bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of acute phase phosphoplipids from patients with chronic fatigue syndrome on the sodium channel in tissue culture
Authors: Campora Cara Empey , Hara Cynthia , Kuribayashi Tina , Le Huynh Diana , Pires Amanda , Imada Brian , Hokama Yoshitsugi , Yabusaki Kenichi
Published: June 2021

Acute phase phospholipids isolated from the sera of patients with clinically diagnosed chronic fatigue syndrome (CFS) were examined for effects on the sodium channel of mouse neuroblastoma cells in tissue culture after studies suggested a structural correlation between CFS lipids and the lipophilic marine toxin, ciguatoxin. The neuroblastoma cell bioassay is a directed cytotoxicity assay that records sensitive changes in the sodium channel of neuroblastoma cells in the presence of sodium channel activators or inhibitors. The ciguatoxins are characterized by their ability to cause the persistent activation of voltage‐sensitive sodium channels, leading to increased cell Na+ permeability. Ciguatera fish poisoning in humans is characterized by neurological, gastrointestinal, and cardiovascular manifestations that are similar to the symptoms of CFS.

Several lipids were examined for sodium channel activity: ciguatoxin, cholesterol, cholesterol myristate, and the phospholipids isolated from CFS patients as well as antagonists gluconate, ursodeoxycholic acid, d‐ribose, and anti‐CTX antibody. Results indicate that cholesterol and cholesterol myristate have weak but observable interactions at the sodium channel as activators, and that CTX and CFS lipids both have strong activity as sodium channel activators. However, studies revealed that none of the potential antagonists evaluated were effective at blocking the activity of the lipids at the sodium channel.

[Supported in part by National CFIDS Foundation, and the Hokama‐Yagawa Fund]
METHYLATION STATUS OF NEUTRAL ENDOPEPTIDASE GENES DOWN‐REGULATED BY DIESEL EXHAUST PARTICULATES IN HUMAN AIRWAY EPITHELIUM
Authors: Wong Simon S. , Sun Nina N. , Burgess Jefferey L. , Lantz R. Clark , Witten Mark L.
Published: June 2021

Our previous study has reported that epithelial neutral endopeptidase (NEP, a 90–110 kDa zinc‐dependent, type II integral membrane protein) expression was down‐regulated by diesel exhaust particulates (DEP) in a concentration‐dependent manner. In addition, sequence analysisof the NEP promoter reveals a CpG island in the type II promoterregion containing five Sp1 elements and juxtaposed to a region containing multiple Alu repeats. To determine the possibility thathypermethylation of the NEP promoter involved a NEP downregulation following exposure to DEP, we conducted a series of experiments in BEAS‐2B cells, including concentration‐effect and time‐course design (7 days). Methylation‐specific PCR primers were designed to discriminate between methylated and unmethylated alleles after bisulfite treatment. Our data show that there is no evidence of methylation in DNA samples either from the dose‐effect (0, 5, 10, 20, 40, 60 μg/ml DEP for 24 hours or from the time‐course designs (2, 4, and 7 days at the concentrations of 40 μg/ml DEP), suggesting that methylationof the tested NEP 5′ CpG island region could not be associated with loss of NEP expressionin epithelial cells after DEP exposure. However, a more comprehensive analysisof epithelial specimens after long‐term DEP exposure is needed to fully rule out the effect of NEP promoter methylation (Supported by HEI).
Positive regulation of cell migration and invasion by VASP via Rac1 pathway in human mammary epithelial cells
Authors: Han Guoge , Wei Lei , Zhang Yimin , Fan Biao , Wang Yongping , Dong Huimin , Zhou Xuan
Published: June 2021

Objective: To study the role of cytoskeleton associated protein vasodilator‐stimulated phosphoprotein (VASP) on migration of human mammary epithelial tumor cells and its mechanism. Methods: The expression level of VASP in HUMEC, MCF‐7 cells, MDA‐MB‐231 cells was detected by RT‐PCR and Western Blotting. The total Rac1 expression level was detected by Western Blotting and activities of Rac1 were examined by GST Pull‐down assays. The relatively high invasive MDA‐MB‐231 cells was intervened with Rac1 siRNA Expression Cassette segments. Accordingly the cells invasive migration ability was analyzed in wound‐healing assays (2D assays) and transwell invasion assays (3D assays). Results: Compared with HUMEC and MCF‐7cells, VASP was highest expressed in MDA‐MB‐231 cells at mRNA and protein level. Accordingly MDA‐MB‐231 cells revealed the highest invasive migration capacity in 2D and 3D assays. Rac1 siRNA Expression Cassette down‐regulated the mRNA and protein expression level of VASP, which caused a marked reduction in 2D and 3D migration capacity in MDA‐MB‐231 cells (P<0.01). Conclusion: The higher expression level of VASP contributed to higher invasive migration capacity of human mammary epithelial cells which was controlled by Rac1 pathway. (This work was supported by National Natural Sciences Foundation of China under Grant No.30770966)
DNMT3b overexpression contributes to a hypermethylator phenotype in human breast cancer cell lines
Authors: Risher J. Devon , Rivenbark Ashley G , Jones Wendell D. , Coleman William B.
Published: June 2021

To determine if a hypermethylation defect occurs in human breast cancer, the expression profile and promoter methylation status of 64 methylation‐sensitive genes was evaluated among 12 human breast cancer cell lines. Unsupervised cluster analysis of gene expression patterns revealed two groups of cell lines that possess distinct methylation signatures: (i) hypermethylator cell lines, and (ii) low‐frequency methylator cell lines. The hypermethylator cell lines are characterized by high rates of concurrent methylation of six genes (CDH1, CEACAM6, CST6, ESR1, LCN2, and SCNN1A), whereas the low‐frequency methylator cell lines do not methylate these genes. Hypermethylator cell lines coordinately overexpress (R=0.71) total DNMT activity (∼2‐6‐fold higher) and DNMT3b protein levels (∼2‐4‐fold higher) compared to normal breast MCF12A cells. In contrast, most low‐frequency methylator cell lines possess DNMT activity and protein levels that are indistinguishable from those of MCF12A cells. These observations combine to strongly suggest that: (a) a subset of human breast cancer cell lines express a hypermethylator phenotype related to overexpression of DNMT activity, and (b) overexpression of DNMT3b protein significantly contributes to the elevated DNMT activity observed in breast cancer cell lines that express this hypermethylator phenotype. Support: NIH CA78343.
Gene expression analysis of TLR9 in myeloid and lymphocytic leukemias
Authors: Webb Rachel N , Cruse Julius M , Lewis Robert
Published: June 2021

