bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of Drugs with Dual Dopamine Transporter and Sigma1 Receptor Activity
Authors: Kopajtic Theresa , Cao Jianjing , Hiranita Takato , Tanda Gianluigi , Newman Amy H. , Katz Jonathan L
Published: June 2021

Prior results suggest that rimcazole and its analogues decrease cocaine self administration (SA) through actions mediated by antagonist effects at sigma1 receptors (σ1Rs) along with blockade of the dopamine transporter (DAT). The present study compared several analogues of rimcazole for the optimum ratio of σ1R to DAT affinity for effects on cocaine SA. SH‐324, which had a 2‐fold σ1R/DAT affinity ratio, was approximately 3‐fold more potent than rimcazole and SH‐328 in attenuating cocaine SA. The latter two compounds had respective σ1R/DAT affinity ratios of 9.14 and 0.11, suggesting that the optimum ratio lies between those values. Rimcazole analogues with varying affinities and selectivities for DAT and σ1Rs were prepared (Cao et al. 2003; 2008). The hydroxylated linking chain analogue, JJC 2‐010 [(±)3‐(4‐(3‐(bis(4‐fluorophenyl)amino)propyl)piperazin‐1‐yl)‐1‐phenylpropan‐1‐ol], and its N‐benzyl analogue, JJC 2‐006, had a σ1R/DAT affinity ratios of 13.04 and 0.27, respectively. Ester analogues of JJC 2‐010 were prepared which had σ1R/DAT affinity ratios varying from 16.34 to 0.86. In contrast to rimcazole, these novel compounds had high affinity for the DAT ranging from 1.72 to 51.3 nM, and affinity for σ1Rs ranging from 24.9 to 292 nM. Selected compounds with a range of σ1R/DAT affinity ratios will be tested for their cocaine antagonist effects in various in vivo procedures indicative of abuse liability.
Growth Associated Protein‐43 Modulates Dopamine Transporter Function
Authors: Guptaroy Bipasha , Zhang Minjia , Gnegy Margaret
Published: June 2021

Dopamine transporters (DAT) are integral membrane proteins that regulate re‐uptake of released dopamine from the synapse and its efflux in response to drugs such as amphetamine (AMPH). Our laboratory has demonstrated that protein kinase C β (PKC β) can regulate AMPH‐induced DAT‐mediated DA efflux. Further, AMPH increases phosphorylation of the growth associated protein‐43 (GAP‐43) at its PKC substrate site (ser‐41) in rat synaptosomes. Our aim in this study was to determine whether GAP‐43 is involved in AMPH‐stimulated DA efflux, using HEK 293 cells stably expressing hDAT. We found that in transfected cells, GAP‐43 caused a 67% enhancement of AMPH mediated DA efflux. There was no concomitant increase in DA uptake in these cells. Phosphorylation of GAP‐43 at its PKC phosphorylation site was enhanced in the presence of AMPH in cells transfected with GAP‐43. We have previously shown that PKC β is associated with the dopamine transporter. Preliminary studies using immpunoprecipitations in GAP‐43 transfected HEK‐hDAT cells indicate that GAP‐43 may not be associated with DAT in these cells. Our studies establish a functional role for GAP‐43 in AMPH‐mediated DA efflux through DAT. Funded by DA011697.
Substrate specificity of rat Na+/taurocholate cotransporting polypeptide
Authors: Sheng Zhonghua , Alnouti Yazen , Hagenbuch Bruno
Published: June 2021

Human Na+/taurocholate cotransporting polypeptide (NTCP) has recently been proposed to be a multispecific transporter that mediates uptake of xenobiotics including some statins in addition to its prototypical substrate (bile acids). In rat basolateral plasma membrane vesicles Na+‐dependent taurocholate uptake was competitively inhibited by several glitazones, suggesting that these drugs could be substrates of rat Ntcp. Therefore we used CHO cells that stably express rat Ntcp to screen for drugs and other xenobiotics that inhibit sodium dependent taurocholate uptake and therefore might be substrates of Ntcp. We tested selected inhibitors as substrates using LC‐MS/MS analysis. We confirmed that troglitazone and rosiglitazone are strong inhibitors of rat Ntcp (IC50 = 13 μM for troglitazone, 8 μM for rosiglitazone). Furthermore, we determined that several additional drugs including losartan, ritonavir, saquinavir, fluvastatin, ketoconazole, diclofenac and carbenoxolone inhibited Ntcp mediated taurocholate transport in a concentration dependent pattern. The so far tested troglitazone, rosiglitazone, ritonavir, saquinavir and pravastatin however are not substrates for rat Ntcp although pravastatin is transported by human NTCP. In conclusion, rat Ntcp is inhibited by numerous drugs but its substrate specificity seems to be more restricted than the substrate specificity of human NTCP.
A cell‐based high‐throughput assay to identify inhibitors of Organic Anion Transporting Polypeptides 1B1 and 1B3
Authors: Obaidat Amanda , Gui Chunshan , Chaguturu Rathnam , Hagenbuch Bruno
Published: June 2021

Organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are expressed at the basolateral membrane of hepatocytes and exhibit broad and overlapping substrate specificity for many endobiotics and drugs. Without specific inhibitors it is almost impossible to determine the contribution of the individual transporters to hepatocellular uptake of shared substrates (drugs). In order to identify such inhibitors of OATP1B1 and OATP1B3, we developed a cell‐based high throughput assay to screen chemical libraries. We used Chinese Hamster Ovary cells that stably express human OATP1B1 or OATP1B3 on 96‐well plates and measured uptake of fluorescein‐methotrexate (F‐MTX). After validating the assay with known inhibitors we screened the pharmacologically well characterized Prestwick library of 1120 compounds. Among the numerous hits were known OATP inhibitors including rifampicin, cyclosporine A and mifepristone. For inhibitors that seemed to be able to distinguish between OATP1B1 and OATP1B3 mediated F‐MTX uptake we determined IC50 values. Estropipate (estrone‐3‐sulfate stabilized with piperazine) turned out to be a most distinctive OATP1B1 inhibitor (IC50 = 0.07 μM vs. 11.2 μM for OATP1B3) while ursolic acid was the most distinctive OATP1B3 inhibitor (IC50 = 3.4 μM vs. 29.6 μM for OATP1B1). In conclusion, this cell‐based assay should allow us to screen larger libraries and identify even more specific inhibitors.
Cancer Chemoprevention: Selective Inhibition of Cytochrome P450 2A13
Authors: Blake Linda C. , Roy Anuradha , Scott Emily E.
Published: June 2021

