bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of Pregnane X Receptor Prevents Lipopolysaccharide/D‐Galactosamine‐induced Acute Liver Failure in Mice
Authors: WANG KUN , Damjanov Ivan , Wan Yu‐Jui Yvonne
Published: June 2021

Pregnane X receptor (PXR) plays crucial roles in xenobiotic and endobiotic metabolism. Recent work has shown that PXR participate in anti‐apoptotic and anti‐inflammatory processes. However, its in vivo function in the pathogenesis of inflammation‐related apoptotic liver injury remains obscure. The role of PXR in liver injury was investigated using lipopolysaccharide (LPS)/D‐galactosamine (GalN)‐induced and TNFα‐dependent acute liver failure mouse model. Our findings demonstrated that PXR‐null mouse liver had a delayed initial response to LPS/GalN characterized by a late activation of Stat3 and NF¿B and reduced TNFα production in comparison with wild‐type mouse liver. However, PXR‐null mouse later developed a vigorous response including strong and sustained activation of Stat3 and JNK1/2, increased production of hepatic pro‐inflammatory cytokines and chemokines, as well as enhanced liver apoptosis and necrosis and increased susceptibility to LPS/GalN‐induced lethality. In addition, activating PXR protected mice from LPS/GalN‐induced acute liver injury and death. The underlying protective mechanisms include inhibition of TNFα, IL‐1β, and IL‐6 production, repression of sustained JNK1/2 activation, increased expression of anti‐apoptotic protein (Bcl‐xL), and blockage of expression of pro‐apoptotic proteins (cleaved caspase‐3 and Bax). Our data indicate that PXR ligands may be useful clinically to prevent TNFα‐dependent liver injury during acute inflammation.
Effects of the butadiene metabolite diepoxybutane on endothelial cell and platelet function
Authors: Dean William L. , Samuel Bankole , Siddiqui Maqsood A. , Sithu Srinivas D. , Hu Chuan , D'Souza Stanley E.
Published: June 2021

Butadiene, is used in the synthesis of rubber and is also found in cigarette smoke and car exhaust. It is a carcinogen and is also associated with cardiovascular disease. The most toxic metabolite, 1,4‐diepoxybutane (DEB), forms adducts with biomolecules. Consequently, we determined the effects of DEB on the function of endothelial cells and platelets. Human endothelial cells (EC) were exposed to 1 ‐10 μM DEB for 3‐7 days. DEB‐exposed EC were examined by FACS for cell surface expression of adhesion molecules, and for leukocyte transmigration. Dose‐dependent increases in ICAM‐1 (25%, P< 0.01 at 5 µM) and VCAM‐1 (30%, P< 0.01 at 5 µM) were observed. A dramatic increase in leukocyte transmigration (125%, P<0.01) through a monolayer of DEB‐treated EC was also observed. Cell proliferation was completely blocked by 4 days at 10 µM DEB and EC developed a morphology resembling senescent cells. However, EC that were treated for 7 days could still form endothelial tubes on matrigel. For platelets, human platelet rich plasma was treated with DEB for 1 hour and the effects on the rates of aggregation were determined by aggregometry. Significant concentration‐dependent increases in the rates of both thrombin‐ (35% P<0.01 at 5 μM) and collagen‐ (22% P<0.01 at 5 μM) induced activation were observed. These results indicate that butadiene exposure can cause inhibition of EC growth while augmenting ICAM‐1, VACM‐1, and platelet activity and retaining EC angiogenic capacity.
Molecular mechanisms of maintaining nervous system architecture
Authors: Hobert Oliver
Published: June 2021

The complex and interconnected structure of a nervous system is largely determined by patterning events during embryonic development. A question that has received little attention in the past is whether the architecture of the nervous system is maintained simply by those factors that have initially patterned the nervous system or, alternatively, whether dedicated mechanisms exist that ensure the sustained integrity of the nervous system. Our microsurgical and genetic analysis in the nematode C. elegans has revealed that nervous systems indeed employ dedicated molecular mechanisms that maintain the positioning of neuronal structures. We have found a set of Immunoglobulin‐domain containing proteins whose sole function appears to be to ensure the maintenance of positioning of axonal tracts and cell bodies in various regions of the nervous system. These proteins include the C.elegans homolog of the human disease gene L1, several secreted, small Ig domain proteins, a gigantic basement membrane protein, and, surprisingly, also the fibroblast growth factor receptor.
GATA3 regulation of human neuroblastoma stem cell activity
Authors: Shi Huilin , Ding Han‐Fei
Published: June 2021

