bluuebunny/crossref_metadata_2025 huggingface dataset:
California Table Grape Consumption Reduces Adiposity, Hepatic Triglycerides, Lipogenic Gene Expression, and Abundance of Sulfidogenic Bacteria in Mice Fed Butter Fat
Authors: Baldwin Jessie , Collins Brian , Wolfe Patricia , Shen Wan , Chuang Chia‐Chi , Zhong Wei , Cooney Paula , Gaskins H Rex , McIntosh Michael
Published: June 2021

This study examined the extent to which consuming polyphenol‐rich, California table grapes (3 or 5%, w/w) reduces adiposity, hepatic steatosis, markers of inflammation or lipid metabolism, or impacts gut microbiota in male C57BL/6J mice fed a butter‐rich diet for 11 weeks. Total body and inguinal fat content were reduced in mice fed grapes at both levels compared to their high‐fat, sugar controls. Liver weights, triglycerides, and expression of lipogenic Gpat1 were decreased in mice fed 5% grapes compared to 5% controls. Mice fed 3% grapes had lower hepatic mRNA levels of the lipogenic genes Pparg2, Scd1, Fabp4, and Gpat1 compared to 3% controls. In white adipose tissue (WAT), mice fed 5% grapes had decreased mRNA levels of the lipogenic gene Agpat2 compared to controls. Although grape feeding had only a minor impact on markers of inflammation in WAT or intestine, 3% grapes decreased the intestinal abundance of sulfidogenic Desulfobacter spp. and the Bilophila wadsworthia‐specific dissimilatory sulfite reductase gene, and tended to increase the abundance of the beneficial bacterium Akkersmansia muciniphilia compared to controls. Notably, grape feeding attenuated the high fat‐induced impairment in localization of the intestinal tight junction protein ZO‐1. These data indicate that some of the adverse health consequences of consuming a diet rich in saturated fat can be attenuated by table grape consumption. Funded by the California Table Grape Commission.
Mice Lacking β‐catenin In Liver Develop Hepatic Fibrosis In Response To Iron Overload
Authors: Preziosi Morgan , Singh Sucha , Ganz Tomas , Monga Satdarshan
Published: June 2021

Dysregulated Wnt/β‐catenin signaling and iron overload disorders like hemochromatosis have been implicated in hepatocellular carcinoma. We sought to identify a potential link between Wnt/β‐catenin signaling and hepatic iron metabolism by subjecting mice to an iron‐overload diet. Hepatocyte‐specific β‐catenin knockout (KO) and littermate control (CON) mice were subjected to a 10,000 ppm iron diet for three months. Mice on the iron diet showed larger liver weight to body weight ratios than their counterparts on the control diet, which was accompanied by an increase in proliferation. Steatosis was observed in all groups after iron, corresponding to an increase in triglycerides and cholesterol esters. Interestingly, KO mice after iron diet had notably more fibrosis and inflammation compared to CON. In addition, a greater number of macrophages containing iron‐laden hepatocytes were evident in KO. Taken together, these results suggest β‐catenin‐deficient mice are more prone to injury, inflammation, and fibrosis in response to iron overload. Thus, this may be a useful model to study human hemochromatosis since most mouse models exposed to high iron do not exhibit hepatic fibrosis.
Efficacy of Dopamine D3 Receptor Antagonist SR 21502 in Reducing Opioid Tolerance and Dependence
Authors: Eon Victoria , Giuvelis Denise , Ananthan Subramaniam , Bilsky Edward
Published: June 2021

Dopamine (DA) D3 antagonists modulate the addictive and rewarding properties of both stimulant and opioid drugs by deactivating the drugs' effects on learning and motivation centers in the brain. SR 21502 (SR), a novel DA D3 receptor antagonist, has been shown to be effective in reducing conditioned place preference and cue‐induced relapse in rodent models of cocaine addiction. This study investigated SR's ability to mediate opioid addiction without compromising morphine's analgesic efficacy. We co‐administered morphine and SR, twice daily for 4 days, to assess the ability of SR to reduce morphine‐induced tolerance and dependence. Results showed that SR was able to reduce morphine‐induced tolerance by producing less of a rightward shift in the dose‐response curve. Standard morphine tolerance produced a 12.4 rightward shift where co‐administration with SR produced a 6.9 fold shift. In the assessment of chronic dependence using naloxone to induce withdrawal, we found that SR treated mice exhibited less vertical jumping and had a significantly lower output of urine and feces during a 20 min observation period. It was also observed that the laxation of SR treated mice produced well‐formed fecal boli versus diarrhea that was seen following standard morphine treatment. These findings support our hypothesis that co‐administration of SR throughout morphine treatment would be an effective approach at identifying an equally efficacious lower‐risk treatment option. This research also provides a promising approach at minimizing negative side effects, including tolerance, gastro‐intestinal and addictive tendencies, which may improve patient compliance to these types of therapies.
Evaluation of the Anti‐Inflammatory and Analgesic Activities of Leaf‐Extracts of Sterculia tragacantha in Rats
Authors: Abatan Matthew , Mogbojuri Olufunke
Published: June 2021

Pilot toxicity studies using 200, 1000 and 3000 mg/kg doses of the aqueous extract of Sterculia tragacantha in albino rats did not produce significant (P< 0.05) adverse effects on haematological and biochemical parameters. Doses of the chloroform extract at 100 and 200 mg/kg caused inhibition of increase in paw volume at 1 hr (9.9%), 2 hrs (10.7%) and 3hrs (14.0%); whereas the 200mg/kg dose caused 1 hr (13.4), 2 hrs (16.4) and 3hrs (20.6%) inhibition in paw oedema.

Evaluation of the n‐hexane extract of S. tragacantha produced 53.9 and 68.8% inhibition of writhing pain at 100 and 200 mag/kg doses respectively; the 100 and 200 mg/kg doses of the chloroform extract also produced 50 and 79.3% inhibition writhing pain respectively. Pooled chromatographic fractions of the chloroform extract with common Rf gave crystals which when tested at 25mg/kg dose gave a 35.4% inhibition of inflammation whereas the same dose produced 79.1% inhibition of writhing pain.