4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) is one of the most prevalent and carcinogenic compounds in tobacco. NNK is selectively activated by human cytochrome P450 2A13 (CYP2A13) to form two carcinogens that generate DNA adducts. CYP2A13 is found in the respiratory tract where NNK‐derived adducts can result in lung cancer. CYP2A13 is 93.5% identical to the hepatic cytochrome P450 2A6 (CYP2A6), which does not efficiently metabolize NNK but does metabolize other xenobiotic compounds. Thus a selective inhibitor of CYP2A13 could be used as a chemopreventative for tobacco users. High throughput screening was used to identify small molecule scaffolds that selectively bound CYP2A13 over CYP2A6. One such scaffold was benzylmorpholine. A small library of benzylmorpholine analogs was generated and has been evaluated for differential ligand binding (Kd) and inhibition of human cytochrome P450 2A enzymes. Examination of these results is revealing selective compounds that could be used for the reduction of lung cancer related to DNA damage in tobacco users.
Kinetics and Molecular Interactions in the Rapid Disulfation of Raloxifene (RAL) by Human Sulfotransferase (SULT) 1E1
Authors: Cook Ian Thomas , Kadlubar Susan Nowell , Falany Charles Napoleon
Published: June 2021

Sulfation is an important Phase 2 conjugation reaction in the metabolism of steroidal compounds and related therapeutic drugs. RAL is sulfated by several human SULTs to form a monosulfate; however only SULT1E1 forms RAL disulfate (RAL‐diS). Kinetic analysis shows that formation of RAL‐diS is favored at low physiological concentrations of RAL, whereas at higher concentrations RAL monosulfate formation is increased. To better understand RAL sulfation, the benzothiophene‐ and phenolic‐monosulfates were synthesized and used in the kinetic analysis of RAL‐diS formation by SULT1E1. The conversion of RAL‐phenyl‐sulfate to RAL‐diS has a significantly lower Km and greater Kcat than determined with the benzothiophene‐sulfate. Molecular modeling of the two RAL‐monosulfates in the SULT1E1 active site indicates that the binding free energy and orientation of RAL‐phenyl‐sulfate in the active site is more conducive to catalysis than that of the benzothophene‐sulfate. These data indicate that at nanomolar concentrations RAL is preferentially converted to the phenyl‐sulfate then to the disulfate by SULT1E1. Since the RAL phenyl‐sulfate is resistant to hydrolysis by steroid sulfatase this represents a mechanism for the inactivation of RAL by SULT1E1 in breast and endometrial tissues.
Hepatic Cytochrome P450 Expression during Citrobacter Rodentium Infection in Tumor Necrosis Factor Receptor p55‐/‐ Mice
Authors: Kinloch Ryan D , Morgan Edward T
Published: June 2021

Citrobacter rodentium is the rodent equivalent of enteropathogenic Escherichia coli infection in humans. The proinflammatory cytokine tumor necrosis factor‐α (TNFα) is induced in mice during C. rodentium infection and may be involved in regulating cytochrome P450 (P450) expression during inflammation. In this study, female mice lacking the gene for the p55 receptor for TNFα, as well as their C57BL/6 controls, were orally infected with C. rodentium and sacrificed 7 days later. CYP2B9 and 3A41 mRNA levels were down‐regulated, and CYP2D22, 3A11 and 3A13 mRNA levels remained near control levels in both the C57BL/6 and the TNF p55 ‐/‐ mice. Hepatic CYP4A10 and 4A14 mRNAs were down‐regulated more (less than 20% of control) in the TNF p55‐/‐ mice than in the C57BL/6 mice, (50% of control). Similarly, CYP2D9 mRNA levels were more highly induced in TNF p55 ‐/‐ (18‐fold) than in C57Bl/6 (2‐fold) mice, although CYP2D protein levels were near control expression levels for both strains. CYP2E1 and 3A25 displayed similar trends in which their mRNA levels were down‐regulated only in the C57BL/6 but not in the TNF p55‐/‐ mice. CYP2E1 protein expression levels followed the same trend as its mRNA. The data suggest that TNFα may play a role in down‐regulating specific hepatic P450s during C. rodentium infection. Supported by NIH grant DK072372.
Anti‐inflammatory effects of andrographolide: inhibition of the release of cytokines relevant to asthma
Authors: Abu‐Ghefreh Ala'a A. , Canatan Halit , Ezeamuzie Charles I
Published: June 2021

Andrographolide ‐ the major active principle of Andrographis paniculata, has been credited with anti‐inflammatory effects. The possibility that the drug may affect asthmatic inflammation has not been explored. The purpose of this study was, therefore, to investigate the ability of andrographolide to inhibit the release of inflammatory cytokines both in vitro in mouse peritoneal macrophages and in vivo in allergic inflammation in a mouse asthma model. The drug significantly inhibited LPS‐induced TNF‐α and GM‐CSF release from mouse macrophages. The IC50 values were 0.6 μM for TNF‐α and 3.3 μM for GM‐CSF, and maximal inhibitions were 77% and 94%, respectively. The drug was as efficacious as dexamethasone, but about 10 times less potent. The effect correlated with reduced expression of mRNA for the two cytokines. In the in vivo study in allergic mice, the drug significantly inhibited allergen‐induced elevation of TNF‐α and GM‐CSF in the bronchial fluid and nearly abolished the influx of lymphocytes and eosinophils. These results provide evidence that andrographolide is an effective anti‐inflammatory drug with the potential to be used for the treatment of asthma as well as other inflammatory diseases.
Nuclear factor erythroid 2‐related factor 2 (Nrf2) activators inhibit pro‐inflammatory cytokine production by murine T cells
Authors: Rockwell Cheryl Elizabeth , Fields Patrick , Klaassen Curtis
Published: June 2021