Neuroblastoma (NB) is a common childhood malignant tumor of the sympathetic nervous system. BE(2)‐C cells, a human NB cell line enriched with tumorigenic stem cells, can be induced to undergo either neuronal or glial differentiation. We use BE(2)‐C cells as a system to identify the genes that regulate NB stem cell activities, such as self‐renewal and differentiation. One of these genes is GATA3, a zinc‐finger transcription factor with an essential role in sympathetic development. Downregulation of GATA3 by siRNA promotes BE(2)‐C cell proliferation and overexpression of GATA3 decreases the proliferation of BE(2)‐C cells. GATA3 regulates cell proliferation through a pathway involving cyclin D1, as evidenced by an increase in the expression of cyclin D1 in GATA3 knockdown cells. In addition, GATA3 knockdown induces BE(2)‐C cells to undergo glial differentiation, as indicated by an increase in the expression of GFAP, a glial cell marker, and a decrease in the expression of neuronal marker SNAP25, whereas GATA3 overexpression promotes neuronal differentiation of BE(2)‐C. GATA3 knockdown also downregulates Phox2B and Mash1, two transcription factors that are expressed in neuronal progenitor cells and are essential for sympathetic development. Together, these finding suggest that GATA3, Phox2b and Mash1 may function in a regulatory network in the control of NB cells in a stem/progenitor cell state.

This work was supported by the NCI grant CA124982 (H.‐F.D.).
CD49d, Zap‐70, and CD38 Expression in Chronic Lymphocytic Leukemia
Authors: Singleterry Will L , Cruse Julius M , Lewis Robert E , Coker John A , Suggs Jeanann L
Published: June 2021

Zeta‐chain (TCR) associated protein kinase 70kDa (Zap‐70), and CD38 expression are predictors of IgVH mutation and prognosis in chronic lymphocytic leukemia (CLL). Previous studies indicate that Zap‐70 and CD38 are usually positive in cases of CLL with non‐mutated immunoglobulin variable region genes (IgVH), and may be used to predict IgVH mutation status and prognosis. Usually, cases of CLL positive for Zap‐70 or CD38 indicate a worse prognosis. In the present investigation, 62 cases of CLL were evaluated for CD38 expression, and 32 cases were evaluated for both Zap‐70 and CD38 expression. Of the 62 cases, 40% positively expressed CD38. Of the 32 cases evaluated for Zap‐70, 65% were positive, while only 35% were positive for CD38 expression; the remaining cases were negative for CD38. More recently CD49d, an integrin molecule, has been added as a predictor of prognosis in CLL. The results of this study show that Zap‐70 expression may be a better indicator of the mutational status of IgVH and prognosis of CLL than CD38 expression, and that CD49d represents an improved prognostic capability in CLL cases. In addition, CD38 negativity does not necessarily indicate that IgVH mutation has occurred. These data point to the need for a broader study to evaluate the significance of Zap‐70 and CD38 expression as indicators of IgVH mutation status, and of CD49d in the prognosis of CLL patients.
Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation
Authors: Liu Bowen , Paranjpe Shirish , Bowen William , Bell Aaron , Luo Jianhua , Mars Wendy , Michalopoulos George
Published: June 2021

