bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting human solid tumor xenografts with ex vivo expanded endothelial progenitor cells
Authors: Lucas Trevor , Untergasser Gerold , Abraham Dietmar , Hofer Erhard , Gunsilius Eberhard , Aharinejad Seyedhossein
Published: June 2021

Cellular therapy involves the capability to transplant autologous, non‐transformed cell populations that migrate to sites of tumorogenic development to deliver targeted, anti‐tumor therapy. Exploiting biological processes obligatory to tumorgenicity such as angiogenesis and vasculogenesis, cellular therapies have the potential to circumvent the development of treatment resistance associated with the instability of cancer cell genomes. In this study we investigated the feasibility of homing modified, human, ex vivo expanded endothelial progenitor cells (EPC) to human prostate xenotransplants in immune deficient mice in vivo. Circulating human CD34+/CD133− EPC were isolated, expanded, infected with adenovirus, fluorescently labeled with dilC18 and injected into the blood stream of athymic nude mice bearing CRL‐2505 prostate xenografts. EPC homed to xenotransplants comprised between 1 and 3% of total tumor cells 5 days following injection as assessed by flow cytometry. Histological analysis revealed that EPC were integrated throughout the developing neovasculature but preferentially accumulated in the peritumoral region of the growing tumor. These data suggest that ex vivo expanded EPC can contribute to vasculogenesis in prostate cancer and may represent a developing tool in cancer therapy.
In Vivo Investigation of a Rabbit Model of Aortic Valve Disease
Authors: Hamilton Amanda M , Ronald John A , MacLean Kyle A , Drangova Maria , Khan Zamir , Lacefield James C , Rutt Brian K , Rogers Kem A , Boughner Derek R
Published: June 2021

Aortic valve sclerosis (AVS) is a vascular disease affecting more than 25% of the population over age 65; it advances to lethal aortic stenosis in 5% of people over age 75. Despite significant clinical consequences, no effective treatment exists other than surgical AV replacement. Long‐term low‐level cholesterol feeding in New Zealand White rabbits results in the development of AVS. In vivo magnetic resonance imaging (MRI) and ex vivo ultrasound and histological analysis were used to examine our rabbit model of AV disease. Our MRI technique allows for the examination of individual subjects in vivo. Aortic valve cusps were observed to thicken over time and diseased valves displayed regurgitant flow after 15 months on the cholesterol diet. Histological analysis of diseased valves revealed AVS with human‐like pathological characteristics including extensive thickening, lipid deposition, mineralization, and immune cell infiltration. This rabbit model, coupled with our MRI technique, allows for the serial examination of AV disease progress in individual animals, thereby enabling the investigation of disease treatments including dietary control or statin therapy. Funded by the Heart and Stroke Foundation of Ontario.
Chronic graft‐versus‐host disease and scleroderma pathology differ in endothelial cell phenotype
Authors: Fleming Jo Nadine , Shulman Howard M , Nash Richard , Johnson Pamela , Wight Thomas , Schwartz Stephen Mark
Published: June 2021

Chronic graft versus host disease (cGVHD) has been used as a model for scleroderma (SSc) and interest in research has been focused on comparing the murine models of cGVHD with SSc. The focus of this paper is to examine cGVHD in the context of structural analysis and molecular markers known to be present in SSc. We compared dermal biopsies from SSc patients, cGVHD patients and normal controls using immunohistochemistry, in situ and vascular quantification. We found that although cGVHD shares proteoglycan changes, smooth muscle pathology, and immunologoical changes (including type 1 interferon markers) that we have previously identified in SSc, there is a significant difference in the endothelial cell (EC) phenotype between these two diseases. We used three EC markers to define the endothelial cells in all three sets of biopsies. Vessel quantification showed that although SSc had a significant drop in blood vessel numbers, cGVHD did not. We have previously shown that SSc EC have lost the important canonical EC markers vWF and VE Cadherin. We used two separate methods to determine whether cGVHD lost the vWF and VE cadherin staining in the EC, and both methods showed that the EC in cGVHD retained these markers. We also found that there were areas of microvascular EC proliferation in cGVHD that were not present in the SSc tissues. We speculate that the loss of the canonical EC markers may be important for the process of rarefaction.
CD36 Plays a Role in Murine Thrombosis Induced by Vascular Injury
Authors: Li Wei , Ghosh Arunima , Chen kan , Febbraio Maria , Silverstein Roy
Published: June 2021

We previously showed that the Type B scavenger receptor, CD36 mediates platelet responses to oxidized phospholipids. Using a FeCl3‐induced injury model and CD36 knockout (KO) and wild type (WT) mice, we examined the role of CD36 in thrombosis by in vivo video‐microscopy after injection of Rhodamine 6G to label platelets. While there was no difference in time to thrombosis (TTT) in carotid arteries using 12.5% FeCl3, when a less severe injury was induced by 7.5% FeCl3, there was increased TTT in KOs (p=0.006). We also observed increased TTT in both arterioles and venules of KOs (p<0.05). Mechanistically, we previously showed that platelet signaling via CD36 was mediated by MAP kinases. Here we demonstrate a significantly elongated TTT in the carotid artery in irradiated thrombocytopenic WT mice transfused with JNK inhibitor pre‐treated WT platelets. No effect was observed using KO platelets. Isolated platelets from KOs were less sensitive to activation in vitro by low dose ADP. Immunohistochemical analysis of carotid thrombi showed a significant decrease in the endothelial cell marker CD105 in KOs, suggesting less accumulation of endothelial cell‐derived microparticles. Plasma urokinase type plasminogen activator levels were increased in KO mice after induction of thrombi. By direct injection of the superoxide indicator hydroethidine, we also found that the level of FeCl3 injury induced reactive oxygen species was lower in KOs. We conclude that CD36 plays a complex role in thrombosis via MAP kinase mediated modulation of platelets activation, promotion of reactive oxygen species formation after endothelial injury, and regulation of activation of the fibrinolytic system.
Intrinsic Differences Between In Vivo Endothelial Transcriptional Profiles of Coronary and Iliac Arteries Revealed by Microarray Analysis
Authors: Burridge Kelley Ann , Zhang Ji , Friedman Morton H
Published: June 2021