Nrf2 is a transcription factor upregulating cytoprotective genes in response to cellular stress. Nrf2 mRNA is upregulated in mature CD3+ cells by the Nrf2 activator, tert‐butylhydroquinone (tBHQ). In addition, tBHQ induces expression of the Nrf2 target genes, Ho‐1, Nqo1, and Gclc in wild‐type, but not Nrf2‐null CD3+ T cells, suggesting the induction is Nrf2‐dependent. Furthermore, tBHQ and butylated hydroxyanisole (BHA), another Nrf2 activator, markedly inhibit IFNγ transcription in wild‐type activated T cells, but have little effect upon IFNγ transcription in Nrf2‐null T cells. Likewise, tBHQ inhibits binding to both the AP‐1 and NFκB response elements in wild‐type CD3+ cells, but only modestly inhibits AP‐1/NFκB binding in Nrf2‐null CD3+ cells, suggesting the effects on the AP‐1 and NFκB binding sites are partially Nrf2‐dependent. In addition to its effects upon IFNγ, tBHQ also inhibits production of TNFα and IL‐2 by activated T cells. Collectively, the current studies suggest Nrf2 activation in CD3+ cells markedly inhibits IFNγ transcription, which is likely due in part to decreased binding to the AP‐1 and NFκB response elements. Overall, the present studies suggest Nrf2 may represent a novel regulatory mechanism in mature murine T cells and thus may be a useful target for the development of new anti‐inflammatory therapeutics. (Supported by NIH grants ES09716, ES07079, ES013714, ES09649, and RR021940.)
Novel Cdk inhibitors identified by in silico screening act through a novel mechanism
Authors: Corsino Patrick , Horenstein Nicole , Ostrov David , Rowe Thomas , Law Mary , Aslanidi George , Barrett Amanda , Law Brian
Published: June 2021

Cyclin dependent kinases (Cdks) have been considered to be important targets in cancer therapeutics due to their critical involvement in cell cycle control. Recent genetic knockout studies in mice have indicated that there is a significant level of redundancy between the Cdks that regulate cell cycle progression. Therefore, an inhibitor that affects multiple Cdks would be more effective at inducing cell cycle arrests in a broad spectrum of cancers than an inhibitor that targets only one. This study involves the characterization of a novel series of small molecule inhibitors that target Cdks 1, 2, and 4. These inhibitors were identified by high throughput in silico molecular screening, targeted to a conserved structural site on Cdk2 that is distinct from the ATP binding site. The mechanism of action of these small molecules is the induced reduction of the apparent levels of Cdks 1, 2, and 4 in cells. This decrease in functional Cdk protein is associated with a decrease in Rb phosphorylation as well as E2F‐dependent gene transcription. Multiple lines of evidence including immunofluorescence and ultracentrifugation indicate that the compounds directly induce Cdk1, Cdk2, and Cdk4 protein aggregation.
APS decreased high glucose induced overexpression of PTP1B through inhibiting the activation of ATF6
Authors: WANG Nian , ZHANG Deling , Jin Huan , ZOU Feng , OUYANG Jingping
Published: June 2021

Protein tyrosine phosphatase 1B(PTP1B) which negatively regulated the insulin signaling was considered as a potential target to prevent and treat type 2 diabetes(T2DM). Endoplasmic reticulum(ER) stress was a key link between obesity, insulin resistance, and T2DM. Interestingly, PTP1B located on the cytosolic surface of ER and played an essential role in potentiating ER stress, we hypothesized that PTP1B may be a chaperon of ER stress. Activating transcription factor 6(ATF6) promoted the synthesis of chaperones when ER stress happened. We have found that astragalus polysaccharide(APS) could increase insulin sensitivity probably through decreasing the expression of PTP1B. In this study, we intended to investigate whether ATF6 was involved in this effect. Firstly, we used MTT assay to analyze the cytotoxicity of different concentration of APS and found that APS in a wide range had little cytotoxicity, approximately from 0‐1.6mg/ml; secondly, we used 25mmol/l D‐glucose to treat on cells for 24 hours, followed by APS with different concentrations mentioned above. Results showed that APS at 0.1‐0.8mg/ml decreased the expression of p50‐ATF6, APS at 0.2‐0.8mg/ml were more potential than 0.1mg/ml; while APS at 0.2‐0.8mg/ml reduced the expression of PTP1B and APS at 0.4mg/ml were more effective than 0.2 and 0.8mg/ml. So APS at 0.2‐0.8mg/ml may be the effective concentration range that can not only decrease the expression of PTP1B but also reduce glucose induced ER stress. This positive correlation provided significant information that APS decreased the expression of PTP1B was mediated at least partly through inhibiting the activation of ATF6.
Quantification of Blood Alcohol Concentration Using Alcohol Oxidase Dipsticks
Authors: Petroni Daniel H , George William J
Published: June 2021