Glypicans are heparan sulfate proteoglycans (HSPGs) that are bound to the cell surface through a glycosylphosphatidylinositol (GPI) anchor. Six members of glypican family have been identified in mammals. Our study focused on glypican 3 (GPC3) which is mutated in the Simpson‐Golabi‐Behmel (SGB) syndrome, an X‐linked disorder characterized by pre‐ and postnatal overgrowth. GPC3 is reported to be over‐expressed in human hepatocellular carcinoma (HCC). Whether GPC3 is involved in liver regeneration and hepatocyte proliferation is still unknown. We investigated its role in regenerative liver using the 2/3rd partial hepatectomy (PHx) model in rats. Results showed that GPC3 is up‐regulated in liver starting from day 2 after PHx. GPC3 is also up‐regulated from day 7 in primary hepatocyte cultures, when the process of proliferation slows down. We hypothesize that GPC3 negatively regulate liver regeneration. In vitro study showed that hepatocyte growth is promoted at the end of proliferation when GPC3 is blocked by Morpholino oligos. Further study will include GPC3 transgenic mice screening and study, and in vivo function and growth study. To investigate the signaling pathway involving GPC3, yeast two hybrid assay was performed. The assay results revealed that GPC3 might interact with CD81, a member of tetraspanin and reported to involve in HCV infection and cell proliferation. Our study showed that CD81 level increased from day 2 after PHx, corresponding to the changes of GPC3. Immunofluorescence showed that CD81 and GPC3 co‐localize at day 2 and day 6 after PHx. Co‐immunoprecipitation also showed that CD81 protein co‐precipitates with GPC3. So we hypothesize that GPC3 may be a negative regulator in liver regeneration and hepatocyte proliferation, and may interact with CD81.
Fexofenadine transport and drug‐drug interactions
Authors: Flynn Colleen Ann , Hagenbuch Bruno , Reed Gregory
Published: June 2021

Characterization of human transporter function in vivo requires well‐characterized probe substrates. Fexofenadine (Fex), a safe drug with pharmacokinetics controlled by transporters, may be a useful probe. There is, however, uncertainty regarding the roles of specific transporters in Fex pharmacokinetics. We previously tested a probe drug cocktail in healthy subjects with Fex, buspirone, caffeine, dextromethorphan, and losartan for the measurement of CYP and transporter activities. A Fex‐drug interaction was observed, resulting in a 40% decrease in mean Fex AUC when Fex was given as part of the cocktail. We are now conducting in vitro studies in HeLa cells transfected with human transporters to determine the molecular basis of this interaction. Initial studies with OATP1A2 indicate an interaction between Fex and one or more of the probe drugs. Preliminary results show a Km for Fex of 30 µM, and that losartan appears to be an OATP1A2 substrate. Characterization of the kinetics of these interactions will better define both substrate preferences for OATP1A2 and the role of this transporter in Fex pharmacokinetics. Supported by NIH/NCCAM R21‐AT002907‐03.
Human Cytochrome P450 2A7: Stability and Activity Relative to other Human CYP2A Enzymes
Authors: DeVore Natasha M. , Walsh Agnes A. , Scott Emily E.
Published: June 2021

Humans have three cytochrome P450 (CYP) 2A genes: CYP2A6, CYP2A7, and CYP2A13. CYP2A6 and CYP2A13 proteins metabolize coumarin and other compounds. Although CYP2A7 is 94% identical to CYP2A6, it has been reported that CYP2A7 is an inactive protein. CYP2A7 expressed in vaccinia virus did not yield functional holoprotein as evaluated by spectral analysis, Western blot, and catalytic activity, likely failing to incorporate heme1. Western blot revealed that CYP2A7 protein was produced in COS cells, but this protein did not metabolize coumarin to 7‐hydroxycoumarin2. Our goal is to reevaluate if CYP2A7 is a stable holoprotein capable of metabolizing coumarin or other CYP2A substrates. We have expressed truncated, His‐tagged CYP2A7 in E. coli. This system produces CYP2A7 protein that does incorporate heme, as indicated by a spectral shift in the reduced carbon monoxide difference assay. Generation of purified CYP2A7 protein is enabling new comparisons of human CYP2A stability, ligands, and catalytic activity. Additionally, comparison of the CYP2A7 sequence with the CYP2A6 and CYP2A13 structures suggests substitutions that may be responsible for differences among the human CYP2A enzymes. Research supported by NIH GM76343 (EES).

1Yamano, S.; Tatsuno, J.; Gonzalez, F. J. Biochemistry 1990, 29, 1322‐1329.