Gas chromatographic mass spectrum analysis of the chloroform extract revealed the bioactive compounds to be n‐hexadecanoic acid, 9‐octadecanoic acid and n‐triacontane.

It is concluded that the chloroform extract of S. tragacantha possesses anti‐inflammatory and analgesic properties which may account for the medicinal use.
A Guanidinium Group Containing Dietary Supplement Inhibits ASIC3
Authors: Agharkar Amruta , Smith Rachel , Gonzales Eric
Published: June 2021

Acid‐sensing ion channels are sodium selective channels that are sensitive to extracellular protons, specifically those following ischemia and injury. The peripheral ASIC3 subtype plays an important role in pain perception. Protons and inflammatory mediators can activate or modulate ASIC3 to produce the pain signal, suggesting that ASIC3 is a pharmacological target for pain therapy. The large extracellular domain of ASICs offers multiple sites for interacting with protons and guanidinium group containing compounds. Guanidinium compounds such as 2‐guanidine‐4‐methylquinazoline (GMQ), amiloride, and agmatine are known to modulate the electrophysiological properties of ASIC3. Here we identified a dietary supplement (DS) that shares molecular similarity to these ASIC ligands and modulate ASICs. We utilize whole‐cell patch‐clamp electrophysiology recording to determine the interaction of DS with rat ASIC3 (rASIC3), transiently expressed in CHO‐K1 cells. Our preliminary data suggests that ASIC3 peak current amplitude and steady‐state current is reduced in the presence of DS. In the absence of extracellular calcium, DS reduces the rASIC3 proton sensitivity by shifting pH‐activation profile to lower pH. Future studies will focus on determining the effect of DS on the ASIC3 window current and other ASIC3 properties to resolve the mechanism of action of the DS influence on channel activity.
Inhibition of Poly(ADP‐Ribose) Polymerase Activity Modulates Autophagy Induced by Experimental Spinal Cord Trauma
Authors: Paterniti Irene , Impellizzeri Daniela , Bruschetta Giuseppe , Cordaro Marika , Siracusa Rosalba , Cuzzocrea Salvatore , Esposito Emanuela
Published: June 2021

Although it is evident that spinal cord injury (SCI) induces necrosis and apoptosis, its relationship to autophagy is uncertain. Recently, several studies showed that Poly (ADP‐ribose) polymerase (PARP) activation occurs during apoptosis, and inhibition of PAR formation impairs the apoptotic machinery. Despite their pathogenetic relevance, the involvement of PARP‐1 in autophagy and the regulatory mechanisms underlying energetic derangement during PARP‐1 hyperactivation associated to SCI, are still elusive. The aim of the present study was to investigate the involvement of autophagy after SCI and the role of PARP‐1 in the cell's decision to commit to autophagy following DNA damage. SCI was induced by the application of vascular clips and ABT‐888, a new synthetic inhibitor of PARP, was administered after injury. In the present study the markers of autophagy expression were evidenced by western blot analysis for Beclin‐1, MAPLC3I‐II, p‐AKT and mTOR in neural tissue after SCI and treated with ABT‐888. Taken together our results, clearly detected elevated levels of Baclin‐1, LC3 as well as AIF and Bax after SCI; whereas treatment with ABT‐888 significantly reduced autophagy activation. Thus, through observing the enhanced autophagy expression in neurons soon after contusion injury and the inhibitive effect of PARP inhibition treatment, this study suggests that autophagic cell death may play a role in neuronal death after spinal cord trauma.
Identification of Small Molecule Inhibitors Selective for Type 1 Adenylyl Cyclase
Authors: Brust Tarsis , Watts Val
Published: June 2021

Adenylyl cyclases (ACs) are important integrators of signaling through G protein‐coupled receptors. There are nine isoforms of membrane‐bound ACs that have unique regulatory properties and expression patterns. Adenylyl cyclase 1 (AC1) belongs to a group of ACs that are stimulated by calcium in a calmodulin‐dependent manner. Studies with AC1 knockout mice suggest that inhibitors of AC1 may be useful for treating neuropathic/inflammatory pain, opioid dependence, and preventing neuronal excitotoxicity. However, previous reports in mice lacking both AC1 and AC8 suggest that non‐selective inhibition of AC1 and AC8 would result in significant memory impairments and possibly depression. These observations suggest that AC1‐selective inhibitors have potential in a variety of neurological diseases. Accordingly, inhibition of AC1 with the small molecule NB001 had analgesic properties in animal models of chronic pain in the absence of alterations of hippocampal LTP and fear memory. Unfortunately, the mechanism of action of NB001 remains unknown as it failed to directly inhibit AC1. The present study identified several novel classes of AC1 inhibitors through both chemical library screening and structure‐activity relationship studies. Compounds were initially tested against forskolin and Ca2+ (i.e. A23187) stimulated cAMP accumulation in HEK‐AC1 cells. Robust inhibitors were also evaluated against members from other AC families (i.e. AC2 and AC5) revealing selective AC1 inhibitors. Further comparisons between AC1 and AC8 revealed ligands that showed selectivity for AC1. Subsequent studies determined the effect of these AC1 modulators on Gαi/o‐coupled receptor‐mediated inhibition of cAMP accumulation as well as heterologous sensitization of AC.
Tamoxifen Loaded in Methacrylate Nanoparticles Evaluated in Breast Cancer Tumor Cells Model
Authors: Gomes Anderson , Barros Susan , Almeida Clara , Joanitti Graziella , Azevedo Ricardo , Lunardi Claure
Published: June 2021