Alcohol detection dipsticks have been utilized as a qualitative test to detect the presence of alcohol in oral fluids. The present study was conducted in order to see whether alcohol oxidase dipsticks (Chematics) could be utilized as a quantitative tool to measure blood alcohol concentrations (BAC). Human saliva was utilized and spiked with ethanol standards to create a final solution with concentrations including 200, 100, 50 and 25 mg/dl of ethanol. Verification of these concentrations was done on a Shimadzu 14‐A Gas Chromatograph, equipped with a Supelco 5% Carbowax packed column. The dipsticks were exposed to the ethanol‐saliva solution and placed within a Biorad Gel Doc XR analyzer under fluorescent white light. The absorbance density of the reaction band was then measured using Biorad's Quantity One analysis software (Ver. 4.6.2) and a standard curve was plotted. A linear regression analysis was conducted between ethanol concentration and absorbance density. A significant correlation was found with an r squared value of 0.9917. These results indicate the potential use of alcohol oxidase dipsticks as a non‐invasive quantitative tool to measure blood alcohol levels.
Lodenafil Carbonate: Reproductive and Developmental Evaluation
Authors: Taricano Ingrid , Costa Elaine Rodrigues , Bonamin Leoni , Afiune Jorge , Scivoletto Regina
Published: June 2021

Lodenafil, is one new orally‐active, potent and selective inhibitor of the enzyme phosphodiesterase 5 (PDE5) developed in Brazil, with therapeutically application in erectile dysfunction. The objective of this study was to evaluate the reproduction toxicity and in offspring, evidencing specifically the occurrence of possible toxic effects in the fertility, gestation, maternal behavior and lactation, as well as growth and postnatal development of F1 since its birth, up to 4 days. Wistar rats received daily: metilcelulose, (2 control groups); 5,7mg/kg, 28,4mg/kg and 142mg/kg (2 groups of 10 males and 10 females in each dose).Drug administration occurred during 35 and 14 consecutive days for males and females, respectively. Animals had been observed for detention of possible clinical and behavior alterations, delivery, and other toxic signals including morbidity or mortality, gestation time, litter size number of each sex, total living pups and corporal weight of the offspring.Litter components had been analyzed in the maximum 24 h after the birth, and the evaluation repeated in all first 4 days after‐birth. No remarkable toxicity was found in the offspring even in the presence of maternal toxicity, no significant decrease in body weight gain was observed, decrease in offspring righting reflex and increase in litter size, were observed.

Fellowship

Cristália Produtos Químicos e Farmacêuticos, Instituto Royal
Choline supplementation influences OGTT in the presence of acute hyperhomocysteinemia
Authors: Lopez Anna‐Lee Nicola , Gossell‐Williams Maxine D , Simon Oswald
Published: June 2021

Choline, upon conversion to betaine, plays an integral role in homocysteine homeostasis. There is new evidence that acute hyperhomocysteinemia may play a critical role in impaired insulin secretion from pancreatic beta cells and thus could prevent the normal response to a glucose challenge. Consequently, the aim of this study was to examine the benefit of choline supplementation on plasma glucose concentration in the presence of acute hyperhomocysteinemia. Male Sprague‐Dawley rats divided into groups for choline supplementation, 60mg/kg (n=5), 90mg/kg (n=5) and control (n=4) were treated orally 5 days per week for 3 weeks. Animals were given access to food and water ad libitum. Oral glucose tolerance tests (OGTT) were done at the end of supplementation in the absence or presence of induced acute hyperhomocysteinemia (induction achieved by methionine loading). OGTT without acute hyperhomocysteinemia was not impaired in rats supplemented with choline. Prolonged elevation of blood glucose concentration (8.33mmol/l ± 0.39) was observed in control rats 2hrs after oral glucose in the presence of acute hyperhomocysteinemia; this was prevented in both groups of choline supplemented rats (P<0.001). Therefore, choline supplementation may offer protection from impaired insulin secretion that can occur with acute hyperhomocysteinemia. Source of funding: Office of Graduate Studies and Research, U.W.I. Mona
Investigation of the cytotoxcity of novel jadomycins in drug resistant breast cancer cells
Authors: Goralski Kerry B , Graham Cathy L , Issa Mark , Jakeman David L
Published: June 2021

The drug transporter ABCB1 plays a major role in drug resistance and chemotherapy failure in the treatment of breast cancer. Jadomycins are natural products synthesized by the bacteria S. venezuelae ISP5230 and inhibit the mitotic regulatory protein aurora‐B kinase. The object of our study was to investigate the cytotoxicity of jadomycins towards breast cancer cells. By MTT assay we found that Jadomycin B, DT and G were selectively toxic against MCF7 and T47D breast cancer cells compared to control human kidney (HK2) cells. Jadomycin B, DT and G did not activate ABCB1‐ATPase activity in isolated membrane preparations, suggesting that these molecules may evade the ABCB1 drug resistance transporter. In support of this, Jadomycin B and DT displayed similar cytotoxicity in MCF7 control and taxol resistant MCF7tx400 cells that overexpress ABCB1. Comparatively, MCF7tx400 cells were relatively resistant to the cytotoxic effects of the ABCB1 substrates taxol and etoposide. We conclude that Jadomycin B and DT possess effective chemotherapeutic action against both drug‐sensitive and ABCB1‐expressing drug‐resistant breast cancer cells in culture. This work was supported by the CBCF‐Atlantic chapter.
Cannabinoid receptor‐independent actions of HU210 upon the viability and mobility of PC3 prostate cancer cells
Authors: Fowler Christopher John , Chung Sui Chu , Persson Emma
Published: June 2021

Both CB receptor‐dependent and independent effects of cannabinoids upon cancer cell viability have been reported in the literature. Here, we reexamined cannabinoid effects upon PC3 cell viability and mobility in view of recent data indicating that prostate cancer CB1 receptor expression is associated with disease severity and outcome (Chung et al., Eur J Cancer, in press).