Phenotypic heterogeneity among endothelial cells (ECs) is particularly relevant to atherosclerosis since the disease occurs predominantly in major arteries, which vary in atherosusceptibility. To explore EC heterogeneity, we used DNA microarrays to compare gene expression profiles of freshly harvested porcine coronary and iliac arteries. Statistical analysis (SAM) revealed 51 genes were differentially expressed in coronary ECs relative to iliac ECs at a 5% false discovery rate. For 42 of these genes, expression levels were greater than 2 or less than 0.5. Consistent with coronary arteries being more atherosusceptible, coronary ECs overexpressed 8 genes known to be pro‐atherogenic and underexpressed 4 genes known to anti‐atherogenic. Gene set enrichment analysis was consistent with SAM findings. More genes were significantly overexpressed in coronary ECs in 10 GO categories. Categories with the largest number of differentially expressed genes were involved in system development, particularly nervous system development. The intrinsic differences found in this study may provide insight into why coronary arteries are highly atherosusceptible and may suggest potential therapeutic targets (NIH HL050442).
In‐vivo Evidence of C‐Reactive Protein‐Mediated Metalloproteinase‐9 Induction from Rat Macrophages: Molecular Insights
Authors: singh uma , Devaraj sridevi , Dasu Mohan R , Afify A , Jialal Ishwarlal
Published: June 2021

C‐reactive protein (CRP) present in higher amounts in the atherosclerotic plaques of acute coronary syndrome (ACS) patients colocalizes with macrophages. Matrix metalloproteinases (MMPs) are known to destabilize atherosclerotic plaques with a causal role of MMP‐9 in the disease. MMP‐9 and CRP correlate strongly in ACS patients. Macrophages are the major source of MMP‐9. A paucity of data exists examining the effect of CRP on MMPs in‐vivo. We examined CRP's effect on MMP‐9 activity in rats. Human (hCRP‐test) and human serum albumin (hSA‐control) injected intraperitoneally (20mg/kg b.w/3 days) led to circulating hCRP levels ∼18 mg/L in test group. Zymography revealed significantly increased MMP‐9 activity in peritoneal macrophages of hCRP rats. To elucidate mechanistic insights, hCRP/hSA (25ug/ml) was injected in the pouches formed by injecting 20 ml sterile air on the backs of rats. Zymography/western blotting (MMP‐9 activity/mass) of pouch exudates revealed increased MMP‐9 which was abrogated by injecting inhibitors to p38 MAP kinase, ERK and NFkB, but not JNK prior to hCRP injection. Further, MMP‐9 is reported to have a NFkB element in its promoter. p65 NFkB activity measured in nuclear fractions of macrophages was significantly increased in hCRP rats. To conclude, NFkB is an upstream event in CRP‐mediated MMP‐9 induction. This study presents an in‐vivo evidence of potential role of CRP in plaque instability.
Apparent pA2 analysis of 5‐HT2A receptor antagonists in rhesus monkeys discriminating DOM
Authors: Li Jun‐Xu , Rice Kenner C. , France Charles P.
Published: June 2021

Discriminative stimulus effects of 1‐(2,5‐dimethoxy‐4‐methylphenyl)‐2‐aminopropane (DOM) have been well characterized in rats and less so in other species. This study used Schild analysis and a cumulative‐dosing procedure to systematically examine the receptor mechanisms that mediate the discriminative stimulus effects of DOM in rhesus monkeys. Four rhesus monkeys discriminated between 0.32 mg/kg (s.c.) DOM and vehicle while responding under an FR 5 schedule of stimulus shock termination. DOM and the 5‐HT1A/5‐HT2A receptor agonist dipropyltryptamine (DPT) increased responding on the DOM lever. M100907 (0.001–0.01 mg/kg), ketanserin (0.01–0.1 mg/kg) and ritanserin (0.01–0.1 mg/kg) shifted the dose‐response curves of DOM and DPT rightward in a parallel manner. Schild analyses were consistent with a simple, competitive interaction and yielded a pA2 value of 8.5 for M100907 with DOM or DPT; a pA2 value of 7.7 was obtained for ketanserin and for ritanserin with DOM or DPT. These results confirm the role of 5‐HT2A receptors in the discriminative stimulus effects of DOM and extend the use of Schild analysis to this discrimination procedure in monkeys, thereby providing the basis for future studies investigating the role of particular 5‐HT receptor subtypes in the behavioral effects of DOM and related drugs. CPF is supported by RCA DA17918.
A delay discounting procedure for rhesus monkeys
Authors: Acheson Ashley , France Charles P.
Published: June 2021

Delay discounting is important in drug abuse research as drug abusers often discount delayed rewards more than non drug users; however, ethical limitations in human research necessitate complimentary studies with other species. An advantage of studying delay discounting in monkeys is that current technologies allow for imaging monkeys while they are performing behavioral tasks. Thus it would be possible to detect delay discounting related brain activation patterns and observe how behavior and activation patterns change as a result of drug exposure, provided suitable tasks are available. Two adult rhesus monkeys have been tested on a delay discounting task in which reinforcer contingencies (i.e. changes in delay to reinforcer delivery) are systematically altered. Subjects were first put on a 2‐choice task where responding on one lever produced a large juice reinforcer (1.25 ml) and responding on the other lever produced a small juice reinforcer (0.125 or 0.25 ml). Once subjects made at least 90% of responses on the lever associated with the larger reinforcer, lever contingencies were switched across days until subjects reliably made at least 90% of responses on the lever that produced that large reinforcer. Next, subjects were tested on a delay discounting task with repeated opportunities to respond for an immediate small juice reinforcer or a large juice reinforcer that was delivered after an increasing delay (0–60 s). Responding for the large reinforcer decreased with increasing delays to its delivery; both monkeys responded predominantly for the large reinforcer more when delays were removed for individual daily sessions. This procedure appears suitable for future studies to assess the effects of drugs of abuse on delay discounting and its underlying neural circuitry.
Dopamine D2/D3 receptor agonists produce antidepressant‐like effects in the rat forced swim test through co‐activation of both receptor subtypes
Authors: Jutkiewicz Emily M , Collins Gregory T , Woods James H
Published: June 2021