This study aimed to investigate the effect of a established cancer drug such as tamoxifen (TMX) when encapsulated into a matrix of copolymer of ethyl acrylate, methyl methacrylate and a low content of methacrylic acid ester with quaternary ammonium groups (EUDRAGIT® RL 100 and RS 100) associated with a fluorescence probe. TMX is used in breast cancer therapy with some side effects such as stroke, and endometrial cancer. This drug delivery system (TMX‐NP) was designed to protect the drug from environmental and to promote a sustained release of the drug and also be tracked by the emission of fluorescence probe. The size and entrapment efficiency were evaluated at different preparation protocols. TMX showed a maximum entrapment efficient of 23% in the proportion of 1.5g of polymer using a emulsion method. The size measurements were compared with the empty nanoparticles and showed values of TMX‐NP = 166.4± 32 nm, and empty NP = 273± 50 nm. The zeta potential of particles was +65 and +38 mV for TMX‐NP and empty NP respectively. The cytotoxicity against the MCF‐7 and 4T1 cells was affected significantly by the released amount of TMX, while empty‐NP exhibit no significant cytotoxicity against breast cancer cells. Supported by FAPDF, CAPES, CNPq, FINATEC and DPP‐UnB.
Engineered Dry Powder Budesonide Particles for Improved Pediatric Inhalation Therapy
Authors: Sullivan Bradley , El‐Gendy Nashwa , Berkland Cory
Published: June 2021

Corticosteroids have been the cornerstone treatment for asthma; however, several challenges exist with current inhaled steroids for pediatric patients. Dry powder corticosteroids are now being prescribed to pediatric patients 蠅 6 years of age with some success; however, many younger patients lack the inhalation energy required to properly fluidize and deagglomerate the drug particles, which leads to improper drug delivery, increases oral steroid exposure, and reduces long term compliance. We tested the hypothesis that engineered budesonide NanoClusters, drug particles with decreased density per surface area, would improve fluidization and deagglomeration efficiency compared to commercially available micronized budesonide and Pulmicort™ powders at flow rates seen in pediatric patients. Micronized budesonide powder was wet milled into micron sized NanoClusters using a Netzsch MiniCer Media Mill and then lyophilized. Budesonide powders were tested at various flow rates in a custom fabricated electronically controlled flow rate adjustable pneumatic device coupled to an Anderson Cascade Impactor. NanoCluster budesonide achieved EFmax (~85%) at a flow rate of 2.5 L/minute, whereas EF of micronized budesonide and Pulmicort™ were only minimally emitted (~2‐3%) at this flow rate. Importantly, fine particle fraction (FPF) of NanoClusters was significantly elevated compared to micronized budesonide and Pulmicort™ formulations. The data suggests that NanoCluster formulation improves budesonide dispersion properties at low flow rates, potentially allowing a lower unit dose formulation of budesonide for children without altering the effective therapeutic dose; increasing overall therapeutic index.
Triclosan Inhibits the Activity of Expressed Human Sulfotransferases (SULTs) Towards Their Diagnostic Substrates
Authors: James Margaret , Ambadapadi Sriram , Falany Charles
Published: June 2021

The antibacterial agent, triclosan [5‐chloro‐2(2,4‐dichlorophenoxy)phenol], is added to several consumer products and has been identified in the blood and urine of over 75% of the US population. Triclosan is known to inhibit human hepatic cytosolic SULT activity with several xenobiotic and steroid substrates. In this study we examined the effects of triclosan on isoform‐selective sulfonation of diagnostic substrates of the major SULT isoforms known to be expressed in human liver and intestine. Expressed recombinant SULTs 1A1, 1A3, 1B1, 1E1 and 2A1 were incubated respectively with 4 μM p‐nitrophenol (p‐NP), 10 µM dopamine, 100 µM tri‐iodothyronine (T3), 1 nM estradiol (E2) or 5 µM dehydroepiandrosterone (DHEA), varying amounts of triclosan and buffer (pH 7.4). The sulfonation reaction was started by adding 10 to 20 μM 3′‐phosphoadenosine‐5′‐phosphosulfate.Triclosan inhibited SULTs 1E1, 1B1 and 1A1 activities towards E2, T3 and p‐NP most potently with IC50 values of 0.017, 2.1 and 3.6 µM, respectively. Triclosan was a weaker inhibitor of SULTs 1A3 and 2A1 towards dopamine or DHEA, with respective IC50 values of 31.1 and 431 µM. This work confirms the high potency of triclosan as an inhibitor of estrogen sulfotransferase, and demonstrates inhibitory interactions with other sulfotransferases. Supported in part by R21 ES020545 and seed funds from the University of Florida.
Endothelial Dysfunction in DOCA‐salt Hypertensive Mice: A Role for nNOS‐derived Hydrogen Peroxide
Authors: Silva Grazielle , da Silva Josiane , Diniz Thiago , Lemos Virginia , Cortes Steyner
Published: June 2021

Aims

investigated the involvement of neuronal nitric oxide synthase (nNOS) and H2O2 in the impaired endothelium‐dependent vasodilatation of mesenteric arteries from DOCA‐salt hypertensive mice (DOCA‐salt).

Methods and results

Myograph studies were used to investigate the endothelium‐dependent vasodilator effect of acetylcholine (ACh), the protein expression by Western blot, H2O2 formation by selective carbon microsensors permeable membrane and the protein localization in the endothelial layer of mesenteric arteries by immunofluorescence approved by ethics committee (protocol 227/08).The vasodilator effect of ACh is strongly impaired in mesenteric arteries from DOCA‐salt (SBP 180± 5 mmHg, Emax 51±2.3%) versus Sham (SBP 128±4 mmHg, Emax 99±1.2%). Nonselective inhibition of NOS strongly reduced the effect of ACh in both DOCA‐salt and Sham mice. Selective inhibition of nNOS and catalase induced a higher reduction in the effect of ACh in Sham than in DOCA‐salt. Production of H2O2 induced by ACh was significantly smaller in vessels from DOCA‐salt and it was blunted after the inhibition of nNOS. The expression of both eNOS and nNOS were significantly smaller in DOCA‐salt, while the phosphorylation of their inhibitory site was increased. The presence of nNOS was confirmed in the endothelial layer of mesenteric arteries from both Sham and DOCA‐salt.