PCA experiments showed that the PC3 cells had measurable, but low CB1 receptor expression, and no clear expression of CB2 receptors. Cell viability experiments, using 1% FBS and incubation times of 1‐6 days, indicated that the CB receptor agonists CP55,940 (0.3 and 1 µM) and HU210 (1 µM), the 2‐AG synthesis inhibitors orlistat (1 µM) and RHC‐80267 (30 µM) and MAFP (0.1 and 1 µM, an inhibitor of 2‐AG metabolism) did not affect cell viability. In contrast 3 and 10 µM HU210 rapidly blocked cell viability. PC3 mobility was assayed at 1% FBS by following the ability of cells to repopulate a scratched area of the well. No effects of CP55,940, orlistat, RHC‐80267, MAFP and 2‐AG itself were seen, whilst HU210 (10 but not 1 µM) and cytochalasin D, which disrupts actin cytoskeletons, completely halted cell mobility. The effect of HU210, due to reduced cell viability, was not blocked by concomitant incubation with AM251 (1 µM) or AM630 (1 µM). Thus under the experimental conditions used here, no CB receptor‐dependent effects upon PC3 cell viability and mobility are seen.
Influence of COX‐2 inhibitor on expression of COX‐2, VEGF, Ang‐1 and Ang‐2 gene in human tongue squamous cell line Tca‐8113
Authors: Wu Yunxia , Hao Fenxiang
Published: June 2021

Through observing the change of COX‐2, vascular endothelial growth factor, angipoietin‐1 and angiopoietin‐2 gene in human tongue squamous cell carcinoma Tca‐8113 cell line by ihibiting the activity of cox‐2 to investigate the affect and mechanism of the selective COX‐2 inhibitor nimesulide in the biological behavior of human tongue squamous cell carcinoma.Human tongue squamous cell carcinoma Tca‐8113 cell were cultured in vitro and treated with NIM for 48h to measure the expression of COX‐2, VEGF, Ang‐1 and Ang‐2 mRNA in Tca‐8113 cells by the semiquantitative reverse transcription polymerase chain reaction analysis.Human tongue squamous cell carcinoma Tca‐8113 cell were treated with NIM for 48h,the expression of COX‐2 ,VEGF, Ang‐1, Ang‐2 mRNA level between the each treated group and the control group was statistical significance(P<0.01), The expression of COX‐2, VEGF mRNA reduced in a concentration depengent manner r= ‐0.963,0.934 . Human tongue squamous cell carcinoma Tca‐8113 cell were treated with NIM,the expression of COX‐2, VEGF, Ang‐1 ,Ang‐2 mRNA level in the each treated group compared with the control group was reduced, and the reduced in a concentration depengent manner.
Modulatory Role of Mammalian Target of Rapamycin in Regulating Endothelial ICAM‐1 Expression and Lung PMN Recruitment
Authors: Rahman Arshad , Minhajuddin Mohd , Fazal Fabeha , Bijli kaiser M.
Published: June 2021

PKC‐δ, PI 3‐kinase, and the downstream target Akt are important signaling intermediates in mediating NF‐κB activation and ICAM‐1 expression in endothelial cells. Here, we show that these pathways also activate mTOR to ensure a tight regulation of NF‐κB activation and ICAM‐1 expression these cells. Exposure of human vascular endothelial cells to thrombin resulted in phosphorylation of mTOR and its downstream target p70S6 kinase in a PKC‐δ‐ and PI 3‐kinase/Akt‐dependent manner. Consistent with this, expression of constitutively active mutant of PKC‐δ or Akt was sufficient to induce NF‐κB activation and ICAM‐1 expression, and co‐expression of mTOR suppressed whereas inhibition/depletion of mTOR augmented these responses. Increasing or impairing mTOR signaling produced similar effects on NF‐κB activation and ICAM‐1 expression induced by thrombin. These data reveal an important role of mTOR as an endogenous modulator that may be engaged in PKC‐δ‐ and PI3K/Akt‐dependent manner to dampen thrombin‐induced NF‐κB signaling of ICAM‐1 expression in endothelial cells. We also determined the in vivo consequence of mTOR overexpression in a mouse model of sepsis. Introduction of the construct encoding mTOR via cationic liposomes caused a marked reduction in lung PMN infiltration in mice challenged i.p. with LPS. Together, these results indicate that mTOR may be a general modifier of lung inflammatory responses.
Postconditioning during reperfusion attenuates myocardial injury without improved mitochondrial oxidative phosphorylation
Authors: Chen Qun , Lesnefsky Edward J
Published: June 2021

Postconditioning (PC) applied during reperfusion (REP) decreases myocardial injury. However, mitochondrial damage occurs during ischemia (ISC). We propose that PC can not improve ISC‐damaged mitochondrial function during REP. In the untreated group (UNT), isolated rat hearts were subjected to 30 min global ISC and 30 min REP at 37°C. In the PC group, 6 cycles of 10 sec ISC + 10 sec REP was applied at the onset of REP. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated at the end of REP. Maximal state 3 rates of oxidative phosphorylation (OXPHOS, nAO/mg/min) were measured. PC decreased LDH release (mu/g/60 min; UNT 1065±182 vs. PC 669±38 n=5 in each group P < 0.05) without improved functional recovery during REP. PC did not improve respiration in SSM and IFM using glutamate, succinate, and TMPD‐ascorbate as complex I, complex II, and cytochrome oxidase substrates, respectively. Thus, cardiac injury can still be attenuated even though OXPHOS are already compromised by ISC. The mechanism by which PC decreases myocardial injury is not through improved OXPHOS.

