bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of the tetratricopeptide repeat (TPR) domain of AGS3 in subcellular localization of the protein
Authors: Vural Ali , Blumer Joe B. , Lanier Stephen M.
Published: June 2021

AGS3 is a receptor‐independent activator of G‐protein signaling involved in unexpected functional diversity for G‐protein signaling systems. AGS3 has 7 TPR motifs upstream of 4 G‐protein regulatory (GPR) motifs, each of which bind and stabilize the GDP‐bound conformation of Giα. The TPR domain is postulated to play an important regulatory role in subcellular positioning of AGS3 through interaction with binding partners perhaps also influencing AGS3 interaction with G‐protein. We asked which regions of the TPR domain may be of particular importance regarding this regulatory role. We examined the subcellular distribution of a series of AGS3‐GFP constructs in COS7 cells. First, we generated constructs with progressive truncations of individual TPR motifs. Then we generated single amino acid substitutions in conserved residues in individual TPR motifs. Modification of individual TPR motifs redistributed AGS3 to punctate structures throughout the cytoplasm. These structures do not colocalize with markers for mitochondria, endoplasmic reticulum, vimentin or EEA1. TPR modifications that led to these punctate structures also increased the amount of expressed protein in the membrane fraction following non‐detergent cell lysis. The punctate structures may represent a protein degradation pathway or define a specific address within the trafficking pathway that have regulatory implications for AGS3.
Crosstalk between Ras/Raf and Tcf signaling following stimulation of the FP prostanoid receptor by prostaglandin‐F2alpha (PGF2a)
Authors: Israel Davelene Davinah , Xu Wei , Chou Chih‐Ling , Hutchinson Anthony , Regan John W
Published: June 2021

PGF2α is an autocrine/paracrine hormone that mediates its effects through the G‐protein‐coupled FP prostanoid receptor. We have previously shown that FP receptors can increase T‐cell factor (Tcf) transcriptional activation by stabilization of cytosolic β–catenin. Using HEK 293 cells stably expressing recombinant human FP receptor, and astrocytoma cells expressing endogenous FP receptors, we now show that stimulation with PGF2α activates a novel cell signaling mechanism involving the activation of Ras and B‐Raf followed by Tcf transcriptional activation. We further show by immunoblot analysis, that the activitation of this signaling pathway induces the expression of CYR61 (cysteine‐rich angiogenic protein 61) and CTGF (Connective Tissue Growth Factor). CYR61 and CTGF are members of the CCN family, which have important roles in the regulation of cell adhesion, proliferation, and survival. Both the Ras/Raf and β‐catenin‐Tcf pathways are dysregulated in cancers and suggest that the FP receptor may be an overlooked therapeutic target in the treatment of cancer. Supported by EY11291.
Regulation of the cGMP signaling pathway in MA10 cells
Authors: Henesy Michelle B , Zhu Bing , Rich Thomas C
Published: June 2021

A great deal of work has been dedicated to understanding NO‐induced cGMP signals. However, relatively little work has been focused on understanding natriuretic peptide‐induced signals. Natriuretic peptides (e.g., ANP) activate particulate guanylyl cyclases leading to increased intracellular cGMP levels. Here we characterize the molecular players regulating cGMP signals in a mouse Leydig tumor cell line, MA‐10 cells. Western blot analysis using antibodies specific for natriuretic peptide receptors A, B, and C, suggest that MA‐10 cells endogenously express NPRA and B, but do not express NPRC. The ANP dose‐dependence of cGMP accumulation are consistent with these findings. Prolonged exposure to ANP results in decreased cGMP production. This suggests the rate of cGMP hydrolysis or receptor desensitization increase with time. In addition, we have measured both phosphodiesterase (PDE) activity and cGMP accumulation in the presence of a variety of type‐specific PDE inhibitors and found that PDE5 is primarily responsible for the hydrolysis of cGMP in these cells. Based upon these and other data, we are developing a quantitative model of the cGMP signaling pathway within MA10 cells. This work was supported by AHA awards 0335084N and 0715101B.
Suppression of IgE‐mediated allergic responses by Rgs13
Authors: Bansal Geetanjali , Xie Zhihui , Rao Sudhir , Nocka Karl , Druey Kirk
Published: June 2021

Mast cells provoke allergic responses through degranulation and release of proinflammatory mediators after antigen crosslinking of the high affinity immunoglobulin E (IgE) receptor (FcƒÕRI). Regulator of G protein Signaling (RGS) proteins negatively control G‐protein‐coupled receptor‐mediated signaling through GTPase accelerating protein (GAP) activity. Here, we show that Rgs13 inhibits allergic responses by physically interacting with the regulatory p85 alpha subunit of PI3K in mast cells and disrupting its association with an FceRI‐activated scaffold complex. Rgs13¡V/¡V mice exhibited increased IgE‐mediated mast cell degranulation and anaphylaxis. Thus, apart from its regulation of GPCRs, Rgs13 inhibits immune receptor‐induced signalosome assembly in MCs. Abnormal Rgs13 expression or function may underlie some cases of idiopathic anaphylaxis or disorders of amplified MC activity.
Novel electrophilic omega‐3 fatty acid derivatives are produced by activated macrophages
Authors: Groeger Alison , Schopfer F. J. , Bonacci G. , Khoo N. , Freeman B. A.
Published: June 2021

Docosahexaenoic acid (DHA) is an ω‐3 fatty acid that reduces the risk of heart disease, colon cancer and neurocognitive disorders, with mechanisms of action including the enzymatic and free radical induced oxidation of DHA to signaling mediators. We report a new LC‐MS/MS‐based analysis that identifies the formation of novel electrophilic fatty acid derivatives (EFADs) using β‐mercaptoethanol (BME) trapping and monitoring for the neutral loss of BME (m/z 78). MS analysis of IFNγ and LPS‐activated macrophages (RAW264.7 and THP‐1) reveals that EFADs are mono‐oxidized products of DHA and docosapentaenoic acid. DHA constitutes less than 3 mol % of total fatty acid content and accounts for 90% of detected electrophilic lipids. EFAD production is dependent on the activation of prostaglandin‐H synthase‐2 and independent of phospholipase‐A2 and 5‐lipoxygenase. The formation of electrophilic fatty acids plays an important role in cell signaling by adducting to nucleophilic residues, thus modulating protein function (e.g. NFκB, Keap1/Nrf2 pathways). Here, we report previously undescribed electrophilic metabolites of ω‐3 fatty acids formed by activated macrophages that can be important as autocrine and paracrine signaling mediators during inflammation.
Impact of CaMKII Localization on Function
Authors: Mishra Shikha , Zhang Tong , Brown Joan Heller
Published: June 2021