Conclusion

A reduction in nNOS‐derived H2O2 production in DOCA/salt hypertensive mice partially contributes to endothelial dysfunction. This opens a new perspective for the understanding of the endothelial dysfunction in hypertension.

Financial support: CNPq
Different Responses in CX3CL1/CX3CR1 and TNF‐α/TNFRSF1A Signaling Pathways to Aspirin in Human Umbilical Vein Endothelial Cells (HUVECs)
Authors: Szukiewicz Dariusz , Stangret Aleksandra
Published: June 2021

Antiprostaglandin and antiplatelet actions of aspirin related to non‐equal and irreversible inhibition of cyclooxygenases (COX1, COX2) are widely known. COX‐independent antiinflammatory effects of aspirin were also reported. Acting as the nuclear factor‐kB (NF‐kB)inhibitor on endothelial cells, aspirin may modulate chemokine fractalkine (CX3CL1) signaling through its sole receptor CX3CR1. Thus, aspirin may reveal the opposite effect to TNF‐α, a strong inducer of CX3CL1/CX3CR1 signaling. We examined the effects of aspirin on CX3CL1 and TNF‐α production, as well as CX3CR1 and TNFRSF1A expression. HUVECs were isolated after normal term pregnancies (N = 24) and cultured in vitro. The cultures were exposed to aspirin (dose range: 1.0 – 6.0 mM) during the 7 day period. The mean concentrations of CX3CL1 and TNF‐α in the culture media supernatants were assessed at predefined time points by ELISA. After termination of the cultures, CX3CR1 and TNFRSF1A were immunostained in formalin fixed and paraffin embedded sections. To estimate the mean expression of these receptors, quantitative immunohistochemistry was applied. Aspirin significantly (p < 0.05) reduced production of CX3CL1, and the mean decline in CX3CL1 production was inversely proportional to the significantly (p < 0.05) augmented expression of CX3CR1. An inhibition of TNF‐α synthesis in HUVECs in response to aspirin treatment was also reported. There were no signs indicating upregulation of TNFRSF1A. Since autoregulatory relationships between CX3CL1 and CX3CR1 were proposed, overexpression of CX3CR1 observed in aspirin‐treated HUVECs may be a form of compensatory mechanism.

WUM grant: 2M2‐N‐14
Increased Role for Large Conductance, Calcium‐Activated K Channels (BKCa) in Endothelium‐Dependent Relaxation of Nitrate Tolerant Mesenteric Arteries
Authors: Sane Mukta , Mughal Amreen , Sun Chengwen , O'Rourke Stephen
Published: June 2021

Prolonged exposure to nitroglycerin (NTG) leads to tolerance and impaired endothelium‐dependent vasodilation. Causes of endothelial dysfunction in nitrate tolerant arteries are well studied, but little is known about mechanisms that underlie the responses to endothelium‐derived mediators in nitrate tolerant blood vessels. Here we evaluated the role of BKCa in acetylcholine (ACh)‐induced relaxation of isolated mesenteric arterial rings from rats treated with or without application of transdermal NTG patches (0.6 mg/hr) for 3 days. Nitrate tolerance was confirmed by the impaired relaxation response to NTG (1 nM – 100 μM) observed in rings from rats treated with NTG patches (pD2= 5.6 + 0.1 vs 4.9 + 0.1, Emax= 86 + 7 vs 53 + 5% relaxation; control vs treated, respectively). ACh‐induced (1 nM – 3 μM) relaxation was also impaired in tolerant rings (pD2= 7.1+ 0.1 vs 6.5 + 0.1; control vs tolerant, respectively) Incubation with nitro‐l‐arginine (30 μM) or indomethacin (10 μM) had no effect on the response to ACh in control or tolerant arteries, consistent with the release of a non‐NO, non‐prostanoid endothelium‐derived mediator in both control and tolerant arteries. ACh‐induced relaxation was unaffected by the selective BKCa‐blocker, iberiotoxin (IBT), in control rings, but was nearly abolished in tolerant rings (Emax = 78 ± 9 vs 8 + 8% relaxation; without vs with IBT, respectively). Protein expression of BKCa β‐subunits was increased by 50% in nitrate tolerant arteries, while BKCa α‐subunit expression was unchanged. The data provide evidence that nitrate tolerance unmasks a pivotal role for BKCa in the smooth muscle relaxation evoked by a non‐NO, non‐prostanoid endothelium‐derived mediator in mesenteric arteries.
HIV‐1 Tat Sensitizes Enteric Neurons to Bacterial Proteins
Authors: Ngwainmbi Joy , Brun Paola , Kang Minho , Dewey William , Hauser Kurt , Akbarali Hamid
Published: June 2021

HIV infection is characterized by intestinal mucosal damage leading to increased translocation of bacteria and viruses into the gut wall. This exposes enteric neurons to bacterial and viral proteins. Previously, we showed that HIV‐1 Tat, an early HIV protein, increases enteric neuronal excitability and significantly alters gastrointestinal (GI) motility. In this study, we examined cytokine expression by Tat and lipopolysaccharide (LPS) in enteric neurons and its effect on GI transit. Tat sensitized enteric neuron/glia cultures (isolated from adult mouse ileum) to LPS‐induced cytokine release. Neither 10 nM Tat nor 10 ng/ml LPS induced TNF‐ α release, however, they significantly up‐regulated TNF‐ α when treated in combination. This effect was time dependent with the highest sensitization occurring at 6 h Tat treatment. This sensitization effect was also seen in transgenic mice in which LPS (50 µg/kg) treated Tat+ (Tat expressing) mice had robust increases in TNF‐α (7 fold) and IL‐1β (3 fold) mRNA compared to Tat‐ (litter mates) which showed 1.9 and 1.5 fold increases respectively. Tat (100 nM) significantly increased LPS receptor, TLR4, expression by 3 fold. Pellet propulsion was measured using the gastrointestinal motility monitor. Tat+ mice were significantly more sensitive to LPS induced decrease in colonic transit than Tat‐ mice. LPS (50 µg/kg p.o.) reduced colonic transit by 80% in Tat+ mice compared to 35% in Tat‐ mice. These findings suggest that Tat exacerbates the effects of bacterial proteins and enhances opportunistic infections in HIV AIDS. Supported by F31NS087952, DA024009
Dual Roles of Claudin‐7 in Human Lung Cancer Cell Growth and Metastasis
Authors: Kim Do Hyung , Lu Zhe , Lu Qun , Shea Michael , Chen Yan‐Hua
Published: June 2021