±SEM; *p<0.05 vs. UNT (untreated), TC, time control
Successive changes in hyaluronan (HA) synthesis and fragmentation in the rat post‐ischemic kidney
Authors: Decleves Anne‐Emilie , Caron Nathalie , Voisin Virginie , Bouby Nadine , Flamion Bruno
Published: June 2021

The precise time‐course of HA accumulation and fragmentation in inflammatory tissues remains uncertain. We examined the expression of the main hyaluronidases (HYAL1 and HYAL2) and HA synthases (HAS1, HAS2 and HAS3) by real‐time PCR in the outer [OSOM] and inner stripes [ISOM] of the renal outer medulla up to Day 14 after ischemia/reperfusion (IR) injury. We also assessed hyaluronidase activity by zymography and HA fragmentation by membrane filtration and pseudo‐ELISA. HAS1 was up‐regulated more than 100‐fold 12 h post‐IR, returning to baseline faster in the ISOM than in the OSOM. HAS2 increased only after a 2‐day delay and remained elevated. The mRNA for HAS3 could not be detected. Both HYAL1 and HYAL2 were strongly but transiently (12‐48 h) repressed in parallel with a reduction in hyaluronidase activity. HA accumulation was detected as early as 12 h and increased steadily thereafter. However, the amount of low molecular weight (MW) HA (i.e., HA fragments) decreased by 60% from baseline to Day 1 in the ISOM. Later, HA fragments amassed in both zones (×100 in OSOM and ×5 in ISOM vs baseline). In summary, within 24 h post‐IR, there is a massive activation of HAS1 combined with a repression of hyaluronidases, leading to a pool of predominantly high MW HA. Significant amounts of HA fragments appear later (Day 7‐14) when HAS2 is induced and HYAL1 and HYAL2 levels are back to baseline. HA fragments are possibly involved in regeneration.
Development of combined soluble epoxide hydrolase inhibitors and epoxide analogs with vasodilator activity
Authors: Imig John D , Sudhahar Varadajan , Ravinder Kodela , Campbell William B , Hammock Bruce D , Falck John R
Published: June 2021

Epoxyeicosatrienoic acids (EET) and soluble epoxide hydrolase inhibitors (SEHi) possess cardiovascular protective properties. SEHi are currently being clinically tested for the treatment of hypertension and EET analogs have recently been demonstrated to possess anti‐hypertensive properties. Thus, a series of compounds were synthesized to have EET analog and SEHi activities. Two of these compounds had a low nM IC50 for SEH and possessed vasoactivity similar to 14,15‐EET in bovine coronary arteries. These two compounds were evaluated further in rat mesenteric resistance arteries. Rat mesenteric resistance arteries averaged 293 ± 6 um at 65 mmHg and were preconstricted with U46619 to 164 ± 5 um. Compound A and compound B dose‐dependently dilated the mesenteric resistance artery and this dilation reached 40% at a 10 uM. Compound A was more active and had a greater dilator effect on mesenteric resistance arteries at lower doses. On the other hand, compound B has a lower IC50 (46 nM) for the SEH enzyme. The EET antagonist, 14,15‐EEZE abolished the dilator responses to compound A and B demonstrating EET mimetic activity for the compounds. These data demonstrate the ability to synthesize dual SEHi / EET analogs and provide initial evidence for beneficial cardiovascular properties.
Actinonin, a meprin A inhibitor, protects the renal microcirculation during sepsis
Authors: Wang Zhen , Herzog Christian , Kaushal Gur P , Mayeux Philip R
Published: June 2021

Meprin A, a matrix metalloproteinase highly expressed in the kidney and reported to be an early biomarker of acute kidney injury (AKI), is thought to play an important role in several causes of AKI. To address the role of meprin A in the early microcirculatory dysfunction associated with sepsis‐induced AKI, we studied the effects of the meprin A inhibitor actinonin in the cecal ligation and puncture (CLP) model of sepsis in the mouse. Male C57BL/6 mice (40 weeks) underwent CLP with vehicle (CLPv) or actinonin at 20 mg/kg, ip (CLPact) 30 min prior to CLP. At 7h post CLP, intravital videomicroscopy was utilized to image renal cortical peritubular capillary perfusion. Actinonin prevented the fall in renal capillary perfusion. The percentage of capillaries with continuous perfusion in CLPv and CLPact were 50 ± 9% and 83 ± 10% respectively (P < .05) and the percentage with no perfusion in CLPv and CLPact were 33 ± 10% and 7 ± 2% respectively (P < .05). Capillary perfusion in CLPact was no different from sham mice. Since meprin A is known to activate the proform of IL‐1β, serum IL‐1β was used to assess the inhibitory activity of actinonin. IL‐1β levels decreased in CLPact by 36% compared to CLPv at 7h (P < .05). These data suggest that meprin A may play an important role in the microcirculatory failure associated with sepsis‐induced AKI and that actinonin should be evaluated further as a possible therapeutic agent. Supported by NIH DK075991.
Effects of hemoglobin‐based oxygen carriers (HBOCs) on nitric oxide (NO) levels in the spinotrapezius muscle and mesentery of the rat
Authors: Yannaccone Andrew , Carvalho Helena , Pittman Roland N.
Published: June 2021

Use of HBOCs has been limited by the side effect of increased mean arterial pressure (MAP). It is thought that HBOCs scavenge NO, leading to decreased [NO], arteriolar vasoconstriction, and increased vascular resistance and arterial pressure. Previous microcirculatory studies investigating effects of HBOC infusion have yielded contradictory results. The aim of this study was to compare responses of tissues with different metabolic activity to HBOC injection. Experiments were conducted in the spinotrapezius muscle or mesentery of anesthetized male Sprague‐Dawley rats. Preparations were loaded with 5 μM DAF‐2DA for 45 min. Cytosolic esterases convert DAF‐2DA to DAF‐2 which reacts with an oxygenated derivative of NO to form highly fluorescent DAF‐2T. The DAF‐2T signal was recorded with a digital CCD camera connected to a Zeiss fluorescence microscope. Vessel diameter, MAP and fluorescence were monitored under control conditions and after bolus i.v. injection of HBOC‐201 in 1, 10 and 100 μM plasma concentrations. HBOC‐201 increased MAP. There was vasoconstriction and an increase in DAF‐2T fluorescence in the spinotrapezius preparation, whereas no vasoconstriction or increase in DAF‐2T fluorescence was observed in the mesentery. These results suggest that metabolically active tissues like skeletal muscle have higher [NO] than less metabolically active tissues such as the mesentery. Support: HL18292.
Caveolae by interfering internalization of AT1 receptors regulate constrictions of isolated arterioles to Ang II
Authors: Bagi Zsolt , Koller Akos
Published: June 2021