The delta isoform of Ca2+/Calmodulin dependent protein kinase II (CaMKII) is the predominant cardiac isoform and both nuclear targeted deltaB (dB) and cytoplasmic deltaC (dC) splice variants are present in the heart. We previously reported that transgenic (TG) expression of CaMKII dB induces cardiac hypertrophy while TG expression of CaMKII dC leads to dilated cardiomyopathy and heart failure. Based on immunostaining, we suggested that localization of the two isoforms was distinct and that nuclear CaMKII affects gene expression while cytosolic CaMKII primarily affects Ca2+ handling. Surprisingly, we found that hypertrophic gene expression in vivo and in vitro was affected by both CaMKII dB and dC. Subcellular fractionation of hearts isolated from CaMKII dB and dC TG mice showed that a fraction of the CaMKII dC transgene was present in the nucleus and a fraction of CaMKII dB appeared in the cytosol. When neonatal rat ventricular myocytes were infected with CaMKII dB or dC, similar observations were made. These findings suggest that overexpression of a particular isoform may affect its cellular localization by influencing the organization of the individual subunits within the CaMKII heteromultimer. We are currently investigating the subcellular localization of agonist‐activated CaMKII and examining apoptosis as an endpoint to determine whether isoform selectivity for this response depends on its localization.
Angiotensin II‐induced migration of vascular smooth muscle cells (VSMCs) is mediated by both 72‐KDa spleen tyrosine kinase (Syk) via p38‐MAPK activated c‐Src and by ERK1/2 via c‐Src‐induced EGFR transactivation
Authors: Mugabe Benon Emmanuel , Yaghini Fariborz A , Buharalioglu Cuneyt K , Malik Kafait U
Published: June 2021

We have previously shown that angiotensin II (Ang II)‐induced VSMC hypertrophy is mediated by activation of Syk via p38‐MAPK stimulated phosphorylation of c‐Src. However, Ang II‐induced proliferation of VSMCs is mediated through transactivation of EGFR. The purpose of this study was to examine the contribution of Syk and EGFR in Ang II‐induced migration of aortic VSMCs using the wound healing technique. Ang II (200nM) treatment for 24 hours increased VSMC migration by 100% (p < 0.05). Ang II‐inducedmigration of VSMC and Syk phosphorylation, as determined by WB analysis, was inhibited by piceatannol (10μM), a tyrosine kinase inhibitor, and by transfection of VSMCs with dominantnegative (DN) but not wild type (WT) Syk plasmids. Ang II‐induced VSMC migration and Syk phosphorylation was attenuated by inhibitors of p38‐MAPK (SB202190) (20μM) and c‐Src (PP2) (10μM) and by DN c‐Src but not partially active c‐Src. Ang II‐induced VSMC migration and EGFR phosphorylation was also inhibited by EGFR blocker AG1478 (2μM). ERK1/2 inhibitor U0126 (10μM) attenuated Ang II‐induced cell migration, and ERK1/2 but not EGFR phosphorylation. c‐Src inhibitor PP2, reduced EGFR and ERK1/2 phosphorylation. These data suggest that Ang II stimulates VSMC migration via Syk through p38‐MAPK activated c‐Src, and via ERK1/2 through c‐Src induced EGFR transactivation (Supported by NIH Grant 079109 from HLBI).
Immunolocalization of KATP channel subunits in human radial artery
Authors: Tunstall Radhika R , Grazul‐Bilska Anna , O'Rourke Stephen T
Published: June 2021

Sirolimus is an antiproliferative and immunosuppressant agent that is widely used to prevent restenosis in drug eluting stents. Though little is known about its effects on vasomotor tone, previous studies in our lab demonstrated that sirolimus causes relaxation of isolated human arteries. This response is blocked by glyburide, suggesting a role for KATP channels in sirolimus‐induced vasorelaxation. To more fully explore the mechanism underlying the smooth muscle relaxing effect of sirolimus, the present study was designed to investigate the expression and localization of KATP channel subunits in human radial arteries. Radial artery segments were obtained from patients undergoing coronary artery bypass graft surgery and prepared for immunohistochemistry and immunoblot studies. Expression of Kir 6.1, Kir 6.2 and SUR 2A, SUR 2B subunit proteins was detected by Western blot analysis in human radial arteries. Confocal microscopy imaging of cryosections of human radial arteries revealed immunofluorescent labeled KATP channel subunits in the smooth muscle layer. Kir 6.1 and Kir 6.2 subunits co‐localized with SUR 2 subunits in smooth muscle cells labeled with a fluorescent antibody to smooth muscle actin. These results indicate that human radial artery smooth muscle cells express KATP channels, which may mediate the vasorelaxant effect of sirolimus in human arteries. (Supported by American Heart Association)
Effects of epoxyeicosatrienoic acids on volume‐activated chloride channels via cyclic GMP pathway in rat mesenteric artery
Authors: YANG CUI , Vanhoutte Paul M , Man Ricky YK , Leung George PH
Published: June 2021