Claudins are a family of tight junction (TJ) membrane proteins involved in a broad spectrum of human diseases including lung cancer. We found that claudin‐7 is a unique TJ membrane protein in that it has a stronger expression at the basolateral side of the membrane than at the apical side in lung cancer cells. To investigate the function of claudin‐7 in lung cancer, we suppressed claudin‐7 expression in HCC827 human lung adenocarcinoma cells using lentivirus shRNA technology. Suppression of claudin‐7 expression in HCC827 cells (KD cells) greatly increased the rate of cell proliferation. The phospho‐ERK1/2, phospho‐Bcl‐2, and survivin were all upregulated in KD cells. In addition, these claudin‐7‐supressed KD cells showed a significantly reduced expression level of integrin β1 and focal adhesion kinase (FAK). The functional analysis revealed that the cell attachment, cell migration, and cell invasion capabilities were all decreased in KD cells compared to those of control cells. The defect in cell attachment is most likely mediated through the integrin β1 signaling pathway since FAK is a downstream target of integrin β1 and its expression is regulated by integrin β1. However, transfection of integrin β1 cDNA into KD cells (β1‐reexpressed KD cells) did not fully restore the cell attachment defect. Only after transfection of claudin‐7 back to KD and integrin β1‐reexpressed KD cells, did the expression level of focal adhesion proteins increase and the cell attachment function restore. We conclude that claudin‐7 plays dual roles in both lung cancer cell growth and cell‐matrix adhesion in human lung cancer cells. This study was supported by National Institute of Health grants ES016888 and HL085752.
Cod Liver Oil Supplementation Effects on 2,3‐ Diphosphoglyceric Acid Blood Levels in Atherogenic Rabbits
Authors: Beard Elizabeth , Michals Edward
Published: June 2021

2,3 Diphosphoglyceric Acid (2,3‐DPG) promotes release of oxygen from hemoglobin in the blood of higher animals, and may reduce atherogenic plaque development in hyercholesterolemic rabbits. Eicosapentanoic acid (EPA) converts arachidonic acid in cell membranes to Prostaglandin I‐3, which inhibits platelet aggregation and reduces blood clot formation. Atherogenesis and increased blood vascular fibrin clot formation can be induced in rabbits by feeding a diet containing 2% cholesterol for a month. Blood clot formation at the intimal surface in the aorta reduces oxygen availability which leads to lipid infiltration and plaque development. The effect of feeding atherogenic rabbits cod liver oil (CLO) containing 10% EPA, was studied here to determine its effect on blood levels of 2,3 Diphosphoglyceric Acid (2,3‐DPG).

For a month, 12 white, New Zealand strain, male rabbits were fed atherogenic diet (AD) of 92% Purina Rabbit Chow(PRC), 6% Corn Oil and 2% Cholesterol. In the next 8 weeks, 6 rabbits were fed a control diet (CD) of PRC and 6 continued to be fed AD. Half of the CD and AD groups receied 0.8ml CLO daily. Blood 2,3‐DPG means after 2 weeks were: 1.51ug/ml in (CD ) vs 1.75 in the (CD + CLO) and 1.39 in ( AD) vs 1.81 in ( AD+ CLO) animals. After 8 weeks the means were 2.12 (CD) vs 2.35 (CD+CLO), and 1.51 (AD) vs 1.82 (AD+ CLO). The ( CD+CLO ) animals had the highest 2,3 DPG levels of all groups and the least aortal lipid infiltration. (AD+CLO) 2,3 DPG levels were significantly higher than in (AD) controls and they had lower lipid infiltration in their aortae. Blood plasma cholesterol levels in the AD groups were significantly lower in the ( AD+CLO ) vs the (AD) control rabbits.
Tobacco Smoke and E‐Vapor Initiate Kupffer Cell Inflammatory Responses
Authors: Rubenstein David , Yin Wei
Published: June 2021

Cardiovascular risk factors instigate cardiovascular diseases through numerous mechanisms. Recently, we have shown that ingested cardiovascular risk factors can initiate Kupffer cell innate immune responses, leading to platelet and endothelial cell inflammatory/thrombotic reactions. Here, we aimed to investigate the role of tobacco smoke, a well‐known risk factor, and e‐vapor, a source of fine particulate matter and oxidative stressors, on Kupffer cell activities. We hypothesized that the exposure of Kupffer cells to tobacco smoke or e‐vapor would initiate innate immune responses. Immortalized Kupffer cells were cultured and exposed to mainstream or sidestream tobacco smoke extracts or e‐vapor extracts prepared from different nicotine containing e‐juice formulations. Kupffer cell complement deposition, complement receptor activation, cytokine release and viability was quantified as a function of extract exposure. Platelets were subjected to Kupffer cell conditioned media, to determine the indirect activation of platelets by Kupffer cells. We observed a significant increase in Kupffer cell innate inflammatory responses after exposure to physiologically relevant concentrations of tobacco smoke extracts or e‐vapor. As nicotine concentration in e‐juice increased, the activity of Kupffer cells also increased. Kupffer cell viability was reduced after exposure to tobacco or e‐vapor extracts. Platelets exposed to Kupffer cell conditioned media also showed an increased aggregation potential towards a thrombin receptor antagonist. Platelet activities were at least partially mediated by an enhanced expression of GPIIb/IIIa. Thus, these data suggests that Kupffer cells are responsive to non‐ingested cardiovascular risk factors and that these signals can be indirectly passed to platelets.
Caveolin‐1 is a Negative Regulator of Adam17 in Adipose Tissue Vascular Endothelium
Authors: Dou Huijuan , Romero‐Lucas Maritza , Bagi Zsolt
Published: June 2021