We hypothesized that caveolae regulate Ang II ‐ induced arteriolar constrictions by modulating the internalization of type 1 angiotensin II (Ang II) receptors (AT1R). Thus to assess the functional availability of AT1R, Ang II (10‐8 M) were administered in a repeated fashion (first and second applications) and the diameter of skeletal muscle arterioles (≈150 μm at 80 mmHg) were measured with videomicroscopy. Compared to the first application, Ang II‐induced constrictions were significantly reduced upon the second application (% max. constrictions: 59±3 vs. 37±2, P<0.05). Incubation of arterioles with concanavalin A (0.1 mM for 2 hours), known to inhibit AT1R internalization, had no effect on Ang II‐induced constrictions upon the first application (65±7%), but further significantly diminished constrictions to the second application (9±5%). Incubation of vessels with methyl β‐cyclodextrin (1 mM for 2 hours), known to disrupt caveolae, resulted in maintained Ang II‐induced arteriolar constrictions upon repeated applications (61±3 vs. 69±7, N.S.). We propose that internalization of AT1R is essential to restore active receptors on the surface of vascular smooth muscle cell, making them available for further stimulation and that caveolae by interfering with internalization limit AT1R availability and thus constrictions. (OTKA T48376, K71591, F48837; ETT 364/2006, and AHA 0855910D, 0735540T, USA)
Coupling between cochlear fibrocytes and microvessels of spiral ligament in guinea pig cochlea
Authors: Nuttall Alfred L. , Shi Xiaorui
Published: June 2021

The dynamic regulation of oxygen and the supply of glucose to match changes in metabolic demand are critical to the maintenance of cochlear homeostasis. Although spiral modiolar artery autoregulation is one control mechanism for the reaction to systemic blood pressure and energy changes of the whole cochlea, another local mechanism may be active at the level of capillaries. With transmission electron microscopy combined with fluorescence confocal imaging, we found that fibrocytes are uniquely positioned and spatially distributed near pre‐capillaries and post‐capillaries of the spiral ligament. Inter‐connected fibrocytes were positive to Na+/K+ ATPase β1 and connexin 40. With fluo‐4, a fluorescent calcium indicator, we found that connected fibrocytes contained relatively high calcium signals in comparison with other cells in cochlear lateral wall. Photolysis of the caged divalent ion chelator NP‐EGTA in fibrocytes resulted in calcium signal propagation to neighbor cells, including the vascular cells: pericytes and endothelial cells. The connection between the fibrocytes and the vascular cells suggests a "fibro‐vascular" coupling in the cochlear microvessel system in the spiral ligament. This signal pathway may be important for the local blood flow control to meet cellular metabolic needs.

Supported: NIDCD DC 008888, NIDCDDC 00105, DC 005983
A Systems Model of HIF1α Dynamics in Ischemia
Authors: Qutub Amina Ann , Popel Aleksander S.
Published: June 2021

During ischemia, a cell responds to changing oxygen levels by activation of a transcription factor, hypoxia‐inducible factor 1 (HIF1). HIF1, in response to hypoxia, upregulates genes involved in angiogenesis, cell growth, glycolysis and inflammation. To characterize this process, we used computational modeling to predict HIF1 signaling during ischemia, on multiple biological levels.

We developed models based on extensive published experimental data on HIF1α in different microenvironments. Three intracellular models consist of ordinary differential equations that represent HIF1α hydroxylation, succinate product inhibition and reactive oxygen species effects. We also designed an experimentally‐based network model of HIF1α signaling pathways related to inflammation and angiogenesis; this model includes HIF1 modulation by NFκB, TGFβ, VEGF, and insulin. Finally, we developed a cellular rule‐based model that relates hypoxia to blood vessel formation. In these models, we studied the conditions of hypoxia and ischemia, for different durations.

Model results predict relative expression of HIF1α in ischemic environments, intracellularly and at the tissue level. Results suggest an optimal window for therapeutic intervention following the onset of ischemia; during this time, inflammatory response could be minimized, while angiogenesis would still occur. This window depends on levels of HIF1α and H2O2 intracellularly, and the modulation of signaling responses in adjacent cells and tissue.

In stroke, peripheral artery disease or wound‐healing, minimizing inflammation while maximizing angiogenesis is a means to restore health. The combined HIF1 systems models predict how this can be achieved through regulation of hypoxic pathways.
Mechanisms underlying smooth muscle Ca2+ waves in cremaster muscle arterioles
Authors: Ella Srikanth Reddy , Davis Michael J , Meininger Gerald A , Yang Yan , Dora Kim A , Hill Michael A
Published: June 2021

Vascular smooth muscle cells (VSMC) exhibit a number of spatially and temporally restricted Ca2+ signals including sparks, waves, flashes and sparklets. In the present studies Ca2+ waves were measured in VSMC of myogenically active rat arterioles with an aim to understand their mechanism and relationship to mechanical forces. Waves were measured in Fluo‐4 loaded VSMC via high‐speed confocal imaging. The number of cells exhibiting waves and their frequency increased linearly with increasing intraluminal pressure. Ca2+ waves persisted despite inhibition of myogenic tone by nifedipine (1µM), adenosine (10µM) and ML7 (10µM). Ryanodine (10µM), 2APB (30µM) a putative IP3 receptor blocker, and cyclopiazonic acid (CPA; 30µM) abolished Ca2+ waves with a critical role of the sarcoplasmic reticulum (SR). Ryanodine and CPA caused vasoconstriction while 2APB caused dilation. Thus, Ca2+ waves are not dependent on active myogenic constriction but are influenced by pressure‐induced distention/tension and cyclic release/uptake of Ca2+ by the SR. Collectively, the data are consistent with mechanical force impacting on the Ca2+ filling state of the SR prior to pressure‐dependent opening of voltage‐gated Ca2+ channels.
Blocking fast calcium‐dependent isometric force development unmasks a slow calcium‐independent contraction when porcine pulmonary artery (PPA) is depolarized
Authors: Patel Hiten M , McIntyre Nathan , Allawzi Ayed M , Kwon David M , Pelaez Nancy J
Published: June 2021