Previous studies by flow cytometry were directed towards determining whether the presence of several Toll‐like receptors (TLR) differed significantly in the context of leukemia compared to their presence in a normal immune system. A statistically significant increase in the expression intensity of TLR9 in CD3+ cells of leukemic samples was detected when compared to normal controls (P<0.001). The aim of the current study is to determine whether this difference at the protein level is due to differential gene expression of TLR9. Reverse transcription and real‐time PCR were performed on fourteen leukemia samples, three normal controls, and one positive control. The leukemia samples included several subtypes of both the myeloid and lymphocytic lineages. Fold changes of TLR9 relative to beta‐actin were determined for each sample and found to range from 1.9–2.3. However, the magnitude of TLR9 gene expression did not seem to coincide with leukemic status. Therefore, further analysis incorporating both a larger population of samples and evaluation of additional signaling molecules is needed to determine whether the upregulation of TLR9 in CD3+ leukemic cells might either be a response to or stimulus for the proliferation and survival of malignant hematopoietic cells.
Positive selection of arginine in α‐defensin: Attenuation of bactericidal activity by Lys→Arg substitutions is dependent on peptide primary structure
Authors: Llenado R. Alan , Weeks Colby S. , Cocco Melanie J. , Young Steven , Qu Xiaoqing , Ouellette Andre J.
Published: June 2021

Mammalian α‐defensins are endogenous cationic antimicrobial peptides that contain an average of nine times more Arg than Lys. To investigate the functional role of such high Arg content, Arg residues in the mouse Paneth cell α‐defensin cryptdin‐4 (Crp4) and rhesus myeloid α‐defensin RMAD4 were completely replaced with Lys to prepare (R/K)‐Crp4 and (R/K)‐RMAD4, respectively. Arg mutagenesis of Crp4 attenuated peptide bactericidal activity against several species and the kinetics of (R/K)‐Crp4‐mediated E. coli ML35 cell permeabilization were markedly slower than Crp4, needing a 20 min peptide exposure to detect permeabilization. In contrast to the attenuating effects of Arg to Lys replacements on Crp4, corresponding mutations in RMAD4 improved both microbicidal activity against certain bacterial species and the ability to permeabilize live E. coli. Therefore, although Arg→Lys substitutions in Crp4 delay E. coli cell permeabilization and extend exposure times required to induce bacterial cell death, comparable mutagenesis of RMAD4 does not attenuate either activity. Collectively, these findings are inconsistent with the positive selection of Arg in α‐defensins solely to confer selective advantage through superior antibacterial function. Supported by NIH grants DK044632 and AI059346.
Coronary artery endothelial phenotype differences related to atherosusceptibility emerge from a multi‐arterial site analysis in adult swine
Authors: Civelek Mete , Manduchi Elisabetta , Riley Rebecca , Grant Gregory R , Stoeckert Christian J , Davies Peter F
Published: June 2021

Atherosclerosis originates as focal arterial lesions having a predictable distribution to regions of disturbed blood flow. Endothelial cells (EC) play a key role in the initiation of lesions. Having previously reported a significant association of EC phenotype with the flow environment in a 3‐region comparison in vivo (Passerini et al, 2005), we extended this observation to 13 distinct coronary and non‐coronary regions (7 susceptible, 6 protected) in 87 adult swine. Site‐specific EC RNA was linearly amplified and hybridized to porcine microarrays. Hierarchical clustering, Pearson correlation calculations, and differential gene expression analyses identified a significant degree of heterogeneity of EC phenotypes as a function of site and hemodynamic environment. Most notably, we observed distinct coronary EC transcript profiles relative to non‐coronary regions. Within coronary artery athero‐susceptible regions, 262 features were differentially expressed relative to atheroprotected regions (25% false discovery rate). NIH DAVID functional annotation of these features identified protein metabolism, apoptosis, and anti‐oxidant activity as enriched themes. Gene Set Enrichment Analysis also identified 140 gene sets (out of 548) as upregulated, including inflammation and immune response, with core genes included within the DAVID results for these regions. Supported by the NHLBI.
Extracellular Matrix Gene Changes in Cardiac Fibroblasts Stimulated with Transforming Growth Factor
Authors: Dai Qiuxia , Escobar G. Patricia , Lindsey Merry L
Published: June 2021

The cardiac fibroblast (CFbs) is the primary cell type that responds to injury stimuli by secreting extracellular matrix (ECM). Excessive ECM accumulation results in fibrosis, which can impair left ventricular (LV) function. Therefore, understanding the connection between CFb activation and ECM accumulation will provide insight into how CFbs regulate LV function. Accordingly, we examined CFb ECM gene response to TGFβ stimulation. Primary fibroblasts were isolated from the LV of C57/BL6J mice (n=4). Paired cultures were incubated in serum free media without (control) or with TGFβ (10 ng/mL) for 24 h. The mouse ECM and adhesion molecule RT2 profiler PCR array was used to determine gene expression levels of 84 ECM and adhesion molecule genes. Of these genes, 18 were differentially expressed with TGFβ stimulation. In the 7 upregulated genes, secreted protein acidic and rich in cysteine (SPARC), was the most upregulated protein, increasing 60.2% from −1.4±0.1 to −2.3±0.1 normalized threshold cycles (ΔCt) (p<0.001). In the 11 downregulated genes, tissue inhibitor of metalloproteinase 2 (TIMP‐2) and vascular cell adhesion molecule 1 (VCAM‐1) were the most downregulated proteins, decreasing 45.8% and 48.4%, respectively. Combined, these data indicate that 10ng/mL TGFβ stimulation activates a pro‐fibrotic CFb phenotype by increasing ECM gene expression.
Characterization of dopamine D1, D2, and D3 receptor function in adult rhesus monkeys exposed to cocaine in utero
Authors: Hamilton Lindsey R , Gage H Don , Calhoun Tonya L , Nader Michael A
Published: June 2021