Dopamine D2/D3 receptor agonists have been shown to have antidepressant actions in clinical and preclinical studies; however, the dopamine receptor subtype that mediates the antidepressant effects is unknown. The present study investigated the antidepressant‐like effects of dopamine D1‐, D2‐, D3‐, and D4‐preferring agonists in the rat forced swim test and the role of different dopamine receptor subtypes in these behavioral effects. The dopamine D3‐preferring agonists (PD‐128,907, pramipexole, quinpirole, and apomorphine), the D2‐preferring agonist (sumanirole), and the dopamine D1 agonist (SKF81297), but not the D4 agonist (ABT‐724), decreased immobility in the forced swim test, indicating an antidepressant profile of action. The D2 antagonist L‐741,626 and the D3 antagonist PGO1037, but not the D1 antagonist SCH23390, attenuated the antidepressant effects of the D2/D3 agonists. These data suggest that antidepressant actions of dopamine D2/D3 agonists require the activation of both D2 and D3 receptor subtypes and blockade of either receptor subtype eliminates the antidepressant effects. This work was supported by USPHS grants DA00254, DA020669, T32 GM07767, T32 DA07267, and T32 DA07268.
Effects of food restriction on dopamine clearance and on behavioral effects of dopaminergic drugs in rats
Authors: Sevak Rajkumar , Koek Wouter , Owens Anthony W , Galli Aurelio , Daws Lynette C , France Charles P
Published: June 2021

Changes in nutritional status and in circulating insulin can affect dopamine (DA) neurotransmission. This study examined the effects of food restriction (10 g/day) on DA clearance and on behavioral effects of DA drugs such as amphetamine (locomotion, conditioned place preference [CPP]), the DA receptor agonist quinpirole (yawning), and the DA receptor antagonist raclopride (catalepsy). Because repeated treatment with amphetamine can restore DA clearance in hypoinsulinemic rats, the present study also examined the effects of amphetamine on DA clearance and on the behavioral effects of quinpirole and raclopride in food‐restricted rats. Amphetamine increased locomotion similarly in free‐feeding and in food‐restricted rats; however, amphetamine produced CPP in free‐feeding and not in food‐restricted rats. Food restriction reduced DA clearance, which was restored by repeated treatment with amphetamine or by free feeding. Food restriction reduced sensitivity to quinpirole and raclopride; normal sensitivity was restored by free feeding, and not by repeated treatment with amphetamine. These data support a role for insulin in modulating DA neurotransmission and demonstrate that the effects of drugs acting on DA systems vary markedly under different physiologic (feeding) conditions. Supported by DA17918 (CPF), DA14684 (AG), and DA018992 (LCD).
Rewiring Cocaine Addiction: N‐Acetylcysteine Reverses Impaired Plasticity After Chronic Cocaine
Authors: Moussawi Khaled , Kalivas Peter W.
Published: June 2021

Neuroplasticity in the cortico‐striatal circuits contributes to the increased vulnerability to relapse and compulsive drug seeking in cocaine addiction. The details and mechanisms of these neuroadaptations are not fully elucidated. In this study, we test the ability of prefrontal‐accumbal synapses to express long term potentiation (LTP) or long term depression (LTD) after chronic cocaine. Rats were trained to self‐administer cocaine for 10 days, followed by 3 weeks of extinction, and field potentials recorded in the nucleus accumbens after prefrontal cortex stimulation, in vivo, under urethane anesthesia. Chronic cocaine impaired both LTP and LTD in rats treated with cocaine as compared to yoked saline or naïve rats. Systemic injection of N‐acetylcysteine (NAC), shown previously to block reinstatement, restored both LTP and LTD. Group II metabotropic glutamate receptor (mGluR) antagonist LY341495 selectively inhibited the ability of NAC to restore LTP, while the mGluR5 antagonist (MPEP) selectively inhibited the ability of NAC to restore LTD. Thus, chronic cocaine impairs accumbens plasticity, possibly by altering mGluR regulation of glutamate transmission. Support provided by DA015369.
Role of phosphodiesterase 8A (PDE8A) in lipid metabolism in the liver
Authors: Shimizu‐Albergine Masami , Patrucco Enrico , Tsai Lisa , Campbell Jean S. , Beavo Joseph A.
Published: June 2021

