permeability occurs due to activation of p38 MAP kinase and disruption of junctional VE‐Cadherin. Supported by NIH R01HL098215, R21AA020049, and the ABMRF/Foundation for Alcohol Research.
Substrate and stretch regulate muscle‐resident mesenchymal stem cells to promote myoblast proliferation (673.1)
Authors: De Lisio Michael , Jensen Tor , Sukiennik Richard , Huntsman Heather , Boppart Marni
Published: June 2021

Mesenchymal stem cells (MSCs) reside in a variety of tissues and provide a stromal role in regulating progenitor cell function. Current studies focus on identifying the specific factors in the niche that can alter the MSC secretome, ultimately determining effectiveness and timing of tissue repair. The purpose of the present study was to evaluate the extent to which substrate and mechanical strain regulate MSC quantity, gene expression and secretome. MSCs (Sca‐1+CD45‐) isolated from murine skeletal muscle via FACS were seeded onto laminin‐(LAM) or collagen‐1‐(COL) coated membranes and exposed to a single bout of mechanical strain (10%, 1 Hz, 20 min). MSC proliferation was not directly affected by substrate or strain; however, the genetic expression of growth and inflammatory factors and ECM proteins were down‐regulated in MSCs grown on COL in a manner independent of strain. In conditioned media (CM) experiments, the combination of strain and LAM induced secretion of factors from MSCs that increased myoblast quantity 24 hours post‐treatment (5.6 fold; p<0.05), while this response was delayed in myoblasts treated with CM from MSCs grown on COL. Here, we demonstrate that exposure to COL, the primary ECM component associated with tissue fibrosis, down‐regulates MSC gene expression, and delayed their ability to stimulate myoblast proliferation.

Grant Funding Source: Supported by the Ellison Medical Foundation
Characterization of particle‐endothelium interaction using particles functionalized with dual antibodies in a complex synthetic microvascular network (674.3)
Authors: Lamberti Giuseppina , Tang Yuan , Kiani Mohammad , Wang Bin
Published: June 2021

The interaction between leukocytes and receptors on the endothelium play a key role in the early response to tissue injury. Research has shown that drug carrier particle rolling velocity increases dramatically with the increase of carrier size which affects their final attachment efficiency. The objective of this study is to characterize the rolling and adhesion profile of different size functionalized particles in a synthetic microvascular network (SMN).

Functionalized particles were prepared by coating their surfaces with different ratios of antibodies against adhesion molecules ICAM‐1 and E‐selectin. Our modified Geographic Information System (GIS) approach was used to digitize the microvascular networks and generate the SMN on PDMS. The rolling velocity and adhesion profile of functionalized 2 & 10 µm particles was quantified in the SMN pre‐coated with TNF‐α activated human umbilical vein endothelial cells under shear conditions from 0 ‐ 280 sec‐1.

Single antibody coated 10 µm particles roll at a higher speed than its 2 µm counterparts. Particle adhesion increased significantly with decreasing shear in SMNs. Increasing particle size from 2 µm to 10 µm did not change its binding efficiency significantly in SMNs, which is different from what have been reported in the traditional parallel plate flow channel.

Particles functionalized with both a rolling molecule and an adhesion particle can more realistically mimic the leukocyte‐endothelium interaction in a SMN. These findings have important implications for better understanding the mechanisms behind inflammatory pathologies and for optimizing the design of carriers for targeted drug delivery.

Grant Funding Source: Supported by Susan G. Komen for the Cure foundation
Systems biology framework for studying hyperglycemia‐induced endothelial dysfunction (676.19)
Authors: Patel Hemang , Chen Juan , Kavdia Mahendra
Published: June 2021

Current understandings of endothelial dysfunction mechanism have failed to provide guidelines for a system wide treatment of endothelial dysfunction. In this study, we investigated and compared the effects of high glucose on HUVEC and HMVEC using a systems biology approach. Following high glucose exposure, gene expression levels of several redox enzymes, ROS neutralizing enzymes, and oxidative stress activated transcription factor were compared in both endothelial cell types. The functional changes of superoxide (O₂‾), hydrogen peroxide (H₂O₂), and mitochondrial membrane polarization (MMP) were measured in endothelial cell. Results illustrated a consistent increase of O₂‾ levels, in both endothelial cells. However, there was a variable change in underlying gene expression profile and H₂O₂ and MMP levels, during high glucose exposure in these two endothelial cells. The results showed that a hallmark increase of O₂‾ in endothelial cells across human vasculature could be a result of variable involvement of gene expression and functional changes. Our findings highlighted a unique framework for hyperglycemia‐induced endothelial dysfunction. Identifying genomic phenotype and corresponding functional changes in hyperglycemic endothelial dysfunction will provide a suitable systems biology approach for understanding underlying mechanisms and possible effective therapeutic intervention.