2Ding, S.; Lake, B. G.; Friedberg, T.; Wolf, C. R. Biochem J 1995, 306, 161‐166.
Reactive oxygen species and the protective effects of glutathione and N‐acetyl cysteine on mercuric chloride toxicity in Neuro‐2A Cells.
Authors: Becker Alan , Soliman Karam F
Published: June 2021

The present study was designed to investigate reactive oxygen species (ROS), which are generated by mercuric chloride (HgCl2) in Neuro 2A cells, and to determine the protective effects of various antioxidants on Neuro‐2A cells. HgCl2 was shown to produce 1.9M and 9.2M hydrogen peroxide (H2O2) at 5mM and 10mM HgCl2, respectively. N‐acetyl‐cysteine (NAC) and reduced glutathione (GSH) were shown to reduce H2O2 and provide strong antioxidant properties, which may account for their added protective effect in HgCl2 toxicity. Superoxide increased at low concentrations of HgCl2 (1M to 5M) up to 388% to 450% compared control. Pretreatment with NAC, protected the Neuro‐2A in a dose dependent manner to 74% versus the LD10 value for HgCl2 at 5M, while GSH and CYS provided 95% and 48% protection, respectively, against HgCl2 LD10. Superoxide dismutase (SOD) did not significantly provide protective effects against HgCl2; however it reduces superoxide production slightly, suggesting that H2O2 was partially produced intracellularly. These results demonstrate that H2O2 and superoxide are generated in response to low levels of HgCl2. The antioxidant properties of GSH and NAC which are utilized in GSH synthesis and reduce H2O2 production effectively protect the Neuro‐2A cell from the toxicity of HgCl2. (Supported by a NIH grant NCRR: RR 03020)
Effect of Microcystis aeruginosa Lipopolysaccharide (LPS) on Neonatal Rat Brain Microglia Cytokine and Chemokine release
Authors: Aldulescu Monica , Frenkel Jeffrey , Hall Mary L , Glaser Keith B , Berry John , Mayer Alejandro M
Published: June 2021

Neuroinflammation has been hypothesized to involve release by brain microglia (BMG) of the inflammatory cytokines interleukin 1 α(IL‐1α) and interleukin 6 (IL‐6), the chemokine macrophage inflammatory protein 2 α(MIP‐2), as well as the anti‐inflammatory cytokine transforming growth factor β1 (TGF‐β1). We have reported that M. aeruginosa LPS (MaLPS) stimulated BMG to release TNF‐α, TXB2, O2− and MMP‐9 (The Toxicologist 102(1):254, 2008). The purpose of this investigation was to determine the effect of MaLPS on BMG priming and release of IL‐1α, IL‐6, TGF‐β1, MIP‐2 and lactate dehydrogenase (LDH) in vitro. MaLPS (1.075 x 105 endotoxin units/mg) was isolated from M. aeruginosa strain UTCC 299 by hot phenol/water extraction. BMG isolated from neonatal rats were treated in vitro with MaLPS or Escherichia coli LPS (EcLPS) for 17 hours. IL‐1α, IL‐6, TGF‐β1 and MIP‐2 were determined by immunoassay, and LDH by enzyme activity. Results were the following (n=3‐4): LDH: release was observed at > 0.1 ng/mL MaLPS; IL‐6, & MIP‐2: concentration‐dependent release was detected at > 0.1 ng/mL MaLPS; IL‐1α: release was observed at > 1 ng/mL; and in contrast no release TGF‐β1 was observed. MaLPS appeared less potent than EcLPS. We conclude that MaLPS stimulates rat BMG cytokine and chemokine release in a concentration‐ and time‐dependent manner. Further characterization of BMG response to MaLPS at both the functional and molecular level is ongoing in our laboratories. Supported by Midwestern[P1] University and Florida International University.
Discovery and Characterization of a Novel Subtype‐Selective M1 Allosteric Agonist for the Treatment of Alzheimer's Disease
Authors: Lebois Evan P , Bridges Thomas M , Kennedy J Phillip , Xiang Zixiu , Jadhav Satyawan B , Yin Huiyong , Jones Carrie K , Conn P Jeffrey , Weaver C David , Lindsley Craig W
Published: June 2021