In fetal rhesus monkeys, prenatal cocaine exposure has been shown to increase dopamine (DA) D1 and D2 receptor densities within the striatum (Fang et al., 1997), but little is known about the long‐term effects of prenatal cocaine exposure on the DA system. In the present study, D2 receptor availability in the striatum, as measured by positron emission tomography (PET), was not different in adult rhesus monkeys that had been exposed to cocaine (n=10) in utero (Morris et al., 1996) compared to controls (n=8). The ability of a high‐efficacy D1 agonist, SKF 81297 (0.03–0.3 mg/kg), to elicit blinking was then characterized. Preliminary data (n=3) indicates that there is a dose‐dependent increase in blinking, with cocaine‐exposed monkeys appearing slightly more sensitive than control monkeys. To study the D3 receptor system, quinpirole‐induced yawning was examined. The prenatally cocaine exposed male monkeys exhibited increased sensitivity to the stimulating effects of quinpirole compared to controls. The prenatally cocaine exposed female monkeys did not differ from controls. These preliminary data suggests that gestational cocaine alters DA receptor function in adult male monkeys. This is particularly striking when one considers the prenatal exposure was over 13 years ago. Whether these neurochemical changes influence the behavioral vulnerability to self‐administer cocaine remains to be studied. DA 10584
Role of brain‐derived neurotrophic factor in the ventral tegmental area in social stress‐induced psychomotor sensitization using virus‐mediated gene‐transfer
Authors: Fanous Sanya , Bass Caroline , Ren Xianghui , Terwilliger Ernest , Hammer Ronald , Nikulina Ella
Published: June 2021

Brain‐derived neurotrophic factor (BDNF) is a known mediator of brain plasticity and is produced by dopamine neurons in the ventral tegmental area (VTA). The VTA is involved in the reinforcing effects of abused drugs, and manipulation of VTA BDNF modulates drug‐directed behavior. Sensitization, a progressive increase of locomotor response to successive drug exposures, results from neuroadaptive changes in brain thought to parallel those involved in addiction. Intermittent social stress augments locomotor response to psychostimulants, known as cross‐sensitization. Recently, we found that intermittent social stress increases VTA BDNF, which may be important for adaptations leading to cross‐sensitization. Here we use adeno‐associated virus (AAV)‐mediated gene transfer to alter VTA BDNF in male rats. We show intra‐VTA infusion of AAV‐BDNF or AAV‐siRNA(bdnf) effectively increases or decreases VTA BDNF, respectively, and increases or decreases locomotor response to amphetamine, respectively. In addition, over‐expression of VTA BDNF augmented sensitization after a single stress, and knockdown of VTA BDNF attenuated sensitization after intermittent stress. Taken together, these data indicate that VTA BDNF plays a significant role in persistent sensitization after stress, and may be important for understanding the relationship between stress and addiction
Differential behavioral and neurochemical effects of nicotine treatment in adolescent and adult rats
Authors: McQuown Susan , Dao Jasmin , Belluzzi James , Leslie Frances
Published: June 2021

Epidemiological studies have established that drug use in adolescence is initiated with tobacco and alcohol and progresses to illicit drugs. These findings have led to the hypothesis that alcohol and tobacco are ‘gateway’ drugs that sensitize reward pathways to the effects of illicit drugs like cocaine. We have previously shown that subchronic nicotine pretreatment enhances acquisition of cocaine self‐administration in adolescent, but not adult, rats (McQuown et al., 2007). The present study was designed to determine whether sensitization to cocaine's reinforcing effects was accompanied by an increase in locomotor response. In addition, we probed for underlying neurochemical changes in the dopamine system caused by this nicotine pretreatment. Male Sprague‐Dawley rats at P28 or P86 were given daily i.v. injections of nicotine or saline spaced 1 min apart for four days. At P32 and P90, one group of rats was sacrificed for radioligand binding. The second group received i.v. cocaine (2 × 0.2‐ or 0.5 mg/kg/0.1 ml) or saline infusions and were placed in a novel locomotor chamber. A third group was treated 3 days with i.v. cocaine or saline after a 30 min habituation in the locomotor box. At P36 or P95, all animals in this group were given a challenge of low dose cocaine and locomotor activity was observed. Nicotine pretreatment had no effect on locomotor response to acute cocaine‐induced locomotor activity at either age. Repeated cocaine exposure produced locomotor sensitization to both doses of cocaine in nicotine‐pretreated animals (p < 0.05). However, only nicotine‐pretreated adolescents expressed a sensitized response on challenge day. Neurochemical analyses revealed age‐specific changes in dopamine D3 receptors. Overall, these findings provide evidence of a differential effect of nicotine on adolescent and adult brain.
The forced‐swim test in juvenile rats: A model to screen antidepressant efficacy for childhood and adolescent depression
Authors: Reed Abbey L , Happe H Kevin , Petty Frederick , Bylund David B
Published: June 2021

We tested 21‐day old male Sprague‐Dawley rats in the forced‐swim test (FST) to screen compounds for antidepressant efficacy. Selective serotonin reuptake inhibitors (SSRIs) prevented behavioral despair, similar to results obtained with adult rats. Treatment with tricyclic antidepressants (TCAs) did not prevent behavioral despair in day 21 rats. This is in contrast to adult studies which show TCAs as effective for decreasing behavioral despair in the FST. The drug response in juvenile rats for the FST is similar to the response of children and adolescents to antidepressant treatment, as they respond to SSRIs but not TCAs. Novel compounds shown to have antidepressant‐like effects in adult animal models of depression have been screened using this newly developed model for possible activity in childhood and adolescent depression. The monoamine oxidase inhibitor, tranylcypromine, was able to decrease immobility and increase swimming behavior in juveniles. In adults tranylcypromine decreases immobility but increases both swimming and climbing behaviors. Other compounds effective at decreasing immobility in the adult FST were not effective in the juvenile FST. Screening drugs for antidepressant effects in the juvenile FST can narrow the focus of further efforts to develop compounds that would be useful for treating childhood and adolescent depression.