Grant Funding Source: NIH R01 HL084337
Protection against depressive symptom‐induced impairments to cerebral vascular reactivity in female versus male rats (676.8)
Authors: Stanley Shyla , Brooks Steven , Chantler Paul , Butcher Joshua , D'Audiffret Alexandre , Frisbee Jefferson
Published: June 2021

Chronic stress/depression alters vascular reactivity and can impact perfusion within target organs. In addition, they also can alter cerebral structure and mass ‐ leading to the question of associations between cerebrovascular reactivity and outcomes. To address this, we imposed 8 weeks of unpredictable chronic mild stress (UCMS) on rats of both genders at 9 weeks of age. In both genders, UCMS caused profound depressive symptoms, including reductions in chronic and stimulus‐induced grooming, learned helplessness and anhedonia, with elevated plasma cortisol. However, these behaviors were more severe in females. Endothelial‐dependent dilation (acetylcholine) was impaired in middle cerebral arteries (MCA) in both genders owing to a reduced NO bioavailability and increased production of ROS and TxA2. There was also a mild increase in serotonin‐induced and myogenic constriction of MCA from males. In females, alterations to constrictor and dilator reactivity were blunted, as were elevated ROS and constrictor prostanoids. These results suggest that female rats may have a protection against depression‐induced alterations to cerebrovascular reactivity.

Grant Funding Source: Supported by National Institutes of Health and American Heart Association
Modeling electrical signal propagation in microvascular networks (678.12)
Authors: Lee Pilhwa , Pradhan Ranjan , Carlson Brian , Beard Daniel
Published: June 2021

A bidomain formulation was developed to simulate propagation of electrical signals in endothelial cells of microvascular networks. The method is used to stimulate spatiotemporal signal propagation and attenuation in arterioles and capillaries, and analyze data from experiments on isolated vessels. A single‐vessel (capillary) segment model is parameterized based on conducted response data of isolated endothelial cells tube from mouse skeletal muscle feed arteries on changes in endothelial cell membrane voltages in response graded electrical stimulation. Simulation of the resulting model facilitates determination of appropriate boundary conditions to apply along the length in simulating signal propagation in whole capillary network. The model fits well to the observed spatial attenuation of the depolarization of endothelial cells, with activated/inactivated calcium‐activated potassium channels of the membrane, and predicts the electrical length constant dependency on acetylcholine. Simulations of realistic network topologies are then conducted to determine the effective time and space scales for metabolic signaling in the microvascular networks, especially with the vasodilator acetylcholine. This framework is a foundation for further study of spatiotemporal aspects of the electrical conduction in various microvascular networks.

Grant Funding Source: U01 HL118738‐01A1
The role of brain angiotensin converting enzyme on the nocturnal thirst and sodium appetite in ovariectomized rats (683.6)
Authors: Reis Luis , Lepletier Ailin , Mecawi Andre , Fonseca Fabricia , Araujo Iracema , Almeida‐Pereira Gislaine , Antunes‐Rodrigues Jose
Published: June 2021

We investigated the role of brain angiotensin converting enzyme (ACE) on nocturnal fluid intake (water and 0.3 M NaCl solution) in ovariectomized (OVX) and estradiol benzoate‐treated (EB‐OVX) rats placed in metabolic cages. We performed peripheral (100 mg/Kg) and central (50 ng/animal) captopril (CAP) administration with or without angiotensin I (ANG I; 100 ng/animal) microinjection. Both CAP administration routes reduced basal water and salt intake in OVX and EB‐OVX groups. Peripheral CAP administration inhibited fluids intake in both groups submitted to sodium depletion (sc furosemide 20 mg/Kg followed by 24 h low sodium diet) while icv CAP decreased fluids ingestion only in the EB‐OVX group. Following furocap protocol (sc furosemide 10 mg/Kg plus sc CAP 5 mg/Kg), icv CAP decreased fluids intake in both groups. Hypothalamic ACE‐1 mRNA expression was unaltered in both protocols comparing OVX vs EB‐OVX. EB replacement increased hypothalamic ACE‐2 mRNA expression in furosemide protocol, despite it has been lower in furocap than in furosemide protocol. Our results suggest that EB replacement attenuates sodium appetite in OVX rats due to the modulation of the brain ANG II activity. The increased ACE‐2 expression induced by EB replacement could be a plausible explanation taking into account other angiotensins produced in the brain may exert a counteracting ANG II signalling.

Grant Funding Source: Supported by FAPERJ & CNPq
Idiopathic benign paroxysmal positional vertigo (ip‐bppv)of the true principal pathology and medical treatments (684.2)
Authors: Tanioka Hisaya , Kaga Kimitaka
Published: June 2021

PURPOSE: 1) What is the true principal pathology of IP‐BPPV? 2) Where is in the free otoconia in the membranous labyrinth? 3) How is the morphological change of the vestibular end‐organs in the affected region? 4) What kind of conditions about the cerebral vasculature in BPPV? 5) How to treat BPPV? SUBJECTS and METHOD: Six unilateral affected patients were entered into this study, and follow‐up CT studies in the recovery period were performed. The axial CT data was applied to create 3D images on the work station for post processing.