Epoxyeicosatrienoic acids (EETs) are one of the proposed candidates of EDHF. It is known that volume‐activated chloride channels (VACCs) play important roles in regulating cell membrane potential, so we hypothesize that VACCs may be involved in EETs‐induced vasorelaxation. Rat mesenteric arteries were dissected out and the arteries rings were relaxed by acetylcholine (ACh, 1 nM to 1 mM) in the presence of L‐NAME and indomethacin. It was of interest to note that the ACh (1 μM)‐induced relaxation was reduced by about 60% when the arteries were precontracted by 60 mM KCl instead of phenylephrine (1 μM). In addition, the ACh (1 μM)‐induced relaxations were almost abolished by EETs antagonist (14, 15 epoxyeicosa‐5(Z)‐enoic acid, 1 μM). Whole cell patch clamping demonstrated that VACC currents in primary cultured rat mesenteric arterial smooth muscle cells were partly inhibited by EETs. However, the inhibition effects were abolished by cyclic GMP (cGMP) antagonists (Rp‐cGMP, 100 μM), and cGMP‐dependent protein kinase inhibitors (KT‐5823, 0.3 μM). Our results suggest that EETs‐induced relaxations of mesenteric arteries were partly mediated by inhibition of VACCs through cGMP pathway. (Supported by the Research Grant Council of Hong Kong)
Exposure to H2O2 rapidly down‐regulates L‐type calcium channels in A7r5 cells
Authors: Kharade Sujay V , Sonkusare Swapnil , Fletcher Terry W , Macmillan‐Crow Lee Ann , Rusch Nancy J
Published: June 2021

An increasing number of studies have shown that oxidative stress is an important contributor to the pathogenesis of cardiovascular diseases including hypertension. The effects of oxidant stress may include the modulation of voltage gated ion channels. For example, recent evidence suggests that reactive oxygen species (ROS) upregulate L‐type Ca2+ channels (CaL)in cardiac cells, but the impact of ROS on CaL channel expression in vascular smooth muscle cells (VSMCs) is unknown. This study was designed to decipher the effect of oxidative stress imposed by H2O2 on the expression and function of the CaL channel pore‐forming α1C subunit in VSMCs. We used A7r5 cells (derived from embryonic rat thoracic aorta), which abundantly express CaL. The cells were serum starved for 24 hrs, and treated with H2O2 (50 â€” 200 μmol/L) for 30 mins before the H2O2 was washed and the cells harvested 2 hrs later. Western blotting suggested a down‐regulation of the α1C subunits in response to H2O2 treatment. This observation correlated with preliminary patch‐clamp data, which showed a reduced CaL channel current density in H2O2 ‐treated cells (−2.4 ±0.3 pA/pF Vs −4.3 ± 0.9 pA/pF in control cells). Thus, the A7r5 cells that express CaL channels in abundance may be a useful model to define the effects of ROS on vascular CaL channel expression and function. Supported by R01 HL064806‐07 (NJR).
An ethanol extract of Polygonum multiflorum (EPM) suppresses atherogenesis in rat with atherogenic‐diet
Authors: Choi Deok Ho , Kang Dae Gill , Kim Eun Ju , Kim Hye Yoom , Kim Jin Sook , Lee Ho Sub
Published: June 2021

Hyperlipidemia and hypercholesterolemia are critical pathogenic factors for the development and maintenance of atherosclerosis. This study was designed to investigate whether the ethanol extract of Polygonum multiflorum (EPM) suppresses atherogenesis in rats with atherogenic‐diet (AD). Treatment of AD with low (100 mg/day/kg) or high (200 mg/day/kg) doses of EPM for 8 weeks markedly attenuated plasma level of triglyceride (TG) and augmented plasma level of high density lipoprotein (HDL)‐cholesterol. Treatment of EPM also lowered the systolic blood pressure (SBP) in AD‐rats. Furthermore, the impairment of acetylcholine (ACh)‐induced vascular relaxation of aortic rings in the AD‐rats was blocked by chronic treatment of EPM. In aortic tissue, treatment of EPM suppressed the AD‐induced increase in expression levels of intracellular cell adhesion molecule (ICAM)‐1, vascular cellular adhesion molecule (VCAM)‐1, and E‐selectin as well as matrix metalloproteinase (MMP)‐2. The decreased expression of endothelial nitric oxide synthase (eNOS) induced by AD was recovered by administration with EPM. Taken together, the present study suggests that chronic treatment of EPM prevents the development of atherosclerosis in the atherogenic‐diet rats.
An ethanol extract of Buddleia officinalis suppresses high glucose‐induced vascular smooth muscle cell proliferation
Authors: Lee Yun Jung , Kang Dae Gill , Moon Mi Kyoung , Cao Li Hua , Hwang Sun Mi , Kim Jin Sook , Lee Ho Sub
Published: June 2021

Diabetes mellitus is a well‐established risk factor for vascular diseases caused by atherosclerosis. In the development of diabetic atherogenesis, vascular smooth muscle cell proliferation is recognized as a key event. Thus, we aimed to investigate whether an ethanol extract of Buddleia officinalis (EBO) suppresses high glucose‐induced proliferation in primary cultured human aortic smooth muscle cells (HASMC). [3H]‐thymidine incorporation and MTT assay revealed that incubation of HASMC with high concentration of glucose (25 mM) increased cell proliferation. The expression levels of cell cycle protein were also increased by treatment with high glucose. Pretreatment of HASMC with EBO significantly attenuated the increase of high glucose‐induced cell proliferation. EBO also suppressed p38 and JNK phosphorylation. EBO suppressed high glucose‐induced matrix metalloproteinase (MMP)‐2/9 expressions and activities in dose‐dependent manner. In addition, EBO suppressed NF‐κB promoter transcriptional activity in a high glucose condition. Taken together, the present data suggested that EBO could suppress high glucose‐induced atherosclerotic processes via inhibition of p38, JNK, NF‐κB, and MMPs signal pathways in HASMC.
Shaker Kv1 channel‐MAGUK complexes are down‐regulated in cerebral arteries of hypertensive rats
Authors: Joseph Biny K , Rhee Sung W , Thakali Keshari , Rusch Nancy J
Published: June 2021