The liver plays a pivotal role in lipid metabolism. Storage of lipids in hepatocytes is used as a means of energy preservation. However, an excess accumulation of neutral lipids in hepatocytes is often a sign of obesity, insulin resistance and type2 diabetes. Intracellular cyclic AMP levels are tightly regulated by cyclic nucleotide phosphodiesterases (PDEs) as well as its formation through the activation of adenylyl cyclase. Recent studies have demonstrated that inhibition of the cyclic AMP response element‐binding protein CREB, one mediator of cyclic AMP signaling pathways, causes increased hepatic triglyceride content, leading to a fatty liver phenotype (Herzig et al., Nature 426:190 (2003)). This study suggests that cyclic AMP/CREB pathway might regulate lipid metabolism in the liver. We have observed that PDE8A, a newly identified cAMP‐specific PDE isoform, is expressed in hepatocytes. Under fasting condition, triglyceride levels were increased in the liver of wild‐type mice, but this increase is abolished in PDE8A knockout mice. This data suggests that higher cyclic AMP levels in a compartment regulated by PDE8A can have a negative effect on lipid accumulation either through decreased lipid synthesis and/or increased lipid breakdown. We also find that hormone stimulated hepatocytes isolated from PDE8A knockout mice showed higher levels of PKA‐dependent protein phosphorylation compared to the wild‐type cells. Therefore, we speculate that the lipid accumulation in the liver is modulated by cAMP‐dependent processes that are regulated by PDE8A. Supported by NIH grant DK21723.
Human FP prostanoid receptors regulate HIF‐1α through activation of a ROS mediated non‐canonical Wnt signaling pathway
Authors: Chou Chih‐Ling , Xu Wei , Hutchinson Anthony J , Israel Davelene D , Regan John W
Published: June 2021

Hypoxia inducible factor‐1α (HIF‐1α) is an important transcription factor in the regulation of angiogenesis and is up‐regulated in many cancers. Here, we report that stimulation of the human FP receptor with prostaglandin F2α (PGF2α) activates the non‐canonical Wnt signaling by a mechanism involving the generation of the superoxide anion (O2‐). This activation of non‐canonical Wnt signaling leads to the stabilization of HIF‐1α and stimulation of luciferase reporter gene activity under the control of a hypoxia response element (HRE). Transfection of cells with a dominant negative mutant of T‐cell factor‐4 (TCF‐4) abolishes FP receptor mediated HIF‐1α up‐regulation and stimulation of HRE luciferase activity. These results provide a novel mechanism for the stimulation of angiogenesis under normoxic conditions.

Supported by EY011291
Identification of two sites of PRAS40 phosphorylated by mTOR complex 1
Authors: Wang Lifu , Harris Thurl E. , Lawrence John C.
Published: June 2021

The rapamycin‐sensitive mTOR complex 1 (mTORC1), containing mTOR, raptor and mLST8, has recently been found to associate with PRAS40 (Proline‐Rich Akt Substrate 40). PRAS40 is able to be phosphorylated directly by mTORC1 and one phosphorylation site, Ser 183, has been identified. Here, Phosphor‐peptide two‐dimensional mapping in conjunction with mutational analysis showed that in addition to Ser183, mTORC1 also phosphorylated Ser 212 and 221 in PRAS40 when assayed in vitro, and mutation of all three residues to Ala completely eliminated mTORC1‐mediated phosphorylation of PRAS40. All three sites were confirmed to be phosphorylated in vivo by 32P‐orthophosphate labeling and peptide mapping. Furthermore, we show that mutation of Ser 221 to Ala reduces the interaction with 14‐3‐3 to the same extent as mutation of Thr 246, the Akt‐phosphorylated site. It has been shown that overexpression of PRAS40 inhibits mTORC1‐mediated phosphorylation of S6K and 4E‐BP1, and mutation of Thr 246 to Ala enhances this effect. In this study we find that mutation of Ser 221 to Ala also increases the inhibitory activity of PRAS40 towards mTORC1. We hypothesize that the phosphorylations of PRAS40 by mTORC1 might be a required step to signal mTORC1 activation to downstream substrates such as S6K and 4EBP1.
Discovery of relaxant flavonols without antioxidant activity
Authors: Qin Cheng Xue , Williams Spencer J , Hughes Richard A , Woodman Owen L
Published: June 2021

3′,4′‐dihydroxyflavonol (DiOHF) is an antioxidant and vasorelaxant that reduces injury after ischaemia and reperfusion. We synthesized a series of compounds based on DiOHF with pharmacophore modifications at the 3′ and 4′ positions on the B ring and examined the biological activity for the 18 novel flavonols [4′‐substituted flavonols (H; OH; OMe; Me; Cl; CF3; C(CH3) 3); 3′‐substituted flavonols (H; OH; OMe; Me; Cl; CF3) and 4′‐hydroxy‐3′‐substituted flavonols (H; OH; OMe; Me; Br; CF3; C(CH3) 3)]. Each flavonol was examined for its ability to relax rat isolated aortic rings precontracted with KCl (30 mM). The most active flavonols (Rmax>80%) were those with a hydroxy group at the 4′ position (eg, DiOHF, pEC50=5.30±0.07, Rmax=102±1%; 4′‐OH‐3′‐OCH3 flavonol, 5.35±0.03, 98±2%; 4′‐OCH3 flavonol, Rmax=37±4%). The antioxidant activity of each flavonol was then assessed by lucigenin‐enhanced chemiluminescence and assays to assess endothelial dysfunction due to oxidant stress. With the exception of DiOHF, none of these flavonols (at 10‐5 M) were able to significantly scavenge superoxide radical or prevent pyrogallol or xanthine/xanthine oxidase‐induced endothelial dysfunction. The discovery of vasorelaxant flavonols without antioxidant activity, such as 4′‐OH‐3′‐OCH3 flavonol, will assist in investigating the mechanism of flavonol‐induced cardioprotection.
CB2 receptor agonists attenuate TNF‐α induced human vascular smooth muscle cell proliferation and migration
Authors: Rajesh Mohanraj , Mukhopadhyay Partha , Bátkai Sandor , Haskó György , Huffman John , Mackie Ken , Pacher Pal
Published: June 2021