Support: MH66959(DBB); HFF(HKH).
Hypotension in RGS5‐deficient mice
Authors: Cho Hyeseon , Park Chung , Hwang Il‐Young , Han Sang‐Bae , kehrl John H.
Published: June 2021

RGS5, a potent GTPase activating protein for Gi and Gq, and a member of the RGS family proteins is highly expressed in pericytes and present in vascular smooth muscle cells. In this study, we demonstrate that targeted deletion of Rgs5 in mice induces low blood pressure that is greater in female than male mice without concomitant cardiac dysfunction. The aorta of RGS5‐deficeint mice is dilated compared to that of littermate control mice without altered vessel thickness. Consistently, phosphorylation of vasodilator‐stimulated protein and ERK are enhanced in response to sodium nitroprusside or sphingosine‐1‐phosphate in aortic vascular smooth muscle cells prepared from Rgs5−/− mice. In addition, these mice are lean compared to littermate controls. Our study along with previous studies demonstrating that the protein stability of RGS5 is likely under the control of nitric oxide via the N‐end rule pathway suggest that RGS5 may balance vascular tone by attenuating vasodilatory signaling in vivo in opposition to RGS2, another RGS family member known to inhibit GPCR‐mediated vasoconstrictor signaling. Blocking the functionor expression of RGS5 may provide an alternative approach to treat hypertension.
Regulation of G‐protein coupled receptor activities by platelet‐endothelial cell adhesion molecule, PECAM‐1
Authors: Yeh Jiunn‐chern , Otte Laura , Frangos John A
Published: June 2021

Previously, we have shown that G protein alpha subunit Gq/11 is rapidly activated in primary HUVEC upon fluid shear stress stimulation. Here we show that Gq/11 forms a complex with PECAM‐1. In order to understand the role of PECAM‐1 in this complex, we determined the critical regions of PECAM‐1 involved in this interaction. By expressing truncated forms of PECAM‐1 in HEK293 cells, we found that the cytoplasmic domain of PECAM‐1 is not required for its association with Gq/11. Domain swapping of PECAM‐1 with intracellular cell adhesion molecule 1 (ICAM‐1), a protein that does not form a complex with Gq/11, provides evidence that the extracellular domain of PECAM‐1 is critical for this interaction. This result also suggests that PECAM‐1 does not directly interact with Gq/11. Co‐expression of bradykinin receptor B2 (BKRB2), a Gq/11‐coupled receptor, with PECAM‐1 enhances PECAM‐1‐Gq/11 complex formation, suggesting an interaction between PECAM‐1 and BKRB2. Co‐immunoprecipitation experiments indicate that these two molecules indeed form a complex when expressed in HEK293 cells. Activation of ERK1/2 by bradykinin in HUVEC is enhanced when PECAM‐1 expression is inhibited by transfection of small interference RNA against PECAM‐1. Taken together, our results provide evidence of PECAM‐1 interaction with BKRB2 and of its possible role in regulating GPCR and G protein functions.

Supported by NIH grant HL40696.
Effect of elevated cAMP on macrophage differentiation
Authors: Hertz Angie L , Bender Andrew T , Smith Kimberly C , Amieux Paul S , Beavo Joseph A
Published: June 2021

Macrophages (Mφ) can be differentiated from monocytes (MO) through exposure to various cytokines, such as GM‐CSF and M‐CSF. Increased cAMP levels can have long‐lasting effects on the final phenotype of a differentiating MO, as well as more immediate effects on the inflammatory functions of activated Mφs. Previous studies using elevated cAMP have focused on the blunted inflammatory response of the treated Mφs to LPS challenge, but have not looked at the effects of cAMP on monocytic differentiation with GM‐CSF. We show here that MO to Mφ differentiation in the presence of elevated cAMP leads to a greatly altered final phenotype. This phenotype is currently being characterized using cDNA microarrays, real‐time PCR, FACS and ELISA techniques. Overall, Mφ differentiation is not affected, as indicated by no change in key surface Mφ markers. However, we see large fold increases in the message and protein levels of the CXCL family of chemokines, among other genes. Since the major cyclic nucleotide phosphodiesterase in these cells is PDE4A, a cAMP‐degrading PDE, these results are likely to have major ramifications for the use of PDE4 inhibitors as anti‐inflammatory agents.
Epac is an integrator of pro‐and anti‐fibrotic signals in cardiac fibroblasts
Authors: Aroonsakool Nakon , Yokoyama Utako , Patel Hemal H , Lai N Chin , Jennings Michelle M , Roth David M
Published: June 2021

Cyclic AMP (cAMP) regulates many biological processes but its role in tissue remodeling is not well understood. Actions of cAMP occur via protein kinase A (PKA) and by Exchange protein activated by cAMP (Epac). We hypothesized that Epac and PKA play distinct roles in cAMP‐mediated effects on cardiac fibroblasts (CF), which regulate repair and remodeling in the heart. We found that the Epac activator, Me‐cAMP, stimulated, while the PKA activator, Phe‐cAMP, inhibited CF migration, as assessed by a Boyden chamber method. siRNA‐mediated disruption of Epac1 blocked agonist‐induced migration of CF while a PKA inhibitor enhanced migration. Activation of either Epac or PKA inhibited collagen I and III mRNA expression. Pro‐fibrogenic agonists (TGFβ1, angiotensin II and endothelin) decreased Epac1 mRNA expression and Epac‐induced migration. Overexpression of Epac1 inhibited TGFβ1‐induced collagen synthesis and Epac1 protein expression decreased at the border zone of rats with myocardial infarction. In summary, 1) Epac promotes while PKA inhibits migration of CF; 2) Epac1 expression is decreased by pro‐fibrotic agonists and by myocardial infarction and 3) Increased expression of Epac1 decreases collagen synthesis. Thus, Epac1 appears to integrate pro‐and anti‐fibrotic signals in the heart.