RESULT: 1) The utericular macula increased volume rather than both the non‐affected side and recovery period. 2) The affected ampulla and cupula showed enlargement and thickening rather than recovery period. 3) The free otoconia was identified at the affected semicircular duct by 3D virtual histology, on recovery, the free otoconia disappeared. 4) The affected semicircular duct was relatively wide diameter rather than that of normal one. 5) The cerebral vasculature showed hyperdense on CT such as hyperdense basilar sign.

TREATMENT: About two days bed rest and medications cured patients of BPPV.

The medications were drugs for increased blood flow and a sedative.

CONCLUSIONS: IP‐BPPV was caused by poor cerebral blood circulation such as infarction. The volume of otoconia increased in affected inner ears. This cause may be secondary to poor turn over or over product.
Single unit recordings from putative presympathetic neurons in the rostral ventrolateral medulla of sedentary and physically active rats (686.35)
Authors: Huereca Daniel , Mueller Patrick
Published: June 2021

A sedentary lifestyle has been linked to the development of cardiovascular disease (CVD). The RVLM is believed to contribute to CVD through direct activation of sympathetic preganglionic neurons. Our previous work showed that sedentary rats exhibit enhanced sympathoexcitation after glutamate activation of the RVLM compared to active counterparts. We have also recently shown increased dendritic arborization in spinally‐projecting C1 RVLM neurons from sedentary rats. We hypothesized that the RVLM exhibits increased excitability due to structural and functional differences in sedentary versus active rats (14 weeks wheel running). To our knowledge, we are the first group to have performed in vivo single unit recordings from putative presympathetic RVLM neurons in sedentary and active rats to examine functional differences in presympathetic RVLM neurons. Preliminary results from Inactin‐anesthetized rats indicate that individual presympathetic RVLM neurons from sedentary rats appeared to have higher resting firing rates (14.0±5.1 Hz, n=4) compared to active rats (8.7±3.0 Hz, n=4). RVLM neurons also appeared to have greater responses to acute increases in blood pressure (∆‐11.4±5.3 Hz vs. ∆‐5.6±1.4 Hz, respectively), but decreases in blood pressure did not appear to elicit different responses (∆+3.1±1.4 Hz vs. ∆+2.9±0.3 Hz). Though our current sample size does not yet permit us to draw definitive conclusions, these preliminary data support our hypothesis and suggest that a sedentary lifestyle may increase sympathetic nerve activity by causing an increase in the activity of individual presympathetic RVLM neurons.

Grant Funding Source: HL096787, WSU Rumble Fellowship
Klotho suppresses epithelial‐mesenchymal transition in adriamycin nephropathy (690.8)
Authors: Takenaka Tsuneo , Inoue Tsutomu , Miyazaki Takashi , Hayashi Matsuhiko , Suzuki Hiromichi
Published: June 2021

Klotho interacts with various proteins to alter their function. Experiments were performed to assess how klotho protects kidney from its injury. Adriamycin (5 mg/Kg) was injected into rats to induce nephropathy. Human recombinant klotho (K, 30 μg/kg/day), 4‐benzyl‐2‐methyl‐1,2,4‐thiadiazolidine‐3,5‐dione (GSK3β blocker, 0.2mg/kg/day, T) or vehicle (A) was administered. Rats untreated with adriamycin were used as the control (U). Animals were killed 8 weeks later. Renal expressions of Wnt1, GSK3β, TGFβ1, Twist and collagen I were assessed with RT‐PCR against GAPDH. Results were shown in Table. Our data indicate that WNT is involved in the pathogenesis of adriamycin nephropathy. Furthermore, the present findings provided the evidence that the inhibition of WNT contributed to glomerular protective actions of klotho. Finally, our results suggest that klotho suppresses epithelial‐mesenchymal transition by inhibiting TGFβ as well as WNT signaling.

Results

U
A
K
T

Alb/Cr
8±2
316±39*
152±21*#
163±19*#

CCr
1.1±0.1
0.9±0.1
1.0±0.1
1.0±0.1

Wnt1
0.2±0.1
3.6±0.3*
2.3±0.3*#
2.5±0.4*#

GSK3Î²
0.1±0.1
0.8±0.2*
0.4±0.1*#
0.4±0.1*#

TGFÎ²1
1.2±0.2*
0.2±0.1
0.7±0.1*#
0.6±0.2:#

Twist
0.1±0.1
1.3±0.2*
0.6±0.1*#
1.1±0.2*$

Collagen I
0.3±0.2
2.3±0.4*
0.7±0.2*#
1.4±0.3*#$

Alb/Cr: Albuminuria(mg)/Creatinine(g), CCr: creatinine clearance (ml/min/g.kidney.wt). *: p<0.05 vs U, #: P<0.05 vs A, $: p<0.05 vs K.

Grant Funding Source: Japanese Ministry of Education and Culture (25461229)