Voltage‐gated K+ (Kv) channels consist of pore‐forming α1.2 and α1.5 subunits and are an important dilator influence in rat small cerebral arteries. The presence of a class 1 PDZ binding domain in the C ‐terminus of α1.2 and α1.5 raises the possibility that these subunits bind to membrane‐associated guanylyl kinases (MAGUK). The aims of this study were to: a) determine if vascular smooth muscle cells (VSMCs) of rat cerebral arteries express the MAGUK, Post Synaptic Density 95 (PSD 95), b) determine if PSD 95 complexes with α1.2 and α1.5, and c) examine if the elevated tone of cerebral arteries of hypertensive rats correlates with a loss of PSD 95, α1.2 and α1.5. Using Western blots and confocal microscopy, we report for the first time that PSD 95 is expressed in VSMCs of rat cerebral arteries. Furthermore, coimmunoprecipitation studies suggest that α1.2 associates with PSD 95. The expression level of PSD 95 was examined in cerebral arteries of two rat models of hypertension. Compared to normotensive rats, cerebral arteries from genetically hypertensive rats and aortic‐banded rats expressed less PSD 95, and the loss of PSD 95 correlated to fewer functional Kv1 channels. Thus, cerebral arteries of hypertensive rats may have a deficit of Kv1 channel‐PSD 95 complexes, suggesting that abnormalities of vascular MAGUK scaffolding proteins are a feature of hypertension. Supported by HL‐59238‐08 (NJR) & AHA predoc 0715570Z (BKJ).
Distributions of alpha 2 adrenoceptor subtypes in porcine coronary vasculature
Authors: Ho Eva Y.W. , Leung George P.H. , Man Ricky Y.K. , Vanhoutte Paul M. , Ng K.F.J.
Published: June 2021

Alpha 2‐adrenoreceptor agonists affect vascular tone of coronary vasculature by multiple mechanisms. One of the mechanisms is the direct action on endothelial α2 adrenoreceptors (mainly α2A), which induces an endothelial‐dependent vasorelaxation via release of nitric oxide. We sought to investigate the subtypes of α2 adrenoceptors and their relative abundance in coronary blood vessels of different sizes (arteries vs arterioles). Result of RT‐PCR clearly demonstrated that mRNA of α2A and α2B adrenoceptors was present in coronary arteries but mRNA of α2C‐adrenoceptors was absent. In contrast, mRNA of α2B and α2C adrenoceptors was present in subendocardial myocardium (which contains coronary arterioles) but mRNA of α2A adrenoceptors was not. While mRNA of α2B adrenoceptors were detected in both coronary arteries and subendocardial myocardium, quantitative analysis revealed that its expression was higher in coronary arteries. Immunohistochemical studies showed that α2A and α2B adrenoceptor proteins were located mainly on the endothelial cells of coronary arteries but none was visualized in coronary arterioles. Distributions and cellular localizations of α2C adrenoceptor proteins had not yet been studied since specific antibody was not available. This preliminary data suggests that coronary blood vessels of different sizes may respond differently to pharmacological agents.
Lysosomal Targeting of Acid Sphingomyelinase in the Formation of Lipid Raft‐Redox Signaling Platforms of Coronary Arterial Endothelial Cells
Authors: Jin Si , Yi Fan , Jia Sujie , Thomas Cindy , Li Pin‐Lan
Published: June 2021

Lipid rafts (LRs) are emerging as an important cellular signaling mechanism currently. Recent studies have demonstrated that LRs are able to cluster NADPH oxidase subunits to form a LR‐redox signaling platform in response to death receptor activation. However, the mechanisms are still poorly understood. In the present study, we examined the role of a sortilin‐mediated lysosomal targeting and trafficking of acid sphingomyelinase (ASM) in bovine coronary arterial endothelial cells (CAECs). By confocal microscopy and detection of fluorescence resonance energy transfer (FRET), ASM was found to be targeted on lysosomal membrane via sortilin in response to FasL stimulation as shown by detection of ASM with sortilin or Lamp‐1, a lysosomal membrane marker. The FRET efficiency between these molecules was over 20% compared to 5% in control cells, indicating a FasL‐stimulated lysosomal targeting of ASM via sortilin. After CAECs were transfected by an siRNA of sortilin, this FasL stimulated targeting of ASM was abolished and therefore LRs clustering could not be formed, resulting in failure of LR‐redox signaling platform formation, as shown by substantial reduction of LR clusters containing NADPH oxidase subunits gp91phox or p47phox. This was also confirmed by Western blot analysis of isolated LRs membrane fractions, showing that sortilin siRNA efficiently prevented FasL‐induced translocation of gp91phox or p47phox into LR microdomains. Consequently, FasL‐induced superoxide production via NADPH oxidase in LRs clusters was also abolished by sortilin siRNA, as measured by electron spin resonance (ESR) spectrometry. It is concluded that lysosomal targeting of ASM through sortilin is present in CAECs and this targeting mechanism is attributed to the formation of LR‐redox signaling platforms in these cells in response to FasL.
Activation of AKT and endothelial nitric oxide synthase by BMP2 and BMP4 in bovine pulmonary artery endothelial cells
Authors: Bauer Philip Michael , Oran Max , Wertz Jeffrey W.
Published: June 2021

Mutations in the bone morphogenetic protein (BMP) receptor‐2 (BMPR2) are causally linked to familial pulmonary arterial hypertension (PAH). The mechanistic link between loss of BMPR2 signaling and development of PAH, however, remains unclear. Recently it was demonstrated that BMPR2 signaling promotes survival in pulmonary artery endothelial cells (ECs). Activation of the protein kinase AKT and its downstream target endothelial nitric oxide synthase (eNOS) are known to promote EC survival. We hypothesized that treatment of ECs with the BMPR2 ligands, BMP2 or BMP4, would result in activation of AKT and eNOS and result in nitric oxide (NO) release from ECs. Serum starved ECs were treated with vascular endothelial growth factor (VEGF; 50 ng/ml), BMP2 (30 ng/ml) or BMP4 (30 ng/ml) for 0, 5, 15, or 30 min. Western blot analysis revealed that both AKT and eNOS were phosphorylated in response to BMP2 and BMP4 similar to the AKT/eNOS activator VEGF. Likewise BMP2 and BMP4 treatment resulted in increased NO release from ECs as determined by nitrite accumulation in the cell culture medium. These data provide the first evidence that BMP signaling leads activation of this important survival pathway in ECs and represent a possible mechanism by which loss of BMPR2 signaling could lead to the pathogenesis of PAH. This work was supported by seed funds provided by the Department of Surgery, University of Pittsburgh School of Medicine.
Hsp90 inhibitors prevent LPS‐induced endothelial hyper‐permeability by inhibiting pp60src activation and maintaining adherens junction proteins
Authors: Chatterjee Anuran , Snead Connie , Catravas John D
Published: June 2021