This work was supported by grants from NIH (2P01HL066941‐060005), AHA, VA and the Ellison Medical Foundation.
A Key Role for Phosphoinositide 3‐Kinase in the Regulation of LPS‐ and TNF‐á‐induced CD44 Expression in Human Monocytic Cells
Authors: Mishra Jyoti Prasad , Mishra Sasmita , Kumar Ashok
Published: June 2021

Alteration in the levels of CD44 expression on monocytic cells by endotoxins and immunoregulatory cytokines may modulate the migratory potential of immune cells to inflammatory sites and the development of immune responses. LPS and the proinflammatory cytokine, TNF‐α act as important regulators of CD44 expression in monocytic cells. We have previously demonstrated that LPS‐ and TNF‐α‐induced CD44 expression is regulated by two independent signaling pathways in human monocytic cells (Mishra et al. JBC, 2005). LPS‐induced CD44 expression is regulated by the JNK‐activated Egr‐1 whereas CaMK‐II‐activated AP‐1 regulates TNF‐α‐induced CD44 expression. In this study, we show that PI 3‐kinase (PI3K) constitutes a key downstream component of both the signaling pathways involved in the regulation of LPS‐ and TNF‐α‐induced CD44 expression. Our results suggest that the JNK‐activated PI3K regulates LPS‐induced CD44 expression through the activation of Egr‐1 whereas TNF‐α induces CD44 expression by a distinct CaMK‐II‐activated PI3K through the activation of AP‐1. Taken together, our results suggest a critical involvement of PI3K in the regulation of LPS‐ and TNF‐α‐induced CD44 expression and hence may represent a potential therapeutic target for inhibiting CD44 expression and consequent CD44‐mediated cell migration, inflammation and autoimmune disorders. This work was supported by NSERC, Canada and OGSST.
Mammalian sprouty 2 protein accumulates during mild and prolonged hypoxic exposure
Authors: Anderson Kimberly M , Patel Tarun
Published: June 2021

Oxygen‐sensing mechanisms allow eukaryotic cells to adapt to fluctuating oxygen levels caused by cellular stresses like hypoxia. Mammalian sprouty 2 protein, a modulator of receptor tyrosine kinase activity, participates in the formation of an iron‐sulfur complex through its conserved cysteine residues. As iron‐sulfur complexes can act as sensors of oxygen as well as form the catalytic centers of enzymes, we asked the question of whether altered oxygen concentrations would affect the function, localization, and/or expression of human sprouty 2 (hSPRY2). Herein, we report that hSPRY2 protein levels were dramatically increased in stably transfected Hela cells exposed to mild hypoxic condition (3% O2) for 24 and 48 hours. While transient expression of hSPRY2 in Hela cells also yielded similar results, expression of proteins like GFP or RSK1, did not show similar increases in protein levels under hypoxia. Degradation studies indicate that the half‐life of hSPRY2 is extended during hypoxic exposure in stably transfected Hela cells. The ability of EGF to stimulate phosphorylation of ERK1/2 in hSPRY2 expressing cells remained unchanged when cells were exposed to hypoxia. However, stimulation with FGF under hypoxic conditions resulted in a shift of the peak of ERK1/2 phosphorylation to an earlier time point in hSPRY2 expressing cells. Exposing cells to the hypoxia mimetics, DMOG, DFO, and CoCl2, for 24 hrs also resulted in increased hSPRY2 levels. Moreover, incubation of cells in DMOG with the lysosomal inhibitor chloroquine resulted in an additional accumulation of hSPRY2 in these cells, suggesting that these two inhibitors additively increase hSPRY2 levels by different mechanisms.
Tanshinone I regulates TNF‐a‐induced expressions of ICAM‐1 and VCAM‐1 through different mechanisms in HUVEC
Authors: Chang Ki Churl , Nizamutdinova Irina Tsoy , Kim Hye Jung , Kim Yeong Shik , Kang Sam Sik
Published: June 2021

We investigated the effect of tanshinone I on TNF‐a‐mediated expressions of VCAM‐1 and ICAM‐1, and the possible molecular mechanisms by which tanshinone I regulates ICAM‐1 and VCAM‐1 expression. Stimulation of human umbilical vein endothelial cells (HUVEC) with TNF‐a resulted in the increase of ICAM‐1 and VCAM‐1 expressions, which were effectively inhibited by pretreatment with tanshinone I (5 ~ 50 uM). Western blot analysis using various protein kinase inhibitors showed that TNF‐a induces ICAM‐1 through p38 and ERK pathway, whereas induces VCAM‐1 through PI3K and PKC pathway as well as p38 pathway. Tanshinone I effectively abolished TNF‐a‐mediated phosphorylation of p38, ERK, PI3/Akt and PKC. Interestingly, as well as NF‐kB activation by TNF‐a tanshinone‐1 significantly inhibited TNF‐a mediated increases of IRF and GATA‐luciferase activity, which binding motifs are known to be existed in the promoter region of VCAM‐1 but not ICAM‐1. Taken together, these results suggest that tanshinone I efficiently regulates TNF‐a mediated expression of VCAM‐1 and ICAM‐1 through regulation of p38 and NF‐kB pathway in common, furthermore it suppresses ICAM‐1 expression through modulation of TNF‐a‐mediated ERK, whereas inhibition of TNF‐a induced VCAM‐1 expression is going through modulation of PI3/Akt, PKC, IRF and GATA binding activity
SHR exhibit greater ANG II induced release of Neuropeptide Y (NPY) from mesenteric bed preparations than normotensive controls
Authors: Byku Mirnela , Macarthur Heather , Westfall Thomas C
Published: June 2021