Vascular smooth muscle proliferation and migration triggered by inflammatory stimuli are involved in the development and progression of atherosclerosis and restenosis. Cannabinoids may modulate cell proliferation in various cell types through cannabinoid 2 (CB2) receptors. Herein, we have investigated the effects of CB2 receptor agonists on TNF‐alpha‐induced proliferation, migration and signal transduction in human coronary artery smooth muscle cells (HCASMCs). HCASMCs were stimulated with TNF‐alpha and smooth muscle cell proliferation was determined by the extent of BrdU incorporation and the migration was assayed. CB2 and/or CB1 receptor expressions were determined by immunoflourescence staining, western, RT‐PCR, real‐time PCR and flow cytometry. Moderate levels of CB2 and CB1 receptors were detectable in HCASMCs compared to the very high levels of CB2 receptors expressed in THP‐1 monocytes. TNF‐alpha triggered up to ∼2.5 fold increase in CB2 receptor mRNA expression and/or protein expression in HCASMCs. Further, TNF‐alpha induced Ras, p38 MAPK, ERK ½, Akt and SAPK/JNK activation, with concordant increase in the proliferation and migration of smooth muscle cells. The CB2 agonists, JWH133 and HU308, dose‐dependently attenuated these effects of TNF‐alpha. Since the above‐mentioned TNF‐alpha‐induced phenotypic changes are critical in the initiation and progression of atherosclerosis and restenosis our findings suggest that CB2 agonists may offer a novel approach in the treatment of these pathologies by decreasing vascular smooth muscle proliferation and migration.
Molecular characterization of specific H1‐receptor agonists histaprodifen and its derivates on bovine aortic H1‐receptors
Authors: Carman‐Krzan Marija , Zorko Matjaz , Schunack Walter
Published: June 2021

We determined the molecular properties of the selective and potent H1‐receptor agonist histaprodifen, its Nα substituted analogues (methyl‐, dimethyl‐, and imidazolylethyl‐ histaprodifen (suprahistaprodifen)) and o′‐, m′‐, p′‐pyridylbutylhistaprodifens. All derivates show high affinity for 3H‐mepyramine labelled bovine aortic H1‐receptor binding sites with the folowing order of potency: suprahistaprodifen >dimethylhistaprodifen >p′‐pyridylbutylhistaprodifen >methylhistaprodifen >m'‐pyridylbutylhistaprodifen >histaprodifen >o′‐pyridylbutylhistaprodifen >histamine. Binding of histaprodifen and all derivates except dimethylhistaprodifen was differentially influenced by the addition of GTP.

All drugs, except dimethylhistaprodifen, were activators of G‐proteins. Their order of potency was suprahistaprodifen >histamine >p′‐pyridylbutylhistaprodifen >histaprodifen >m′‐pyridylbutylhistaprodifen >methylhistaprodifen >o′‐ pyridylbutylhistaprodifen. Their effect was abolished by the addition of the H1‐receptor antagonist triprolidine (10μM). Our data suggest that histaprodifens are potent H1‐receptor ligands with diverse effects in our model system. Our results for the histaprodifen and its derivates are in agreement with their agonistic nature, as previously shown in the functional studies, dimethylhistaprodifen behaved as an antagonist in our study.
Competing Effects Among Protein Kinase G, Chronic Hypoxia, and Postnatal Maturation on 5HT Agonist Affinity in Ovine Carotid Cerebral Arteries
Authors: Pearce William J , Williams James M
Published: June 2021

In ovine arteries, contractile responses to 5HT are differentially influenced by artery type, chronic hypoxia (CH), postnatal maturation (PM), and maximal Protein Kinase G activation (PKG). Here we explore the hypothesis that all of these effects involve competing changes in agonist binding affinity at the 5HT receptor (pKa). pKa was measured using the Furchgott method of partial irreversible blockade with phenoxybenzamine. In cerebral arteries, pKa was similar in fetal (6.3±0.1) and adult (6.5±0.2) arteries. CH increased pKa in both fetal (6.5±0.1) and adult (6.6±0.1) arteries. PKG activation decreased pKa in both fetal (5.8±0.2) and adult (6.3±0.1) arteries. However, PKG activation together with CH had little net effect on pKa in both fetal (6.3±0.3) and adult (6.3±0.2) arteries. Results in carotid arteries were qualitatively different: PM decreased pKa, CH increased pKa in fetal arteries but decreased it in adult arteries, and combined PKG activation and CH had little effect in fetal arteries but decreased pKa in adult arteries. In all cases, changes in pKa were closely correlated with changes in both the Emax and pD2 for 5HT. Together, these results demonstrate that PKG and CH often produce opposing effects on pKa values for 5HT. More importantly, changes in agonist binding affinity play a key role in the effects of artery type, CH, PM, and PKG on contractile responses to 5HT. Supported by NIH HD31226 and HL64867
Pharmacological Intervention Studies Reveal the Protective Role of Coronary Endothelium during the Development of Cardiac Hypertrophy
Authors: Sun Xiaowei , Ku David D.
Published: June 2021

Extensive studies have shown that chronic heart failure/hypertrophy often accompanied by abnormalities in coronary endothelial (CEC) function. In a selective right ventricular hypertrophy (RVH) rat model we noted for the first time that the right CEC function was significantly increased prior to the development of RVH, while the left coronaries in the unaffected left ventricle was not altered. As cardiac hypertrophy developed, marked depression of CEC function was noted. To further investigate the significance of CEC function during the development of RVH, we studied the effects of both early (prevention) and late (rescue) treatments with a natural product (garlic) or a standard statin (rosuvastatin, RS). Oral treatment with 1% garlic supplemented diet either 1 day before or 1 day after the induction of RVH showed significant preservation of CEC function and prevention of RVH. Similar treatments with garlics without its active ingredient, allicin, failed to produce a protective effect. Oral treatment with a water‐soluble HMG‐CoA reductase inhibitor, RS, 1 week before as well as 1 week after the induction of RVH also effectively prevented CEC dysfunction and RVH. Conversely, inhibition of CEC function with L‐NAME treatment exacerbated the development of RVH. Taken together, results of our studies demonstrate that modulation of CEC function provides a novel approach for the management of cardiac hypertrophy.
C‐Type Natriuretic peptide (CNP) release does not contribute to endothelium‐derived hyperpolarizing factor (EDHF) mediated relaxation in porcine coronary artery
Authors: Ng Kwok Fu Jacobus , Leung Susan Wai Sum , Man Ricky Ying Keung , Vanhoutte Paul M
Published: June 2021

Objective: The release of CNP accounts for EDHF‐mediated relaxations in the rat mesenteric artery1 and is Gi protein dependent2. EDHF‐mediated relaxations in the porcine coronary artery are not Gi dependent3. This study investigates whether or not CNP release accounts for EDHF‐responses in this artery.