The muscarinic acetylcholine receptors are a family of five cholinergic GPCRS, designated M1‐M5, located throughout the central nervous system (CNS) and periphery. Due to impaired hippocampal‐dependent learning, the M1 muscarinic receptor has long been implicated in the pathophysiology of Alzheimer's Disease (AD). While the M1 receptor has remained a Phase III clinically‐validated target for obtaining cognitive efficacy in AD, all efforts heretofore have not met FDA approval due to a lack of muscarinic selectivity, giving rise to severe cholinergic side effects such as poor GI tolerance. The present work reports the discovery of the first truly subtype‐selective M1 allosteric agonist, VU0184670 which possesses EC50 and Achmax values of 150nM and 85%, respectively, with exclusive selectivity versus M2‐M5. In addition, VU0184670 was found to robustly potentiate NMDAR current in rat hippocampal slices. Favorable pharmacokinetics were observed following intraperitoneal dosing of an analog VU0357017 in rats at 10 mg/kg, while PanLabs analysis showed no significant off‐target binding at 10μM, providing a clear window with which to perform in vivo studies. The discovery of a potent, truly selective M1 allosteric agonist represents a significant breakthrough for AD treatment and will provide a critical basic tool to allow dissection of M1‐mediated efficacy in other disease states such as Schizophrenia.
LPS‐induced transcriptomic changes are detectable in minimal volumes of rat whole blood
Authors: Grudzien Meagan M , Ditewig Amy C , Jung Paul M , Liguori Michael J , Blomme Eric A , Yang Yi
Published: June 2021

Whole blood is an easily accessible tissue that allows for non‐invasive, real‐time monitoring of compound exposure and effects. Using blood for transcriptional profiling presents technical challenges, particularly because PAXgene tubes require 2.5mL of blood. We have developed a reproducible method of RNA stabilization and isolation to be used for transcriptional profiling from as little as 25μL of whole blood. To demonstrate the validity of this technique, we used an acute inflammation model and treated rats with lipopolysaccharide (LPS, IV) for 2h. Varied amounts of whole blood (25‐500μL) were collected from each rat and immediately added to Qiazol or collected directly into PAXgene tubes. Both techniques yielded abundant, high‐quality RNA. Total RNA (50ng) was processed using the NuGEN Ovation RNA Amplification System, then hybridized to Affymetrix Rat230_2 microarrays. Statistical analysis of microarray intensity values revealed high correlation between blood collected using PAXgene tubes and all volumes of blood added to Qiazol (r = 0.8, p=0.001, FC=1.5). Expression analysis of both methods showed little discordance in IL‐10 signaling, IFN signaling, and LPS‐induced inflammatory response. The evidence from this novel blood collection technique suggests as little as 25μL of whole blood can be used in preclinical toxicology and pharmacology studies for future identification of novel biomarkers.
Estradiol Differentially Modulates the Growth of Oligodendroglial and Microglial Cells
Authors: Schaufelberger Sara Andrea , Gillespie Delbert G. , Barchiesi Federica , Dubey Raghvendra K. , Jackson Edwin K.
Published: June 2021

Purpose

Estradiol (E2) is neuroprotective, however the mechanisms involved remain unclear. Here we investigated the effects of E2 and its metabolites on cultured microglia and oligodendrocytes.

Results

Treatment with E2 differentially modulated growth of microglia (BV2) and oligodendrocytes (Oli‐Neu). E2 significantly induced growth of Oli‐Neu cells and inhibited growth of BV2 cells. In BV2 cells the inhibitory effects of E2 were mimicked by DPN (ER‐β agonist), but not by PPT (ER‐α agonist). Additionally, the anti‐proliferative effects of E2 on BV2 cells were blocked by ICI (unspecific ER antagonist) but not by MPP (ER‐α antagonist). These findings suggest that effects of E2 on BV2 growth are ER‐β mediated. Interestingly, the inhibitory effects of E2 were mimicked by 2‐methoxyestradiol (2ME) and 2‐hydroxyestradiol, two endogenous E2 metabolites with no ER‐affinity. Additionally, 2ME decreased LPS‐stimulated iNOS and COX expression.