NPY is a cotransmitter with NE and ATP in sympathetic nerves. There is evidence for increased activity of the sympathetic nervous system and the renin‐angiotensin system (RAS) as well as a role for NPY in the development and maintenance of hypertension in experimental animal models and in humans. Angiotensin II (Ang II) is known to facilitate sympathetic neurotransmission, and evoke the release of NE. This effect of Ang II is enhanced in blood vessels obtained from Spontaneously Hypertensive Rats (SHR). The objective of this study was to investigate the role of Ang II on the basal and nerve stimulated overflow of NPY from the perfused mesenteric arterial bed of SHR as hypertension develops.

We observed that Ang II (0.01; 0.1 μM) facilitates basal and nerve stimulated overflow of NPY from the mesenteric arterial bed; an effect that is greater in preparations obtained from 4–6, 10–12 and 18–20 week old SHR than age matched normotensive Wistar‐Kyoto (WKY) or Sprague Dawley (SD) rats. Preparations obtained from prehypertensive (4–6 week old) SHR appear to behave similarly to those of 10–12 week old SHR with respect to Ang II induced changes in nerve stimulated NPY overflow. This facilitatory effect of Ang II appears to be mediated by the AT1 receptor. In addition, Captopril administration resulted in decreased NE overflow from SHR preparations, suggesting that the local RAS is active in this model. (Sponsored by NIH HL60260 and AHA)
Ceramide kinase mediates toll‐like receptor 4‐induced inflammation in vascular smooth muscle cells
Authors: Houck Kristy L , Fox Todd E , Kester Mark
Published: June 2021

Sphingolipid metabolites have recently been implicated in toll‐like receptor 4 (TLR4) signaling. Activation of lipopolysaccharide (LPS)‐induced TLR4 signaling is dependent upon production of ceramide by acid sphingomyelinase (Cuschieri et al, Surg Infect, 2007). Yet, the role of ceramide metabolites to contribute to TLR4‐induced inflammatory responses has not been investigated. Specifically, we now hypothesize that TLR4‐induced proliferation and inflammation in vascular smooth muscle cells (VSMC) is dependent upon activated ceramide kinase (CERK) and resulting production of ceramide‐1‐phosphate (C1P). We demonstrate that LPS treatment of A7r5 VSMC leads to elevated CERK mRNA and protein expression. This increased expression of CERK correlates with increased LPS‐induced C1P mass, quantified by LC/MS/MS analysis. We show that LPS‐ and ceramide‐1‐phosphate‐, but not ceramide‐treatment, leads to hallmarks of vascular inflammatory disease, including increased VSMC proliferation, positive remodeling, and production of inflammatory mediators. In addition, LPS‐treated mouse blood samples had increased levels of ceramide‐1‐phosphate at the expense of ceramide. Taken together, our studies implicate ceramide‐1‐phosphate as a potential biomarker and /or mediator of vascular inflammation in diseases such as atherosclerosis or restenosis after angioplasty. These studies were funded, in part, by NIH RO1 HL076789 to MK.
The hydrogen sulfide donor IK‐1001 stimulates neovascularization and improves wound healing
Authors: Pyiochou Anastasia , Papapetropoulos Andreas , Olah Gabor , Wintner Edward , Jeschke Marc , Branski Ludwik , Herndon David , Traber Daniel , Szabo Csaba
Published: June 2021

Here we studied the effects of the novel biological mediator hydrogen sulfide (H2S) on angiogenesis and wound healing. We have utilized IK‐1001, an aqueous formulation of H2S. Treatment of chicken chorioallantoic membranes with H2S (0.24–240 pmol/egg) increased vascular length. In vitro, incubation of human umbilical vein endothelial cells with H2S (6 uM to 600 uM concentration) promoted cell growth. In addition, treatment of the cells with H2S (60 uM) increased their migration and capillary morphogenesis on matrigel. We have next tested the potential effect of hydrogen sulfide in a wound healing model. Rats received a 30% total body surface area dorsal full‐thickness scald burn under deep anesthesia. Starting at 48h post burn, the animals received daily subcutaneous injections at four equally spaced sites in the transition zone between burn eschar and healthy tissue. IK‐1001 (0.1 mg per injection) or saline control was used at each time point. After four weeks, the burn eschar was removed and the remaining wound area as well as the degree of re‐epithelialization determined. Re‐epithelialization was improved in the presence of hydrogen sulfide (24±2% vs. 16±5%, p<0.02). Our results demonstrate the pro‐angiogenetic and wound healing promoting effects of hydrogen sulfide.
Epigallocatechin‐3‐O‐gallate inhibits the angiotensin II‐induced adhesion molecule expression in human umbilical vein endothelial cell via inhibition of MAPK pathways
Authors: Ahn Hee Yul , Chae Yeon Jeong , Kim Chan Hyung , Sachinidis Agapios
Published: June 2021

We investigated the mechanism(s) by which epigallocatehin‐3‐O‐gallate (EGCG), the main catechin, which is derived from green tea, inhibits angiotensin II (Ang II)‐induced elevation of the membrane associated vascular cell adhesion molecule (VCAM)‐1 and intercellular adhesion molecule (ICAM)‐1 in human umbilical vein endothelial cells (HUVEC). Ang II induced a 40% increase of VCAM‐1 and ICAM‐1 in the plasma membrane amount. EGCG (10 to 50 μM) inhibited the effect of Ang II in a dose‐dependent manner toward to control levels. In parallel, the Ang II‐induced elevation of the mRNA expressions of VCAM‐1 and ICAM‐1 in HUVEC was completely inhibited by 50 μM EGCG. On the other hand, EGCG (30 to 50 μM) completely inhibited the Ang II‐induced phosphorylation of ERK (extracellular signal‐regulated kinase) 1/2 and p38 MAPK. In conclusion, EGCG inhibits the Ang II‐induced elevation of VCAM‐1 and ICAM‐1 in the HUVEC plasma membranes via inhibition of the p38 MAPK and the ERK1/2 signaling pathways resulting in an inhibition of the VCAM‐1 and ICAM‐1 transcription.
Role of Ang AT1a receptors in the ACE and cardiovascular responses to a fructose diet
Authors: Cunha Tatiana Sousa , Elased Khalid M , Farah Vera , Morris Mariana
Published: June 2021