Methods: (1) CNP concentration was measured by enzyme immunoassay in bathing solution containing porcine coronary arterial rings where EDHF response was initiated by bradykinin (100 nM). (2) EDHF‐mediated relaxations induced by SFLLRN were compared with those to exogenous CNP in the presence of phosphoramidon (10 μM; known to potentiate CNP induced relaxations).

Results: There was no significant difference in the CNP concentrations between chambers containing rings that did or did not exhibit a EDHF‐mediated response. Relaxations induced by CNP alone were potentiated by phosphoramidon (maximal relaxation 60.7 ± 7.7 % vs 84.1 ± 7.5 %, control vs phosphoramidon, P<0.05) but the EDHF‐mediated responses to SFLLRN were not (maximal relaxation 69.9 ± 9.1 % vs 72.8 ± 9.9 %, control vs phosphoramidon)

Conclusions: Unlike in the mesenteric artery and the heart of the rat, CNP release does not contribute to EDHF‐mediated relaxations in the porcine coronary artery.
Non‐genomic effects of estrogen on human coronary artery smooth muscle are mediated by nNOS via HSP90 complex
Authors: Han Guichun , Fulton David , Ma Handong , Owen Mary , Barman Scott , White Richard
Published: June 2021

Estrogen ameliorates or exacerbates coronary heart disease. Although estrogen dilates coronary arteries from a variety of species, the molecular basis for this acute, non‐genomic effect is unclear. Moreover, we know very little of how estrogen affects human coronary artery smooth muscle cells (HCASMCs). Heat shock protein 90 (HSP90) facilitates neuronal nitric oxide synthase (nNOS, type 1) activity in vivo. We propose that HSP90 is involved in estrogen signaling in HCASMC by facilitating nNOS activity. We have combined molecular and cellular functional studies with protein analysis to identify novel targets of estrogen action in HCASMCs: nNOS and HSP90. Fluorescence studies demonstrated that 17b‐estradiol (E2) increases NO production, and single‐channel patch‐clamp experiments revealed that activation of the NO/cGMP signaling pathway opens calcium‐activated potassium (BKCa) channels in these cells. Western blot revealed nNOS expression, but failed to detect eNOS or iNOS isoforms. Co‐immunoprecipitation studies revealed that E2 stimulates association of HSP90 with nNOS, whereas HSP90 inhibitors, geldanamycin or radicicol reversed the stimulatory effect of E2 on BKCa channels and on NO production, thereby implicating the nNOS/HSP90 complex as an estrogen transduction component. We conclude that estrogen opens BKCa channels in HCASMC by stimulating nNOS via a transduction sequence involving HSP90 forming a complex with nNOS. These findings provide a molecular mechanism that can explain the clinical observation that estrogen enhances coronary blood flow in patients with diseased or damaged coronary arteries.
Agonist‐induced desensitization of rat bladder relaxation by β‐adrenergic agonists
Authors: Vrydag Wim , Michel Martin C.
Published: June 2021

The overactive bladder symptom complex has a high prevalence and reduces quality of life. A potential drug target is the β‐adrenergic receptor, which mediates the detrusor smooth muscle relaxation. Since treatment with β‐adrenergic receptor agonists is symptomatic only, it requires a long term usage and could be limited by desensitization of the receptor. Therefore, we have explored the presence and possible mechanisms of such desensitization in the bladder.

Rat bladder strips were studied in an organ bath after 6 hours pre‐stimulation with isoproterenol, forskolin or vehicle. Following wash‐out relaxation curves were generated against both KCl‐induced tone and passive tension.

A 6 hour pre‐stimulation with isoproterenol desensitized the subsequent relaxation in response to isoproterenol against both KCl and passive tension, but not that to forskolin. In contrast, pre‐stimulation with forskolin did not alter the efficacy or potency of isoproterenol.

We conclude that rat bladder β‐adrenergic receptors are subject to agonist‐induced desensitization despite a proposed involvement of the relatively desensitization‐resistant β3‐subtype. Whether this also occurs with agonists currently in clinical development for the treatment of bladder dysfunction, remains to be established.
Mitochondrial glutathione (GSH) transport in diabetic nephropathy
Authors: Lash Lawrence Harold , Zhong Qing
Published: June 2021

Nephropathy is a serious complication of diabetes, and is associated with oxidative stress and altered mitochondrial function. We previously defined the function of the 2‐oxoglutarate (OGC; Slc25a11) and dicarboxylate (DIC; Slc25a10) carrier in the transport of GSH into renal mitochondria. Using a diabetic rat model (ip injection of streptozotocin (STZ) into male Sprague‐Dawley rats), we tested the hypothesis that diabetic nephropathy is characterized by mitochondrial dysfunction and decreased rates of GSH transport. We characterized mitochondrial function and GSH and dicarboxylate transport in isolated mitochondria from normal and STZ‐induced diabetic rats one month post‐injection (i.e., before the onset of nephropathy). Even at this early stage of diabetes, rates of succinate‐linked State 3 respiration, respiratory control ratios, activities of GSH‐dependent and other mitochondrial enzymes, and intramitochondrial concentrations of GSH were significantly higher in mitochondria from diabetic rats as compared to those from control rats. In contrast, kinetics of GSH and dicarboxylate transport were only slightly altered by diabetes. Mitochondria from diabetic rats, however, were markedly more susceptible than those from normal rats to oxidative injury.
Enhanced resistance to oxidative lung injury by an Nrf2‐ARE inducer in mice
Authors: Cho Hye‐Youn , Miller‐DeGraff Laura , Talalay Paul , Yamamoto Masayuki , Kleeberger Steven
Published: June 2021