Conclusions

Our findings suggest that inhibitory effects of E2 on microglia would limit neuronal injury by decreasing cell growth and production of cytotoxic molecules, whereas pro‐mitogenic effects of E2 on oligodendrocytes would promote neuronal recovery. Finally, the protective effects of E2 can be mediated via both ER‐dependent and ER‐independent mechanisms.
Vaso‐ and athero‐protective effects of Angiotensin (1‐7) and CGP42112 in apolipoprotein‐E deficient mice
Authors: Tesanovic Sonja , Vinh Antony , Widdop Robert , Gaspari Tracey
Published: June 2021

The aim of this study was to investigate the effects of Angiotensin (Ang) (1‐7), a Mas receptor agonist, and CGP42112, an AT2 receptor agonist, in a setting of atherosclerosis. All treatments were investigated using apolipoprotein E‐deficient (ApoE−/−) mice fed a high fat diet for 16 weeks, with chronic treatment administered in final 4 weeks via osmotic mini‐pumps. Treatments were either: saline (vehicle), Ang (1‐7) (24µg/kg/hr) or CGP42112 (1µg/kg/min) alone, as well each agonist in combination with either an AT2R antagonist, PD123329 (10mg/kg/day), or the MasR antagonist, A779 (48µg/kg/hr). Chronic treatment with either Ang (1‐7) or CGP42112 significantly improved endothelial function and decreased lesion development compared to vehicle treated mice. Superoxide levels were significantly reduced and eNOS expression increased with Ang (1‐7) and CGP42112 treatments when compared to vehicle treated mice. Ang (1‐7) effects were reversed with both AT2R and MasR blockede, however, CGP42112 mediated effects were only reversed by the AT2R antagonist. This study indicates that both Ang (1‐7) and CGP42112 improve endothelial function and reduce atherosclerotic lesions via either the AT2 or the Mas receptor, or both. This highlights the importance of gaining a better understanding of the role of AT2 and Mas receptors in atherosclerosis.

NHMRC (Australia)
Paradoxical effects of thymoquinone in prostate cancer cells: Induction of reactive oxygen species.
Authors: Koka P.Sandeep , Mondal Debasis , Mageed Asim B.Abdel , Agrawal Krishna
Published: June 2021

Thymoquinone (TQ) a well known anti‐oxidant is an active ingredient of black seed oil. It has been shown to inhibit growth of prostate cancer (PCa) cells by inhibiting expression of androgen receptor (AR) and E2F‐1 in parental LNCaP cells and its isogenic variant, hormone independent AR expressing C4‐2B cells. However, the precise mechanism of action of TQ remains unexplored, especially in AR naïve (PC‐3) cells as models of hormone refractory disease. Our results demonstrated that TQ inhibits growth of both androgen receptor positive (C4‐2B) and androgen receptor naïve (PC‐3) PCa cells with an IC50 value of 80 and 50 µM at 48 hr, respectively. TQ (100 µM) induced a 3 and 4 fold increase in ROS generation and a decrease in GSH levels by 35% and 45% in C4‐2B and PC‐3 cells, respectively. Treatment of PCa cells with TQ did not activate caspases but resulted in an increase in the levels of apoptosis inducing factor (AIF) indicating a caspase‐independent cell death mechanism. Treatment of PC‐3 cells with TQ (50 µM) up‐regulated GADD45α and down‐regulated BCL‐2 (2 fold) and BAG‐1 (1.5 fold). However, TQ‐induced inhibition of the expression and transactivational activity of AR in C4‐2B cells was not restored upon simultaneous co‐exposure with N‐acetylcysteine (NAC). In both cell types, (NAC) completely inhibited TQ‐induced ROS generation and protected the cells from cytotoxicity suggesting that ROS generation by TQ was primarily responsible for growth inhibition.
Role for FAK expression in adhesion and metastasis of EL4 lymphoma cells.
Authors: Zhang Zhihong , Knoepp Stewart M , Chahal Manpreet S , Xie Yuhuan , Meier Kathryn E
Published: June 2021