A high fructose diet is associated with cardiovascular dysfunction which may be related to activation of the renin angiotensin system. The aim was to test a high fructose diet in mice lacking Ang AT1a receptors, measuring the changes in mean arterial pressure (MAP) and ACE activity. AT1a knockout (KO‐C) and wild type (WT‐C) mice were fed a standard diet (Control). AT1a knockout (KO‐F) and wild type (WT‐F) mice were fed a fructose diet (60% fructose). Blood pressure recordings were made at week 8 and blood was collected. ACE activity was determined in plasma (1 ìL) using a mass spectroscopic (MS) based proteolytic assay. Fructose increased MAP in WT‐F (8% increase vs. WT‐C). Plasma ACE activity was increased in WT‐F as compared to the other groups (1.4±0.3; 0.8±0.1; 0.6±0.06; 0.5±0.06 Ang II/Ang I; WT‐F, WT‐C, KO‐F, KO‐C, respectively). The fructose diet decreased MAP in the KO (24% decrease vs. KO‐C). In the presence of an intact Ang AT1a receptor system, fructose increased ACE and MAP. In the gene deletion model, there was hypotension with no changes in ACE. Results document important interactions between the genetics and diet with an association between plasma ACE and blood pressure.
Reduction of renal interstitial fibrosis and protection of renal function by pentoxifylline in chronic partial ureteral obstruction
Authors: Ma Hongbao , Lee Stephen , Nair Sreekumar , Chou Shyan‐Yih
Published: June 2021

Tubulointerstitial fibrosis (TIF) is a hallmark of progressive renal disease from diverse etiologies and is a reliable marker of disease severity. In the present study, we examined whether pentoxifylline (PTF), a phosphodiesterase inhibitor, reduced TIF in a rat model of chronic partial ureteral obstruction (PUO). Male Sprague‐Dawley rats underwent removal of the right kidney and diversion of the left ureter into the psoas muscle which was split and reapproximated to produce retroperitoneal fibrosis and PUO. Studies were performed in sham‐PUO rats and 30‐day PUO rats receiving PTF (400 mg/liter in drinking water) or no PTF (control) (4 rats in each group). Intravenous pyelogram showed hydronephrosis in PUO rats. Western blot showed that the α‐smooth muscle actin expression (an indicator of renal fibrosis) in the medulla was 1.16±0.40 (densitometry unit) in control rats, significantly greater than 0.37±0.03 in PTF‐treated rats. Histological examination revealed less TIF in kidneys in PTF‐treated rats than in control rats. GFR in PTF‐treated rats was 0.89±0.04 ml/min/g, greater than 0.24±0.16 ml/min/g in control rats (p<0.001). In conclusion, PTF treatment ameliorated renal fibrosis and helped preserve renal function in a rodent model of PUO.
Analysis of mRNA for ABC‐, SLC‐ and SLCO‐transporter in human respiratory epithelial cells
Authors: Ehrhardt Carsten , Gumbleton Mark , Endter Sibylle
Published: June 2021

The role of transporter proteins in drug disposition in the lung remains mostly unknown to date. Here, RT‐PCR is used to determine the presence of mRNA transcripts for drug transporters in human lung epithelial cell models. In the cell lines, A549, Calu‐3 and 16HBE14o‐, expression of mRNA transcripts for all ABC‐transporters tested, except for MRP8, was found, while in hBEpC and hAEpC, mRNA transcripts for all ABC‐transporters were present. When the cell lines were examined for expression of SLC‐transporters, all tested cells were found positive for OCT1, OCTN2 and PEPT2. PEPT1, OCTN1 and OAT4 expression was noted in all cells, except hBEpC. OCT3 was positively identified in A549, Calu‐3 and hBEpC. None of the cells showed positive transcripts for OCT2, OAT1, ‐2 and ‐3. Levels of SLCO transcripts varied between all cell lines tested, although none were found in 16HBE14o‐. Transcripts for OATP3A1 and OATP4A1 were found in all other cell lines. Additionally to A549 and Calu‐3, mRNA for OATP5A1 and OATP1B3 was found in BEAS‐2B and for OATP1A2 in BEAS‐2B and hBEpC, whereas mRNA for OATP1B1 and OATP1C1 was only detected in A549 and Calu‐3. This study shows that there are significant differences in the expression pattern of ABC‐, SLC‐ and SLCO‐transporters between cell types of the respiratory epithelium. These data are essential for the appropriate use and interpretation of in vitro data obtained with the described cell cultures.
Induction of human CYP4A11 gene expression by fasting or peroxisome proliferator activated receptor alpha (PPARa) agonists in transgenic mice
Authors: Savas Üzen , Machemer Daniel E. , Hsu Mei‐ H. , Gaynor Pryce , Lasker Jerome M. , Tukey Robert H. , Johnson Eric F.
Published: June 2021