Aging is associated with increased level of proinflammatory cytokine, TNF. Pericardial adipose tissue (pFat) is one of the important sources of TNF, but very little is known how the release of TNF is regulated in pFat and whether this changes in aging. We tested the hypothesis that a disintegrin and metalloproteinase, ADAM17 (known as TNF converting enzyme or TACE) mediates an enhanced proteolytic shedding of membrane‐bound TNF from pFat in elderly patients. We obtained pFat from 47 consecutive patients (age between 40 and 80 yo) undergoing heart surgery. Immunohistochemistry and immune‐electron microscopy showed that within the pFat the vascular endothelium exhibits a prominent ADAM17 expression, with much less extent in adipocytes. In the superfusates of intact pFat vessels TNF release was significantly higher in older (>70 yo) then in younger (<70 yo) patients, which was inhibited by the ADAM17 inhibitor, TIMP‐3. Correspondingly, we found an age‐related increased ADAM17 activity in pFat vessels. We also found an age‐related decline in caveolin‐1 (Cav1) expression in pFat vessels and observed a reduced co‐localization between Cav1 and ADAM17 in older patients. In primarily cultured pFat endothelial cells genetic deletion of Cav1 increased ADAM17 activity. Thus, we propose that due to the age‐related deficit in Cav1‐mediated inhibition the activity of endothelial ADAM17, hence TNF release from pFAT is elevated in aging.
A Bioinspired Microfluidic Assay for Investigation of the Role of Protein Kinase C‐delta on Leukocyte‐endothelial interactions in Sepsis
Authors: Soroush Fariborz , Pandian Prabhakar , Kilpatrick Laurie , Kiani Mohammad
Published: June 2021

As a significant item in sepsis induced tissue injury, neutrophils excessive migration through endothelium to the tissue has no specific pharmacologic therapies available. Our previous studies showed Protein Kinase C‐delta (PKCδ) as a critical regulator of the inflammatory response is effectively regulate neutrophils migration. Using our established bioinspired microfluidic assay (bMFA), we studied the effect of PKCδ inhibition, under flow conditions, on neutrophil migration. The bMFA has been validated against in vivo data and consists of vascular channels connected with a tissue compartment area through a 3µm porous barrier. Human umbilical vein endothelial cells (HUVECs) were cultured in the network and stimulated with TNFα to mimic the inflammatory response of tissue. The tissue compartment was filled with a chemoattractant (fMLP or IL‐8) and the adhesion and migration of activated human neutrophils were measured in the presence or absence of a PKCδ inhibitor. Our results demonstrate that PKCδ inhibition had no effect on neutrophil adhesion to activated endothelium. In contrast, PKCδ inhibition significantly reduced migration of neutrophils into the tissue compartment by more than 50% (P<0.05) in response to either IL‐8 or fMLP indicating a critical role for PKCδ in neutrophil migration but not adherence. We are currently using this novel platform to ascertain whether targeting PKCδ may offer a unique therapeutic strategy for regulating the signaling cascade that controls neutrophil‐endothelial interaction in sepsis.
Soluble Guanylate Cyclase Activation during Ischemic Injury in Mice Protects against Postischemic Inflammation at the Mitochondrial Level
Authors: Wang Derek , Jones Allan , Wang Walter , Wang Meifang , Korthuis Ronald
Published: June 2021

Previous studies showed that soluble Guanylate Cyclase (sGC) activation with BAY 60 protected against postischemic inflammation when injected as a preconditioning agent in wild type (WT) H129 and heme‐oxygenase‐1 KO (HO‐1‐/‐) mice. This occurred when given both 10 min and 24 hours prior to ischemia (Wang et al. AJP Heart Circ Physiol 305:H521‐H532, 2013). We hypothesized that the same protective effect would occur when BAY 60 is administered during ischemia. When tested we observed reduced postischemic leukocyte rolling (LR) and leukocyte adhesion (LA) to intestinal venules in WT and HO‐1‐/‐ mice, as well as prevention of increased TNF‐α and circulating neutrophil levels that accompany ischemia/reperfusion (I/R). Based on these findings, we further hypothesized that BAY 60 when given during ischemia would prevent intestinal mucosal mitochondrial dysfunction induced by I/R. Mitochondrial integrity was evaluated by the Calcein AM fluorescence method (used to measure mitochondrial permeability), and the JC‐1 fluorescence ratio (used to measure mitochondrial membrane potential). Both mitochondrial permeability and membrane potential were disrupted following I/R, which indicates the formation of mitochondrial permeability transition pores (mPTP). However, both Calcein fluorescence and JC‐1 fluorescence ratio were maintained at control levels in cells from the mice that were treated with BAY 60. Our results indicate that sGC activation during ischemia protects against I/R inflammatory processes and preserves mucosal mitochondrial function by preventing mPTP formation Supported by: HL095486 & AA14945. Bayer Schering Pharma AG provided BAY 60 as a gift.
Inward‐Rectifier K+ Channels in Vascular Smooth Muscle Prevent Myogenic Tone Development in Mouse Bladder Arterioles
Authors: Tykocki Nathan , Heppner Thomas , Bonev Adrian , Nelson Mark
Published: June 2021