Nrf2 is essential in airway protection against oxidative insults via antioxidant response element (ARE)‐mediated induction of antioxidants. To test the hypothesis that enhanced Nrf2‐ARE responsiveness protects lungs from subsequent hyperoxia (>95% O2) exposure, Nrf2+/+ and Nrf2−/− mice were fed with diet containing standardized broccoli sprout extract (SBE) or regular diet for 14 days before air or hyperoxia exposure. In a separate study, the mice were orally treated with sulforaphane (days ‐5, ‐3, ‐1) before hyperoxia exposure. In Nrf2+/+ mice, hyperoxia‐induced lung neutrophilic inflammation, protein edema, and cell necrosis were significantly decreased by SBE treatment. Hyperoxia‐induced body weight loss was significantly attenuated by SBE. Sulforaphane also significantly decreased lung inflammation caused by hyperoxia in these mice. Expression of lung Nrf2 and antioxidant mRNAs was highly elevated in Nrf2+/+ mice by sulforaphane after hyperoxia. SBE enhanced antioxidant expressions in air‐exposed mice but there was no significant effect of SBE after hyperoxia. Lung injury phenotypes of Nrf2−/− mice with suppressed antioxidant expressions were not significantly changed by SBE or sulforaphane treatment after hyperoxia. Results support a preventive role for fortified Nrf2‐ARE pathway in pulmonary oxygen toxicity. Supported by the Intramural Program of NIEHS
Genotype‐phenotype correlations for polymorphisms in cytochrome b5 and NADH cytochrome b5 reductase and hepatic sulfamethoxazole hydroxylamine reduction
Authors: Sacco James C , Trepanier Lauren A
Published: June 2021

NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX‐HA) to the parent drug sulfamethoxazole. SMX‐HA and a spontaneous nitroso metabolite are thought to contribute to sulfonamide hypersensitivity. Variability in SMX‐HA reduction could therefore contribute to the risk of sulfonamide hypersensitivity. The aim of this study was to characterize variability in SMX‐HA reduction in 100 human livers, and determine genotype‐phenotype correlations for polymorphisms in b5R and b5 cDNA. Mean SMX‐HA reduction (200 μM) was 0.59 ± 0.33 nmol/min/mg protein, with a 10‐fold range (0.11–1.23) in 70 livers to date. Vmax for livers with high reduction activity was an order of magnitude higher than livers with low reduction activity.

Three individuals were heterozygous for novel single‐nucleotide polymorphisms (SNPs) in either b5R (R59H, R297H) or b5 (S5A). The subject heterozygous for the R297H had low reductase activity (0.21 nmol/min/mg). Two previously reported synonymous b5 SNPs (C35T and G288A; frequencies: 27% and 29% in this population) were found to be in linkage disequilibrium. Work is ongoing to determine the relationship between non‐synonymous SNPs in b5R or b5 and outlier reduction activities.
Cyclic Conversion of the Novel Src Kinase Inhibitor TG100435 and Its N‐Oxide Metabolite by Flavin‐Containing Monoxygenases and Cytochrome P450 Reductase
Authors: Kousba Ahmed A , Soll Richard , Yee Shiyin , Martin Michael
Published: June 2021

TG100435 is a novel multi‐targeted Src family kinase inhibitor with demonstrated anticancer activity in preclinical species. Potent kinase inhibition is associated with TG100435 and its major N‐oxide metabolite TG100855. The objectives of the current study were to identify the hepatic enzyme(s) responsible for 1) the total metabolic flux of TG100435, 2) the formation of TG100855, and 3) the subsequent metabolism of TG100855. Flavin‐containing monooxygenases (FMO) and cytochrome P450 monooxygenases (P450s) contribute to TG100435 total metabolic flux. TG100435 metabolic flux was completely inhibited by methimazole and ketoconazole, suggesting only FMO‐ and CYP3A4‐mediated metabolism. TG100855 formation was markedly inhibited (>90%) by methimazole or heat inactivation (>99%). FMO3 was the primary enzyme responsible for TG100855 formation. In addition, an enzyme mediated retroreduction of TG100855 back to TG100435 was observed. Interestingly, the retroreduction of TG100855 to TG100435 in recombinant P450 or liver microsomes lacked inhibition by the P450 inhibitors. TG100435 formation in the human liver microsomes or recombinant P450 increased as a function of cytochrome P450 reductase activity, suggesting potential involvement of cytochrome P450 reductase. The results of this in vitro study demonstrate the potential of TG100435 and TG100855 to be interconverted metabolically. This is the first body of work demonstrating the explicit involvement of cytochrome P450 reductase activity in the xenobiotic retroreductive metabolism.
Regulation of ATP‐binding cassette (Abc) transporters by organoselenium compounds
Authors: Arch Dorinda D , El‐Sayed Wael M , Aboul‐Fadl Tarek , Roberts Jeannette C , Franklin Michael R
Published: June 2021