Transgenic mice (CYP4A11‐Tg) were generated to examine regulation of the human CYP4A11 gene by PPARa agonists and fasting in vivo. Expression of the CYP4A11 transgene in mice gave liver and kidney CYP4A11 protein levels similar to those found in the corresponding human tissues. Fasted CYP4A11‐Tg exhibited a 2‐ to 3‐fold increase in hepatic CYP4A11 mRNA and protein as measured by qPCR and Western blots, respectively. Treatment with PPARa agonists, fenofibrate or clofibric acid, produced 2‐ to 4‐fold increases in hepatic CYP4A11 mRNA and protein. Collectively, these data show that fasting and PPARa agonists enhance CYP4A11 gene expression in CYP4A11‐Tg. The role of PPARa in this process was then tested in CYP4A11‐Tg that carried PPARa−/+ or PPARa−/− genotypes. Hepatic P450 4A11 protein and mRNA were induced 2‐fold by fenofibrate in PPARa−/+ CYP4A11‐Tg. Constitutive CYP4A11 levels were dramatically reduced (> 95%) in PPARa−/− females compared to PPARa−/+ females, whereas male PPARa−/− CYP4A11‐Tg retained >60% of the levels found in PPARa−/+ males. As neither CYP4A11 mRNA nor protein was inducible by fenofibrate in male or female PPARa−/− CYP4A11‐Tg, our data suggest that PPARa mediates induction of the CYP4A11 transgene by fenofibrate. Supported by NIH grants HD04445 (EFJ), GM49135 (RHT), and AA07842 (JML).
Pancreatic Tissue Distribution, Pharmacokinetics and Toxicity of Bioactive delta‐tocotrienol, a Potential Anti‐tumor Bioactive Food Component, in Mice
Authors: Husain Kazim , Hutchinson Sean Z , Francois Rony A , Neuger Anthony M , Lush Richard , Sebti Said , Malafa Mokenge P
Published: June 2021

Tocotrienols are natural vitamin E compounds in foods such as cereal. They have been shown to inhibit cancer cell growth and survival. Recently, we observed that δ‐tocotrienol is the most active tocotrienol against pancreatic cancer. The objective of this study was to determine the oral bioavailability of δ‐tocotrienol in mice pancreas and in the tumor. Mice (4–5 weeks old) were treated orally with vehicle (PBS/ethanol extracted olive oil), 50 mg/kg or 100 mg/kg dose of δ‐tocotrienol. Plasma and tissues were isolated at different time points and δ‐tocotrienol was analyzed by HPLC using fluorescence detection. The results show that the peak plasma concentration (Cmax) was 57 μM and the Tmax was 2 h. The maximum concentration of δ‐Tocotrienol was achieved at 2 h in the liver and at 8 h in the pancreas. The δ‐tocotrienol was cleared from plasma and liver within 24 h but had delayed clearance from the pancreas. Interestingly we found that δ‐tocotrienol was 10‐fold more concentrated in the pancreas than in the tumor tissue. We also observed no toxicity due to δ‐tocotrienol administration as documented by normal weight gain of the mice with no mortality. Our data suggests that δ‐tocotrienol is safe, and orally bioavailable in the pancreatic tissue of mice at levels which predicts bioactivity against neoplastic cells. We conclude that δ‐tocotrienol should be investigated for the prevention and treatment of pancreatic cancer.
Interaction of TNF‐α with Angiotensin II contributes to mitochondrial oxidative stress and end organ damage in rats
Authors: Mariappan Nithya , Elks Carrie , Prejean Kayla , Sriramula Srinivas , Francis Joseph
Published: June 2021

A growing body of evidence suggests that TNF‐α and angiotensin II induce oxidative stress and contribute to progression of cardiovascular disease. We examined whether the cross talk between TNF‐αand ANGII contributes to oxidative stress and end organ damage in rats. Rats were treated with TNF‐α (30μg/kg, ip), TNF‐α + losartan (LOS, 1mg/kg, ip), or vehicle for 5 days. Left ventricular (LV) function was measured using echocardiography. Rats were sacrificed and LV tissues were removed for gene expression, EPR, mitochondrial assays, and western blot. TNF‐α treatment resulted in a significant increase in expression of NADPH oxidase subunits and AT‐1R in the LV of rats. EPR measurement showed an increased formation of superoxide, peroxynitrite and total ROS in the cytosol and mitochondria of TNF‐α treated rats. TNF‐α a treatment also resulted in a decrease in fractional shortening and an increase in Tei index, suggesting diastolic dysfunction. Treatment with TNF‐α +LOS attenuated mitochondrial damage, restored cardiac function, and decreased the expression of AT1‐R and NADPH oxidase subunits. These results suggest that TNF‐α a damages both the cell and the mitochondrial membranes by increasing total ROS, thus contributing to end organ damage. The cross talk between RAS and TNF‐α contributes to the pathophysiology of cardiovascular disease.
Role of the protein C pathway in the extra‐intestinal thrombosis associated with inflammatory bowel disease
Authors: Yoshida Hideo , Russell Janice , Stokes Karen , Yilmaz Cigdem , Granger D. Neil
Published: June 2021

Inflammatory bowel diseases (IBD) are associated with an increased risk for thromboembolism. While thrombosis is known to contribute to the morbidity and mortality of patients with IBD, the underlying mechanisms that contribute to the genesis of a hypercoagulable state during intestinal inflammation remain poorly defined. The objective of this study was to determine whether the protein C pathway contributes to the enhanced extra‐intestinal thrombosis that is associated with dextran sodium sulfate (DSS)‐induced colitis in mice. Intravital microscopy was used to monitor thrombus formation in arterioles and venules of the cremaster muscle in colitic wild type (WT) and mutant mice that overexpress the endothelial cell protein C receptor (EPCR). Thrombus formation was induced using the light/dye endothelial injury model and quantified using the time of onset of the thrombus and time to blood flow cessation. Some WT mice were treated with either activated protein C (APC) or an APC neutralizing antibody (APC‐Ab). The DSS‐enhanced thrombosis response was greatly attenuated in the EPCR transgenic mice and in WT mice treated with APC immediately prior to photoactivation. Treatment with the APC‐Ab further promoted the DSS‐enhanced thrombosis response. These findings indicate that an altered protein C pathway contributes to the extra‐intestinal thrombosis that accompanies colonic inflammation. (P01 DK43785)