The absence of calcium in the extracellular solution ([Ca2+]o) abolishes contraction of vascular smooth muscle (VSM) in most arteries. In contrast, we find that VSM from fourth generation PPA contracts in response to high potassium depolarization in both the presence and absence of [Ca2+]o. PPA contractile response is known to involve both [Ca2+]o and intracellular sources. To test if the contraction in Krebs is due to influx of calcium, verapamil (50uM) or nifedipine (5uM) were used to inhibit voltage operated calcium channels (VOCC). Isometric force was measured in isolated PPA and the resulting rate constants were modeled using JSIM software. VOCC inhibitors reduced the contraction rate. No difference in rates was detected between contractions in calcium‐free saline containing 0.1 mM EGTA versus contraction in Krebs with VOCC inhibitors. Surprisingly, PPA contracted in response to high potassium depolarization in the presence of nifedipine to prevent calcium entry through VOCC and the SERCA inhibitor cyclopiazonic acid (10 uM CPA) to deplete calcium stores. Even a calcium ionophore, ionomycin (0.04 mM), did not affect the contraction in calcium‐free saline with EGTA. Perhaps these methods unmask a slow calcium‐independent contraction when PPA is depolarized.
Effect of Co‐culture with Pulmonary Arterial Endothelial Cells on Phenotype of Pulmonary Arterial Smooth Muscle Cells
Authors: Gairhe Salina , Bauer Natalie , Grethoffer William , McMurtry Ivan Fredrick
Published: June 2021

Pulmonary arterial smooth muscle cells (PASMCs) work in concert with pulmonary arterial endothelial cells (PAECs) to maintain the homeostasis of pulmonary arteries. Injury and pathological conditions alter PASMCs to a de‐differentiated/synthetic phenotype, most likely dependent on the endothelium. The interactions between PAECs and PASMCs and the role of these interactions in modulating PASMC phenotype are not yet understood. To this end, we co‐cultured PASMCs with either PAECs or PASMCs, as a control, on a transwell insert such that the cells can make direct contact with each other through 0.4µm pores in the membrane of the insert. After 48 hrs of co‐culture, PAECs and PASMCs were processed for confocal imaging or collected for western analysis. Morphologically, PASMCs co‐cultured with PAECs were more elongated and spindle shaped than control. Also, in co‐culture with PAECs, PASMCs showed higher expression of the contractile smooth muscle cell (SMC) markers than did control. PASMCs co‐cultured with PAEC developed a more contractile‐like phenotype and resembled the characteristics of SMCs in normal pulmonary arteries. Collectively, these results suggest that coordinated interaction between PASMCs and PAECs is important in maintaining a contractile PASMC phenotype, and future experiments will investigate the roles of paracrine factors and direct myoendothelial contact. Supported by NIH P01 HL014985.
Neurotrophins in pulmonary artery smooth muscle
Authors: Meuchel Lucas William , Gates Benjamin , Thompson Michael , Pabelick Christina M , Prakash Y. S.
Published: June 2021

Neurotrophins are growth factors traditionally associated with neuronal structure and function; however, neurotrophins and their receptors have been found throughout the lung, including airway, pulmonary artery and endothelium although their roles within the lung are still under investigation. We tested the hypothesis that brain derived neurotrophic factor (BDNF) enhances pulmonary artery constriction. In endothelium‐intact arterial rings, BDNF (acute 10nM, 1h; chronic 1nM, 18h) did not affect phenylephrine‐induced contraction, but enhanced subsequent ACh‐induced relaxation. These effects were blunted by endothelium‐denudation. In fura‐2 loaded human pulmonary artery smooth muscle cells, acute and chronic BDNF decreased the Ca2+ responses to 10 μM bradykinin. In human pulmonary artery exposed to TNFα (50 ng/mL, 18h), BDNF levels were decreased, compared to human airway, where previous studies under identical treatment resulted in increased levels. These data refute our hypothesis and suggest that neurotrophins, working via endothelial cells, enhance vasodilation in the pulmonary vasculature.

Supported by FAMRI, NIH grant UL1RR024150, and the Department of Anesthesiology, Mayo Clinic.
Caveolae and neurotrophins in pulmonary artery smooth muscle
Authors: Gates Benjamin , Thompson Michael , Meuchel Lucas , Pabelick Christina , Prakash Y S
Published: June 2021

The mechanisms underlying pulmonary hypertension are still being explored. Recent studies suggest a role for caveolae (pit‐like plasma membrane structures), although it is not entirely clear whether they enhance or blunt pulmonary hypertension. The proteins caveolins ‐1, and ‐2, and polymerase I and transcription release factor (PTRF) are essential to forming caveolae. In other tissues, growth factors called neurotrophins (e.g. brain‐derived neurotrophic factor, BDNF) are thought to affect cell proliferation and structure. This study investigated caveolins and neurotrophins in human pulmonary artery. Western analysis of artery from a patient with primary pulmonary hypertension showed decreased levels of caveolin‐1, caveolin‐2, PTRF and RhoA. Exposure of human pulmonary artery to 1 nM BDNF increased protein levels of caveolin‐1 and PTRF. Isolated pulmonary artery smooth muscle cells exposed to BDNF showed decreased [Ca2+]i response to bradykinin (10 nM) and histamine (10 μM), but no change in caveolin or PTRF levels. These data suggest that decreased caveolin levels may contribute to increased pulmonary vascular contractility, while neurotrophins (potentially by increasing caveolin levels) may alleviate excessive constriction. Further studies are required to determine whether neurotrophins affect caveolae in endothelial cells or smooth muscle.

Supported by FAMRI, NIH grant UL1RR024150, and the Department of Anesthesiology, Mayo Clinic.