Endothelial hyper‐permeability leading to vascular leak is an important consequence of sepsis and sepsis‐induced lung injury. We previously reported that heat shock protein (hsp) 90 inhibitor pretreatment improved pulmonary barrier dysfunction in a murine model of sepsis‐induced lung injury. We now examine the effects of hsp90 inhibitors on LPS‐mediated endothelial hyper‐permeability, as reflected in changes in transendothelial electrical resistance (TER) of bovine pulmonary aortic endothelial cells. Vehicle‐pretreated cells exposed to endotoxin exhibited a concentration‐dependent decrease in TER, activation of pp60Src , phosphorylation of the focal adhesion protein paxillin and reduced expression of the adherens junction (AJ) proteins, VE‐cadherin and β‐catenin. Pretreatment with the hsp90 inhibitor, radicicol prevented the decrease in TER, maintained VE‐cadherin and β‐catenin expression, inhibited activation of pp60Src and phosphorylation of paxillin. Similarly, when BPAEC hyper‐permeability was induced by endotoxin‐activated neutrophils, pretreatment of neutrophils and/or endothelial cells with radicicol protected against the activated neutrophil‐induced decrease in TER. These results suggest that the protective effects of hsp90 inhibitors include prevention of pp60Src activaton and maintenance of adherens junction proteins.
Racial differences in the inhibition of calcineurin in stimulated T cells
Authors: Gooch Jennifer L , Roberts Brian R , Kokko Kenneth E , Minshawy Osama El , Tumlin James A
Published: June 2021

The introduction of calcineurin inhibitors (CIs) greatly improved short‐term graft survival but long‐term outcomes have not significantly improved. While multiple factors can effect organ survival, as a group, African American (AA) transplant recipients suffer higher rates of rejection than other racial groups. Despite intense effort, no explanation for this difference has been identified. The aim of this study was to determine if differences in sensitivity to CIs contributes to racial disparities. T cells were isolated from control volunteers and renal transplant recipients and calcineurin stimulation and response to CIs was determined. T cell CaN activity was significantly increased in response to CD3/CD28 co‐stimulation in control but not transplant subjects. Despite comparable blood levels of FK506, T cell activation was markedly higher in AA patients compared to Caucasians (CC). Similarly, both FK506 and cyclosporin A failed to inhibit calcineurin activity in T cells of AA control subjects. Finally, increased stimulation of CaN activity was associated with elevated production of cytokines including IL‐3, IL‐4, and TGFâ. Consistent with this, AA transplant patients had significantly higher serum levels of TGFâ compared to CC. These data suggest that reduced efficacy of CIs at blocking T cell calcineurin activity may contribute to worse long‐term outcomes of AA patients.
Age‐Related Differences in Cisplatin‐Induced Renal Toxicity in Rats
Authors: Espandiari Parvaneh , Zhang Jun , Rosenzweig Barry A , Zhou Yuzhao , Vaidya Vishal S , Schnackenberg Laura , Goering Peter L , Brown Ronald P , Bonventre Joseph V , Herman Eugene H , Beger Richard D , Thompson Karol L , Hanig Joseph , Sadrieh Nakissa
Published: June 2021

A multi‐age rat model was developed and utilized to examine potential age‐dependent differences in drug‐induced renal toxicity caused by cisplatin. Male Sprague‐Dawley rats (10‐, 25‐, 40‐, and 80‐days‐old) were dosed with 0, 1, 3 and 6 mg/kg i.p. cisplatin (cip). The high dose of cip (6 mg/kg) caused: decreased body weight (all ages); decreased kidney weight/body weight (80‐days‐old); moderate to severe renal lesions (tubular necrosis and apoptosis) (all ages except 25‐days‐old); significantly increased serum BUN and creatinine levels (80‐ and 40‐days‐old). This dose also caused increases in the urinary level of the renal biomarkers: kidney injury molecule‐1 (Kim‐1), renal papillary antigen‐1 (RPA‐1) and N‐acetyl‐beta‐D‐glucosaminidase (NAG) in 40‐ (72 hrs) and 80‐ (48 and 72 hrs) day‐old rats; increases only in NAG and RPA‐1 occurred in 25‐day‐old rats (72 hrs). Significant increases in the gene expression levels of 4 nephrotoxicity biomarkers (Kim‐1, Spp1, Lcn2, Clu) were detected in the kidneys from all age groups (except for 25‐days‐old) treated with 6 mg/kg cip. In addition, similar gene expression levels were observed in 40‐ and 80‐day‐old rats given 3 mg/kg cip. Glucosuria and hydroxyprolinuria were detected on day 2 and 3 in 40‐ and 80‐day‐old rats. These findings indicate that certain biomarkers can help identify these age‐related differences in susceptibility.
Aluminum citrate treatment decreases the renal toxicity associated with ethylene glycol poisoning in Wistar rats
Authors: Besenhofer Lauren M , Cain Marie , McMartin Kenneth E
Published: June 2021

Ethylene glycol (EG) is responsible for ∼5000 poisonings a year. EG metabolites, especially calcium oxalate monohydrate (COM), produce the observed renal toxicity. Therapy from accumulated COM is needed for patients who are not diagnosed rapidly. Aluminum citrate (AC) decreases and reverses COM induced cytotoxicity in human proximal tubule (HPT) cells. The current study was designed to show that AC can decrease EG‐induced renal toxicity in vivo. Male Wistar rats were treated with EG (2 g/kg) or an equal volume of water by oral gavage. At 6 h post EG treatment, animals received either 1 mmol/kg AC by oral gavage or 0.1 mmol/kg AC by IV infusion. Urine was collected for 24 h, at which time, the right kidney was perfused with ethidium homodimer, the animal was sacrificed and the kidneys were harvested for analysis. Urine oxalate was decreased in rats treated with EG and AC compared to EG. N‐acetyl‐β‐D‐glucosaminidase (NAG) and γ‐glutamyltransferase (GGT) were measured as markers of nephrotoxicity. While GGT appeared unchanged, NAG appeared to be decreased in AC treated rats. H and E staining indicated the presence of COM crystals in all EG treated animals, but ethidium homodimer histology suggested a decrease in kidney cell necrosis of AC‐treated rats compared to EG controls. The results of this study indicate that AC may be a useful in treating the renal toxicity associated with EG poisoning.
Stereochemical aspects regarding the detoxification of the 4‐hydroxynonenal enantiomers by human glutathione S‐transferase A4‐4
Authors: Balogh Larissa Maria , Roberts Arthur G , Atkins William M
Published: June 2021