Selenium‐containing compounds have demonstrated chemopreventive activity in a variety of chemically‐induced tumor models, but a unifying mechanism for this effect remains elusive. Altered disposition of chemicals and toxic metabolites may be contributing factors. Following dietary ingestion of selenocysteine pro‐drugs and selenocystine (15 ppm Se) for 1 month, mouse hepatic glutathione transferase, quinone oxidoreductase and microsomal epoxide hydrolase activities were elevated by 2‐butyl‐ and 2‐oxoselenazolidine‐4‐carboxylic acids (SCAs) and selenocystine, but not by 2‐cyclohexyl‐, 2‐phenyl‐, and 2‐(2′‐hydroxyphenyl)‐ SCAs. Expression of multidrug resistance‐associated proteins and other Abc transporters were evaluated by qPCR. Of 10 transporters examined, none responded in a pattern similar to the activities of the enzymes capable of being regulated via the Nrf2/ARE mechanism. Abcc3 (Mrp3) was increased only by 2‐butylSCA and no transporters were induced by 2‐oxoSCA or selenocystine. 2‐(2′‐Hydroxyphenyl)SCA was the only compound that induced expression of Abcc2 (Mrp2) and Abcb6. All the selenocompounds reduced the expression of Abcb11. Expression of Abcc1, Abcc4, Abcc5, Abcc6, and Abcg2 and Abca3, was not altered by any compound. Selenocompound regulation of Abc transporter expression appears to be compound dependent and unrelated to the Nrf2/ARE pathway.

Support: GM 058913
The Cyclophilin CyP40 is necessary for the Ligand‐Dependent Aryl Hydrocarbon Receptor Signaling in Human Breast Cancer Cells
Authors: Luu Tony C , Bhattacharya Pompeya , Chan William K
Published: June 2021

The aryl hydrocarbon receptor (AhR) is a ligand‐activated transcription factor that heterodimerizes with AhR nuclear translocator (Arnt) to regulate expression of genes such as cyp1a1. Our previous data from in vitro protein‐DNA interaction studies revealed that a cyclophilin called CyP40 restores the ligand‐dependent AhR/Arnt/DRE complex formation. Although it has been known that CyP40 is involved in Hsp90‐dependent signaling pathways, the role of CyP40 in the AhR signaling has not been established. In order to investigate the role of CyP40 in AhR signaling, we performed CyP40 knockdown studies in MCF‐7 cells to address whether CyP40 is essential for cyp1a1 induction by an AhR ligand. Real‐time PCR data showed that when CyP40 expression in MCF‐7 cells was reduced to 30–40% of its endogenous level by two independent siRNAs, approximately 50% decrease in 3‐methylchloranthrene‐induced cyp1a1 message was observed (p <0.05). In addition, we mapped the essential CyP40 domain for restoring the AhR/Arnt/DRE complex formation by generating three CyP40 deletion constructs as 6‐His fusions. We found that the amino acid residues 186–215 were necessary for CyP40 to restore the AhR/Arnt/DRE complex formation in a gel shift assay. Data from various protein interaction studies suggest that CyP40 may interact with AhR to elicit the CyP40 effect on the AhR signaling. This work is supported by a grant from the National Institute of Health (R01ES014050).
Age‐related influences on markers of inflammation and fibrinolysis
Authors: Acree Luke Sean , Killewich Lois A. , Aston Christopher , Taylor E. Laurette , Fields David , Dinger Mary , Gardner Andrew
Published: June 2021

Purpose: The purpose of this investigation was to determine if age is associated with markers of inflammation (C‐reactive protein [CRP]) and fibrinolysis (fibrinogen, tissue plasminogen activator [t‐PA], and plasminogen activator inhibitor–1 [PAI‐1]), independent of body fat and physical activity (PA) levels. Methods: Healthy males (n=40, 22–89 years) and females (n=42, 21–82 years), who were free of confounding clinical conditions and not taking medications, participated in this cross‐sectional study. Circulating CRP, fibrinogen, t‐PA, and PAI ‐1 were measured and compared with age and subject characteristics. Body composition was assessed by dual‐energy x‐ray absorptiometry and PA was assessed with a step activity monitor. Results: Age correlated with t‐PA (r=0.228, p=0.042), but not with CRP (r=−0.073, p=0.519), fibrinogen (r=0.206, p=0.065), or PAI‐1 (r=0.084, p=0.454). Body‐mass index was correlated with CRP (r=0.304, p=0.006). Percent body fat, total fat mass, percent trunk fat, and trunk fat mass were related (p<0.05) to fibrinolytic markers. Conclusions: While markers of inflammation and fibrinolysis were associated with body fat measures, this study shows that age was associated with t‐PA but not CRP, fibrinogen, or PAI‐1. This suggests that the risks of developing cardiovascular disease associated with inflammation and impaired fibrinolysis are modifiable, not inevitable.
Macrophage Differentiation Involves Activation of the Unfolded Protein Response
Authors: Dickhout Jeffrey G. , Colgan Stephen , Hilditch Brooke , Basseri Sana , Lhoták Šárka , Sood Sudesh K. , Austin Richard C.
Published: June 2021

To determine if the unfolded protein response (UPR) is activated during macrophage differentiation and is cytoprotective to macrophages in atherosclerosis, human peripheral blood monocytes were treated with macrophage colony stimulating factor to induce differentiation. Differentiation was assessed as well as UPR activation. UPR markers upregulated during differentiation included the protein folding chaperones GRP78, GRP94 and calnexin and XBP‐1 mRNA splicing. However, the expression of these UPR markers was temporally distinct from ER stress‐induced UPR, did not saturate as further induction occurred with ER stress and did not involve oxidative stress. A dramatic increase in protein synthesis (31‐fold), measured by 35S‐ methionine incorporation, preceded UPR activation. Cytoprotection against ER stress followed UPR activation or plasmid‐mediated GRP78 overexpression. In early atherosclerotic lesions of apoE−/− mice, GRP78 was markedly increased in resident macrophages, but not monocytes. Our findings demonstrate that UPR activation occurs as a physiological response to increased protein synthesis during macrophage differentiation and is cytoprotective. This may represent an important mechanism for macrophage survival in atherosclerotic lesions, contributing to lesion development and progression. Supported by the Heart and Stroke Foundation of Ontario (T‐5385) and the CIHR (MOP‐74477).