The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to phorbol 12‐myristate 13‐acetate (PMA). In sensitive cells, PMA causes Erk MAPK activation and Erk‐mediated growth arrest; both are mediated by expression of Ras‐GRP. In resistant cells, PMA induces a low levels of Erk activation, without growth arrest. A relatively unexplored aspect of the phenotypes is that resistant EL4 cells are more adherent to culture substrate than are sensitive cells. The roles of the protein tyrosine kinase, focal adhesion kinase (FAK), in EL4 cell phenotype was therefore examined. FAK is expressed only in PMA‐resistant (or intermediate phenotype) cells, correlating with enhanced substrate adherence, while the FAK‐related kinase Pyk2 is more highly expressed in PMA‐sensitive cells. These differences in protein expression are reflected in mRNA levels. A series of studies indicated that FAK does not play a major role in modulating PMA‐induced Erk activation. However, FAK expression enhances cell migration and invasion. In an experimental metastasis model using syngeneic mice, only PMA‐resistant EL4 cells that express FAK form liver tumors. The results demonstrate that the PMA‐resistant phenotype is required for metastasis of this lymphoma cell line. These studies also suggest that, in combination with other factors, FAK expression promotes metastasis of EL4 cells. Supported by NIH CA94144.
Rnd3 accelerates termination of thrombin‐induced Rho activation in endothelial cells.
Authors: Kurtz Kristine M , Breslin Jerome W
Published: June 2021

Rnd3, recently implicated as an endogenous inhibitor of Rho signaling, is present in cultured endothelial cells but its functional significance is unknown. We tested the hypothesis that Rnd3 inhibits Rho signaling in endothelial cells during an inflammatory challenge. We assessed in vivo expression of Rnd3 in rat mesenteric microvessels by immunofluorescence confocal microscopy. RhoA activation was assessed in human umbilical vein endothelial cells (HUVEC) treated with thrombin (1U/ml) for 5, 30, or 60 min. using a commercially available G‐LISA kit. HUVEC were mock‐transfected or transfected with pMAT‐FLAG‐Rnd3, Rnd3 siRNA, or siCONTROL RNA. The results show that Rnd3 is present in vivo within endothelial cells. Thrombin significantly increased Rho activity in all groups treated, however the time course varied, particularly at 60 min. Rho‐GTP levels were significantly lower in pMAT‐FLAG‐Rnd3 cells compared to mock at 60 min. In contrast, in Rnd3 siRNA cells, Rho‐GTP levels at 60 min. were significantly higher than siCONTROL cells. The data indicate that Rnd3 accelerates termination of thrombin‐induced Rho activation, which may serve as an important negative feedback signal for concluding inflammatory signaling in endothelial cells. Supported by NIH RR018766 and a grant from the American Heart Association.
The systemic inflammation of alveolar hypoxia is initiated by a circulating mediator(s) released from alveolar macrophages
Authors: Chao Jie , Wood John G , Blanco V. Gustavo , Gonzalez Norberto C.
Published: June 2021

Alveolar hypoxia (FIO2 = 0.1) produces widespread systemic inflammation in rats. Recent evidence suggests that the inflammation is not triggered by the low systemic PO2, but by activation of mast cells (MC) by a circulating mediator released from activated alveolar macrophages (AM). If this is correct, the following should apply: 1.Neither MC nor resident tissue macrophages should be directly activated by hypoxia and 2. MC would be activated when in contact with hypoxic AM, but not with hypoxic tissue macrophages.

Methods

Rat mesenteric microcirculation intravital microscopy was combined with primary cultures of AM, peritoneal macrophages (PM), and peritoneal MC.

Results

a. Cultured peritoneal MC did not degranulate with hypoxia. b. Hypoxia induced a respiratory burst in AM, but not in PM cultures. c. Immersion of isolated MC in supernatant of hypoxic AM, but not in supernatant of hypoxic PM, induced MC degranulation d. Supernatant of hypoxic AM, but not of hypoxic PM produced MC degranulation and leukocyte‐endothelial adherence in mesentery.

Conclusions

A mediator(s) released from hypoxic AM activates MC and triggers the systemic inflammation. Reduced systemic tissue PO2 and activation of systemic tissue macrophages do not play a role in this phenomenon. The inflammation may contribute to the systemic effects observed in conditions with reduced alveolar PO2.