4‐Hydroxynonenal (HNE) is a toxic unsaturated aldehyde generated during lipid peroxidation that has been implicated in pathological states such as neurodegenerative diseases and cancer. HNE may spontaneously react with glutathione (GSH) to form a conjugate (GSHNE) possessing three chiral centers in the cyclic hemiacetal portion of the structure. The aim of this study is to investigate the significance of the chirality of HNE and GSHNE with respect to metabolism. Three of the GSHNE diastereomers were isolated and sets of distance restraint estimates obtained from rotating‐frame Overhauser enhancement NMR experiments were incorporated into Crystallography and NMR System‐based simulated annealing structure determinations on all the potential stereochemical configurations for GSHNE. Relative energies calculated for each combination were then used to select the best fit stereochemical configurations for each diastereomer. Physiologically, GSTA4‐4 is the predominate enzyme responsible for the conjugation of HNE and GSH. LC/MS analyses using the racemic‐ and enantioisomeric‐HNE substrates demonstrated that GSTA4‐4 conjugates GSH to HNE in a stereoselective manner that is not observed in the spontaneous reaction. Furthermore, similar experiments conducted to compare the different GST isoforms revealed that GSTA1‐1 and GSTP1‐1 are less stereoselective than GSTA4‐4.

Supported by NIH grant GM32165.
Distinct pharmacological activities associated with naturally occurring splice variants of the human constitutive androstane receptor
Authors: DeKeyser Joshua G , Bryant Sharon D , Omiecinski Curtis J
Published: June 2021

The constitutive androstane receptor (CAR/CAR1, NR1I3) responds to chemical modulators to regulate the expression of many hepatic genes, including those related to all three phases of xenobiotic biotransformation. Recent reports from our laboratory have focused on functional analysis of human CAR variants produced by alternative mRNA splicing. In particular, CAR2 and CAR3 contain insertions of 4‐ and 5‐amino acids, respectively, within their ligand binding domains. Results obtained from specific, quantitative RT‐PCR assays demonstrated that the proportions of CAR1:CAR2:CAR3 transcript levels are relatively consistent among individual hepatocyte preparations derived from 7 different human donors. On average the CAR2 insertion occurred in 28.4% of CAR transcripts and the CAR3 insertion was found in 18.3%. Luciferase reporter/transcription assays were used to differentially assess the CAR variants’ ability to activate a promoter derived from the endogenous CYP2B6 target gene. Our results demonstrate that CAR2 possesses a distinct activation profile relative to the CAR1 ligands, (5‐β)‐pregnane‐3,20‐dione, CITCO, meclizine, and clotrimazole. These data are now being used to design pharmacophores for use in ligand‐based virtual screening studies to identify selective CAR2 ligands.

Supported by NIH, GM66411.
Studies on the role of cytosolic sulfotranpsferase SULT1E1 in estrogen signaling in human CA‐HPV‐10 cells
Authors: Kapoor Ruchita , Sheng Jonathan
Published: June 2021

Human cytosolic sulfotransferase SULT1E1 catalyzes the sulfation of estrogens and estrogenic drugs in human hepatic and non‐hepatic tissues. This estrogen‐preferring sulfotransferase isoform could play a regulatory role in intracellular estrogen signaling activities. Previous studies have suggested that estrogens induce the expression of TFRC, a cell membrane transferrin receptor gene. Studies using DNA microarray and quantitative real‐time PCR methods by our group also showed that TFRC expression was down‐regulated by SULT1E1 transfection in human prostate cancer CA‐HPV‐10 cell line. Because human CA‐HPV‐10 cell line is an estrogen receptor positive cell line, the estrogen receptor‐coupled signaling pathways could relate to the molecular mechanism underlying the observed effect of SULT1E1 on TFRC expression. To test this hypothesis, the effect of SULT1E1 transfection on the estrogen receptor‐coupled signaling pathways was examined using an estrogen response element reporter gene assay in the present studies. The results showed that β‐estradiol was able to induce the estrogen response element controlled reporter gene expression in CA‐HPV‐10 cells. This β‐estradiol‐induced signal transduction process was repressed after the cells were transfected with SULT1E1. These results suggest that SULT1E1 may function as an estrogen signaling mediator in human prostate cancer CA‐HPV‐10 cells.
Coordinate Induction of ATP‐Binding Cassette (ABC) Proteins by Bile Acids via Nrf2 is Mediated by Mitogen Activated Protein Kinases (MAPK)
Authors: Tan Kah Poh Hendrick , Ito Shinya
Published: June 2021

Many liver ATP‐binding cassette (ABC) proteins can be induced by bile acids. Because these efflux transporters are capable of extruding bile salts, their induction constitutes a cytoprotective role to limit bile acid accumulation and toxicity. Nrf2 silencing via siRNA in human liver HepG2 revealed that ABC transporters such as ABCC2 (MRP2), ABCC3 (MRP3) and ABCG2 (BCRP) induced by bile acids are dependent on Nrf2. The mechanisms, particularly the involvement of signaling kinases, by which this adaptive response occurs were sought. Using a quantitative multiplex kinase activation assay, we found that lithocholic acid (LCA)‐treated HepG2 had higher phosphoproteins representing activation of mitogen‐activated protein kinase (MAPK), c‐Jun N‐terminal kinase, and phosphatidylinositol 3‐kinase pathways. Inhibition of MAPK, but not the other kinases, significantly mitigated LCA‐induced ABC transporter genes and Nrf2 target genes. Activation of Nrf2 transcription machinery by LCA was prevented by blocking of MAPK as shown by reduced nuclear accumulation of Nrf2 and antioxidant‐response element (ARE) reporter activities. Moreover, exposure to LCA in the presence of inhibitors of MAPK or ABC transporters sensitized HepG2 cells to LCA‐induced cell death. In summary, our findings suggest that MAPK signaling mediates Nrf2 activation and induction of ABC transporter genes by bile acids. (Supported by CIHR)
Establish a novel FRET‐based bioassay for detection of dioxin‐like compounds
Authors: Hsieh Chun‐Hung , Lin Chi‐Iou , Lee Hsinyu
Published: June 2021