The urinary bladder is highly vascularized, and chronic decreases in blood flow are linked to bladder dysfunction. While increases in intraluminal pressure constrict most arterioles (“myogenic tone”), the regulation of bladder vascular diameter is largely unknown. We examined the myogenic and vasoactive properties of mouse bladder feed arterioles to determine how they affect bladder blood flow. Myogenic tone was wholly absent from isolated pressurized bladder arterioles (5‐80 mmHg). This was endothelium‐independent, as inhibition of eNOS (100 µM LNNA) and KCa channels (300 nM apamin and 100 nM charybdotoxin) did not alter diameter. However, arterioles constricted to U‐46619 (1 µM), phenylephrine (10 μM) and depolarizing stimuli (60 mM K+). Pre‐constricted arterioles dilated to carbachol (1 µM) and the IK/SK opener NS309 (1 µM), but not the TRPV4 agonist GSK1016790A (100 nM). The inward‐rectifier K+ (KIR) channel blockers BaCl2 (100 μM) and ML‐133 (5 µM) caused significant constriction, indicative of KIR channel activity. Electrophysiological recordings from isolated bladder arteriolar smooth muscle cells showed significant Ba2+‐sensitive inward currents characteristic of KIR channels, and typical KV channel currents. Furthermore, bladder arterioles from smooth muscle‐specific KIR2.1 knockout mice developed significant myogenic tone at 60 and 80 mmHg. Together, these data support the concept that the absence of pressure‐induced depolarizing conductances leads to KIR channel activation, which then causes membrane hyperpolarization and dilation. This represents a novel mechanism in bladder arterioles that opposes myogenic constriction to maintain blood flow during filling. Supported by NIH R37DK053832.
H2O2‐Induced Dilation in Human Adipose Arterioles: Role of Smooth Muscle K+ Channels
Authors: Nishijima Yoshinori , Zhang David
Published: June 2021

Hydrogen peroxide (H2O2) induces potent vasodilation in human coronary arterioles from subjects with coronary artery disease (CAD) through the activation of protein kinase G‐large conductance Ca2+‐activated K+ (BKCa) channel pathway. Because the vasodilator effect and mechanism of action of H2O2 vary among different vascular beds and species, it remains unknown whether the K+ channel‐mediated dilation is conserved in other systemic vascular beds in humans. We investigated the vasomotor effect of H2O2 and its underlying mechanism in human adipose arterioles. Arterioles (100‐200 μm) obtained from subjects without CAD were cannulated under 60 mmHg and examined for diameter changes using video microscopy. H2O2 (1‐100 µM) induced concentration‐dependent vasodilation, with maximal dilation of 95±1% at 100 µM. H2O2‐induced dilation was endothelium‐independent and markedly reduced by both paxilline, a BKCa channel blocker, and 4‐AP, a voltage‐gated K+ (KV) channel blocker (dilation at 10 μM, 19±13% vs. 72±9% in control, and 26±5% vs. 66±11% in control, respectively). We further investigated which subtype(s) of the KV1 channels (the major KV family in the vasculature) is responsible to H2O2‐induced dilation. Similar to 4‐AP, DPO‐1 (KV1.5 channel‐specific blocker) reduced dilation to 10 μM H2O2 (12±5% vs. 54±7% in control). In addition, the KV 1.3/1.5‐specific blocker Psora‐4 caused a similar rightward shift in H2O2‐induced dilation, but further reduced the maximal dilation to 100 μM H2O2 (58±5% vs. 83±5% in DPO‐1). In conclusion, H2O2 dilates human adipose arterioles from non‐CAD subjects and the dilation is mediated by both KV and BKCa channels in smooth muscle cells. The mechanism by which H2O2 activates K+ channels (especially KV channel) remains to be determined.
sCD40L ‐CD40/VLA‐ 5 Signaling Contributes to AngII‐induced Thrombosis and Inflammation.
Authors: Senchenkova Elena , Russell Janice , Vital Shantel , Yildirim Alper , Granger D Neil
Published: June 2021

AngII‐induced hypertension is associated with accelerated thrombus formation and leukocyte recruitment. The objective of this study was to evaluate the mechanisms underlying the prothrombogenic‐proinflammatory responses associated with chronic AngII infusion. We assessed the contribution of CD40/CD40L to these AngII‐mediated responses and whether/how the generation of soluble CD40L(sCD40L) contributes to the exacerbated thrombosis and leukocyte recruitment. Thrombus formation in mouse arterioles was induced using the light/dye injury model, while firm adhesion of leukocytes was quantified in adjacent venules. Wild type (WT), CD40‐/‐, CD40L‐/‐ , bone marrow chimeric CD40‐/‐ and CD40L‐/‐ mice (CD40‐/‐→WT or CD40L‐/‐→WT) were infused with AngII (1 µg/kg/min for 14 days) via osmotic pumps. Some WT mice and AngII infused WT mice were treated with either recombinant sCD40L, a P‐selectin blocking antibody, or a VLA5 antagonist (ATN‐161). The studies revealed that both AngII‐infused CD40‐/‐ and CD40L‐/‐ exhibited reduced leukocyte adhesion and oxidative stress in venules, and a blunted thrombosis response in arterioles. WT‐AngII mice treated with ATN‐161 were completely protected against the enhanced thrombosis and leukocyte adhesion responses to AngII, while P‐selectin immunoblockade offered only partial protection. Infusion of recombinant sCD40L in either WT, CD40‐/‐, or CD40L‐/‐ mice restored the accelerated thrombus formation elicited by AngII infusion. These findings implicate sCD40‐CD40L/VLA‐5‐dependent signaling in the prothrombotic‐proinflammatory responses elicited by chronically elevated AngII levels (Postdoctoral Fellowship from Malcolm Feist Endowment).
Dietary fats shift mechanisms underlying vasodilation to acetylcholine
Authors: Lamping Kathryn , Nelson Peter , Coppey Lawrence , Yorek Mark , Nuno Daniel
Published: June 2021