Dioxins are group of compounds contain double aromatic ring‐like structure. They are among the most prevalent and toxic environmental pollutants. Accumulation of dioxins in human tissue poses a potential threat to human health. Currently, analytical chemical procedures dominate the dioxin detection protocols. In this study, we established a fluorescence resonance energy transfer (FRET) based dioxin‐detection bioassay. We generated plasmids encoding fusion proteins of aryl hydrocarbon receptor (AHR) and AhR nuclear translocator (ARNT) with CFP and YFP, respectively. The interactions between these two proteins are suitable for FRET experiments. In rat hepatoma cell line, H4IIEC3 cells, transiently co‐expressing AHR‐CFP and ARNT‐YFP showed no FRET signals. Dioxin treatments up‐regulated FRET signals in these transfected cells in a dose‐dependent manner. These results suggest that using FRET as a dioxin‐detection bioassay is successful and suitable for initial screening for dioxin‐like compounds.
Elevated expression of CYP4F3 associated with occupational benzene‐induced hepatotoxicity and induction of CYP4F3 by benzene metabolites in HL60 cells
Authors: Zhao Zhiwei , Bi Youngyi , Xia Ying , Tao Ning , He Xiaoqing , Ma Qiang
Published: June 2021

Benzene is an established human carcinogen and leukemogen. Although the mechanism remains uncertain, an initial metabolism and bioactivation of benzene, especially by cytochrome P450 2E1 in the liver, was considered to be a prerequisite. To further identify the other members of the CYP family associated with benzene‐induced hematotoxicity, a cDNA microarray containing 32 cytochrome P450 genes was used. Seven female shoe‐manufacture workers with different hematological disorders were recruited, and seven age‐ and gender‐matched normal subjects were selected as controls. Total RNA from the two groups was purified, and reverse transcription to cDNAs with concomitant incorporation of fluorescent dCTP. The cDNAs was then hybridized to the microarray chips. Genes with a two‐fold higher/lower expression level were considered to be significant. Our results revealed that six CYP genes (CYP4F3, CYP1A1, CYP27A1, CYP1B1, CYP2B6, and CYP51) were found to be differentially expressed between benzene‐exposed patients and controls. Among these, CYP4F3 gene, which encodes the leukotriene B4 ω‐hydroxylase, was up‐regulated consistently among all patients. To further analyze the mechanism of elevation of CYP4F3, HL60 cells were treated with benzene and metabolites. Benzene metabolite phenol induces CYP4F3 similarly to all‐trans‐retinoic acid albeit at a higher concentration, whereas hydroquinone strongly induces apoptosis. Our study indicated that CYP4F3 is up‐regulated in human peripheral blood cells that is correlated with benzene toxicity and thus, may serve as a new biomarker for exposure and poisoning of benzene.
Vitamin D receptor is expressed in human livers and inhibits CYP7A1 gene transcription
Authors: Han Shuxin , Chiang John Y. L.
Published: June 2021

Lithocholic acid (LCA) is a potent endogenous ligand of Vitamin D receptor (VDR). It is thought that VDR may induce intestinal CYP3A4 to detoxify bile acids and protective against colon cancer. It has been reported that VDR is expressed in the rat but not mouse liver. The objective of this study is to identify VDR in human hepatocytes and to test our hypothesis that VDR may inhibit transcription of the gene encoding CYP7A1, the rate‐limiting enzyme in bile acid synthesis in hepatocytes. Immunoblot and real‐time PCR analyses have detected VDR protein and mRNA expression in HepG2 and human primary hepatocytes. Confocal immunofluorescence has detected VDR in the cytoplasm of hepatocytes. Upon treatment with 1α, 25(OH)2‐VD3 or LCA, VDR is redistributed to the nucleus and cell membrane. Reporter assays showed that 1α, 25(OH)2VD3 and LCA‐acetate decreased human CYP7A1 reporter activities. The region conferring VDR inhibition was localized between −80 and −150 of the human CYP7A1 promoter. VDR decreased hepatocyte nuclear factor 4α (HNF4α) interaction with peroxisome proliferators‐activated receptor γ coactivator 1α (PGC‐1α)/glucocorticoid receptor interacting protein‐1 (GRIP‐1) and trans‐activation of CYP7A1. Co‐immunoprecipitation and GST pull down assays showed that VDR interacted with HNFα. Chromatin immunoprecipitation (CHIP) assay revealed that 1, 25(OH)2‐VD3‐activated VDR interacted with HNF4α and blocked its recruitment of PGC‐1α and GRIP‐1 to the human CYP7A1 chromatin, and resulted in inhibition of CYP7A1 gene transcription. This study suggests that VDR may play a role in protecting liver cells against bile acid toxicity and cholestatic liver injury.
Renal vasoconstrictor and natriuretic effects of acute administration of human recombinant TNF‐α (rTNFα) in mice
Authors: Shahid Mohd , Francis Joseph , Majid Dewan S A
Published: June 2021

Previous studies have implicated a role for TNFα in the pathogenesis of renal injury in hypertension and other chronic conditions. TNFα has also been shown to inhibit ENaC activity in many in‐vitro studies. However, the acute effects of TNFα on renal function in‐vivo are not yet clearly defined. We examined the renal responses to continuous infusion of rTNFα (0.33 μg/kg/min) in anesthetized mice (C57BL/6). Arterial pressure (AP) was recorded from a carotid arterial cannula. Urine was collected from a cannula inserted into the bladder. Renal blood flow (RBF) and glomerular filtration rate (GFR) were determined by PAH and inulin clearance. Following the consecutive two 30 min control urine collections, rTNFα infusion was initiated. After 15 min of stabilization, another two 30 min collections were made. rTNFα (n=6) caused decreases in RBF (7.9±0.3 to 6.4±0.3 ml/min/g) and GFR (1.04±0.06 to 0.62±0.08 ml/min/g) without altering AP. However, there were increases in urine flow (9.3±1.4 to 15.0±2.0 μl/min/g) and sodium excretion (0.8±0.3 to 1.4±0.3 μmol/min/g). These responses were not seen in animals pretreated with a TNFα blocker, etanercept (5 mg/kg, ip; 24 hr before rTNFα; n=5). Pretreatment with tempol (2μg/gm/min; i.v. started 2 hr before rTNFα; n=4) also attenuated the decreases in RBF and GFR but the renal excretory responses to rTNFα remained unaffected. These data suggest that superoxide production mediates the renal vasoconstrictor effect of TNFα while the natriuretic effect may be due to its inhibitory action on ENaC. Supported by grant RO1HL‐066432.
Effects of Fish Oil Concentrate Added to an Atherogenic Diet on Rabbit Tissue Fibrinolytic Activity
Authors: Beard Elizabeth Letitia , Bergens Bryan
Published: June 2021

New Zealand strain, male rabbits were fed atherogenic diet (AD) (2% cholesterol, 6% corn oil and 92% Purina Rabbit Chow) with added concentrated fish oil (MaxEPA), rich in omega 3 fatty acids – eicosapentanoic and docosaheanoic acid ‐ for seven weeks. Five rabbit controls (Group I) were fed AD. Five experimental rabbits (Group II) were fed AD + 2% Max EPA and five (Group III) received AD+ 5% Max EPA. Blood plasma, platelets and lysosomes from lung, liver and kidney were assayed for cholesterol, phospholipid, total protein and plasminogen activator activity (PAA) measured as % fibrin clot lysis. Mean PAA was 9.9% in Group III blood plasma vs 8.5% in Group I. PAA was significantly higher in Group I lung, liver and kidney lysosomal supernate(LS), whole lysosome(WL) and homogenized lysosome(HL) fractions vs Group II and III. Ex.‐Lung LS PAA Group I =8.5%, II =4.7%, III= 2.3%, WL PAA Group I =16.8%, II= 5.3%, III =2.8% and HL PAA Group I = 36.8%, II =3.0% and III= 1.8%. Liver and kidney lysosomal PAA was comparable to lung values. Cholesterol assays of blood plasma : Group I = 660.6, II =886.8 and III = 870.1 mean mg. %, and in platelets: Group I= 746.8, II =549.3 and III =497.5 mg% Platelet glucose uptake assays: Group I =273.6, Group II = 342.6, III =367.6 mg% glucose. Fish oil supplements may counteract PAA lysosomal retention induced by AD and improve platelet sensitivity to insulin in hypercholesterolemic animals.
The effect of chronic degradation of the endothelial glycocalyx on atherosclerosis progression in ApoE knockout mice
Authors: Meuwese Marijn Clara‐Maud , Stroes Erik Sjoerd Gerard , Vink Hans , van den Berg Bernard
Published: June 2021

The protective barrier between flowing blood and the vessel wall is formed by endothelial glycocalyx.

We investigated the effect of chronic damage to glycocalyx through enzymatic degradation of its major component hyaluronan on atherosclerosis progression.

Male ApoE‐KO mice (14 wks) received 10 U/hr of active or inactivated hyaluronidase intravenously via an osmotic minipump for 4 wks, after being on a Western diet for 6 wks. Systemic glycocalyx volume (Vg) was estimated by tracer dilution techniques using labeled erythrocytes and 40kDa dextran. We are evaluating atherosclerotic lesions in the aortic root and arch.

ApoE‐KO tolerated chronic infusion with hyaluronidase well. Within 4 wks Vg was reduced to 9.1 ± 8.9 ml/kg (n=11) as compared to Vg before infusion of 16.0 ± 4.8 ml/kg (n=12, p=0.03) and inactive hyaluronidase infusion of 12.1 ± 5.3 ml/kg (n=6, ns). Diet for 6 wks resulted in fatty streak formation within the aortic root (lesion area 0.5 ± 0.0 mm2). Infusion of hyaluronidase resulted in advanced lesions (area 1.3 ± 0.2 mm2) with intimal thickening, cholesterol accumulation and necrotic cores.

These preliminary results suggest a possible association between reduced glycocalyx and atherogenic acceleration.

Supported by NHF 2005T037 to H. Vink and 2006B08 to E. Stroes
Insulin‐enhanced proliferation and viability of human umbilical vein endothelial cells
Authors: Reilly Frank , Shrader Carl , Bailey Kelly , Konat Gregory , Cilento Eugene
Published: June 2021

This investigation is a follow‐up to our previous in vivo studies revealing that rapid stretch increases gene‐specific tissue insulin in murine skin flaps, coincident with up‐regulation of key angiogenic effectors and enhanced vascular growth and wound healing. In the current study, we used human umbilical vein endothelial cells (HUVECs) as an in vitro model system to determine the role of insulin in endothelial cell proliferation and survival. MTT‐based colorimetric analyses demonstrated that insulin enhances proliferation and viability of HUVECs. Western blots revealed that insulin induces the up‐regulation and activation of mitogenic signaling intermediates in endothelial cells. Activated insulin and insulin growth factor receptors (pIR / pIGFR), protein kinase B (pAkt308, pAkt473) and vascular endothelial growth factor (VEGF) were among the insulin‐responsive intermediates. Responses to insulin were abrogated by the inhibition of pIR / pIGFR or phosphoinositide‐3 kinase (PI3‐K), indicating that insulin‐induced proliferation and survival are mediated through IR / IGFR and PI3‐K effectors. These data provide new knowledge about the beneficial effects of insulin on vascular growth and tissue viability, providing a mechanistic link to improved blood flow and wound healing in acutely stretched skin. (Supported in part by the School of Engineering and Mineral Resources.)