Dietary fatty acids may be an effective target for reversing vascular abnormalities associated with diet‐induced obesity. We tested the hypothesis that dietary fats modify mechanisms underlying vascular responses by altering membrane composition and the contribution from nitric oxide synthase (NOS) and cyclooxygenase (Cox) in vascular responses. We fed male mice (C57BL/6J) normal (13% kcal fat) or a high saturated fat diet from lard (HF, 60% kcal fat) for 12 weeks before substituting half of the saturated fats with n‐3 polyunsaturated fatty acid enriched menhaden oil (MO) while still maintaining the same total kcal from fat. After an additional 6‐9 weeks, weights of mice on HF and MO‐enriched diets were similar and more than normal diet mice. MO‐enriched diet increased percentage of n‐3 long chain fatty acids DHA and EPA over three fold in aorta and the unsaturation index (1.07±0.03 vs 1.63±0.14, p<0.05). Relaxation to acetylcholine (ACh) was similar in aorta from all groups but was reduced by inhibition of NOS only in normal and high fat and not MO fed mice. Inhibition of Cox had no effect on aortic responses in any group. ACh responses were not different in gracilis arteries from HF but enhanced in MO compared to normal diet mice. Inhibition of NOS reduced ACh relaxation only in normal and MO fed mice, but Cox contributed to dilation to ACh in gracilis arteries in MO mice. Relaxation to nitroprusside was not affected by diet. Despite shifts in the contribution from NOS versus Cox in responses, diet did not affect total expression of endothelial NOS or Cox1 in arteries from either group. We conclude that a diet high in unsaturated n‐3 fatty acids improved small artery vascular responses but not large artery, in part, through an increase in the Cox contribution.
Bcl‐2 Expression in Endothelial Cells Impacts Retinal Vascularization and Choroidal Neovascularization
Authors: Sorenson Christine , Zaitoun Ismail , Wang Shoujian , Almomani Reem , Jamali Nasim , Johnson Ryan , Sheibani Nader
Published: June 2021

Bcl‐2 plays an important role in retinal vascular homeostasis. Mice globally lacking Bcl‐2 display attenuated postnatal retinal vascular development and neovascularization.To determine the role Bcl‐2 expression in endothelial cells plays during retinal vascular development, remodeling, and neovascularization, we generated mice carrying a conditional Bcl‐2 allele (Bcl‐2Flox/Flox) and VE‐cadherin‐cre (Bcl‐2EC mice). Bcl‐2EC mice displayed some but not all of the retinal vascular defects previously observed in global Bcl‐2 deficient mice. These mice displayed decreased numbers of retinal endothelial cells, arteries and veins. They also demonstrated premature primary branching of the retinal vasculature but unlike the global knockout mice, spreading of the superficial retinal vascular layer proceeded normally. Choroidal neovascularization was attenuated in Bcl‐2EC mice, although neovascularization accompanying oxygen‐induced ischemic retinopathy (OIR) was not. Therefore, our data suggest that Bcl‐2 expression in the endothelium affects postnatal retinal endothelial cell number and arteriogenesis, but differentially impacts retinal and choroidal pathological neovascularization.
Expression of Thrombospondin‐1 Modulates the Angioinflammatory Phenotype of Choroidal Endothelial Cells
Authors: Sheibani Nader , Fei Ping , Sorenson Christine
Published: June 2021

The choroidal circulation plays a central role in maintaining the health of outer retina and photoreceptor function. Alterations in this circulation contribute to pathogenesis of many eye diseases including exudative age‐related macular degeneration. Unfortunately, very little is known about the choroidal circulation and its molecular and cellular regulation. This has been further hampered by the lack of methods for routine culturing of choroidal endothelial cells (ChEC), especially from wild type and transgenic mice. Here we describe a method for isolation and culturing of mouse ChEC. We show that expression of thrombospondin‐1 (TSP1), an endogenous inhibitor of angiogenesis and inflammation has a significant impact on phenotype of ChEC. ChEC from TSP1‐deficient (TSP1‐/‐) mice were less proliferative and more apoptotic, less migratory and less adherent, and failed to undergo capillary morphogenesis in Matrigel. However, re‐expression of TSP1 was sufficient to restore TSP1‐/‐ ChEC migration and capillary morphogenesis. TSP1‐/‐ ChEC expressed increased levels of TSP2, phosphorylated endothelial nitric oxide synthase (NOS) and inducible NOS a marker of inflammation, which was associated with significantly higher level of NO in these cells. Wild type and TSP1‐/‐ ChEC produced similar levels of VEGF, although TSP1‐/‐ ChEC exhibited increased levels of VEGF‐R1 and pSTAT3. Other signaling pathways including Src, Akt, and MAPKs were not dramatically affected by the lack of TSP1. Together our results demonstrate an important autocrine role for TSP1 in regulation of ChEC phenotype.
Impaired Middle Cerebral Artery and Cerebrovascular Vasodilation in Healthy Aging
Authors: Coverdale Nicole , Badrov Mark , Shoemaker J.
Published: June 2021

This study quantified the effect of age on cerebrovascular reactivity (CVR) and cerebrovascular conductance. Cerebral blood flow velocity (CBFV; transcranial Doppler (TCD)) and cross‐sectional area (CSA) of the middle cerebral artery (MCA; 3T Magnetic Resonance Imaging (MRI)) to measure cerebral blood flow (CBF) (HC; 6% CO2) were measured in healthy populations of young (YA: n=6, 24 ± 2 years, 3 males) and older adults (OA: n=6, 66 ± 6 years, 1 male) during hypercapnia (HC). CVR was calculated as the percent change (%Δ) in CBF per change in end tidal carbon dioxide where the end tidal carbon dioxide was the average of the change during TCD and the MRI studies which were performed on separate days. Cerebrovascular conductance was calculated as the quotient of CBF and mean arterial pressure. CVR was not different between YA (2.0 ± 0.9 at minute one to 3.1 ± 0.9 %/mmHg at minute four) and OA (2.6 ± 1.2 at minute one to 4.0 ± 1.6 %/mmHg at minute four) over the four minutes of HC. The CSA in YA increased from 6.29 ± 0.71 at baseline to 7.16 ± 1.00 mm2 at minute four while OA increased from 6.36 ± 0.92 at baseline to 6.75 ± 1.27 mm2 at minute four (p=0.5). The %ΔCBF was not different between the YA and OA yet the %Δ in CBFV was greater during HC in the OA (p=0.01). However, when %ΔCBFV was normalized to the change in mean arterial pressure there was no longer a difference between YA and OA. Also, cerebrovascular conductance was greater in the YA (p=0.03). Therefore, a greater blood pressure response in the OA may be responsible for their preserved CVR despite evidence for an age‐related impairment in MCA dilation and vascular conductance. Supported by the Canadian Institutes of Health Research.