Supported by NIH HL39443‐18 and AHA 0815652G
Role of proteases in ischemia/reperfusion‐induced arteriolar endothelium‐dependent vasodilatory dysfunction.
Authors: Dai Hongyan , Kalogeris Theodore , Korthuis Ronald
Published: June 2021

Intestinal ischemia and reperfusion (I/R) is associated with leukocyte rolling and adhesion in postcapillary venules, neutrophil infiltration and release of proteases in the interstitium, and endothelium‐dependent vasodilatory (EDD) dysfunction in arterioles. The aim of this study was to determine whether proteases were involved in the genesis of postischemic EDD. To address this issue, the responses of rat mesenteric arterioles (20‐40 µm diameter) to increasing doses of acetylcholine or nitroprusside were evaluated under control conditions (no ischemia) and after 20 min of ischemia and 60 min of reperfusion using intravital microscopy. In some groups, the mesentery was superfused with broad‐spectrum protease or selective matrix metalloproteinase (MMP) inhibitors during reperfusion. Intestinal I/R induced a significant deficit in endothelium‐dependent arteriolar responses to acetylcholine, but not nitroprusside. Postischemic EDD dysfunction was largely abolished by treatment with broad‐spectrum protease or selective MMP inhibitors. Comparable results were noted in isolated mesenteric arterioles (80‐140 µm) obtained from MMP‐9 knockout mice that had been subjected to mesenteric I/R. In summary, our data suggest that proteases, including MMP‐9, play an important role in the development of I/R‐induced arteriolar endothelium‐dependent vasodilator dysfunction. The work was supported by HL‐082816 from the NIH.
Proteomic analysis of the myoendothelial junction
Authors: Heberlein Katherine , Best Angela K , Isakson Brant E
Published: June 2021

Myoendothelial junctions (MEJ) are cellular projections through the internal elastic lamina originating from the vascular smooth muscle cell (VSMC) or endothelial cell (EC) layers that may serve as a distinct signaling domain, however the proteins that make up this domain are unknown. Using a vascular cell co‐culture model (VCCC) (Isakson et al., 2005), we demonstrate for the first time isolation of proteins specifically localized to the MEJ in vitro. Proteins from VSMC, EC and MEJ fractions were labeled separately with Cy5, Cy3, and Cy2 dyes respectively and subject to 2D‐DIGE analysis. Proteins within the MEJ fraction that displayed a minimum 2.5 increase in fluorescence intensity when compared to EC or VSMC fractions as determined by DeCyder software were chosen for mass spectrometry analysis and were compared to the NCBI protein database. Of the proteins identified at the MEJ, plasminogen activator inhibitor‐1 (PAI‐1) was shown to be one of the most abundant. Preferential expression of PAI‐1 at the MEJ was confirmed in vitro using immunoblotting and immunocytochemistry on the VCCC, as well as in vivo using transmission electron microscopy in multiple vascular beds. Based on this data we conclude that proteomic analysis of MEJ fractions in vitro provides valuable insight into possible protein interactions at the MEJ in vivo. Supported by NIH RO1 088554 and an AHA SDG.
Differential Effects of GRB2‐SOS, JNK, STAT and Akt Down Stream Signaling to EGFR Tyrosine Kinase in Mice Coronary Arteriolar Myogenic Tone
Authors: Abd Elmageed Zakaria , Amin Ali , Palen Desiree I , Matrougui Khalid
Published: June 2021

Background

In a previous report, we showed that epidermal growth factor receptor (EGFR) tyrosine kinase is essential in the development of microvascular myogenic tone (MT). We determined the mechanism of how EGFR tyrosine kinase contributes in the development of mice coronary arteriolar MT focusing on down stream signaling to EGFR: GRB2‐SOS, Akt, JNK and STAT.

Methods and results

Freshly isolated coronary arterioles from C57/BL6 mice were mounted in an arteriograph and stimulated by 25 to 100 mmHg of intraluminal pressure. We performed pressure‐active diameter, passive diameter with and without inhibitors of EGFR tyrosine kinase, GRB2‐SOS, Akt, JNK and STAT. Pressurized coronary arteriolar under 25 and 75 mmHg ± EGFR tyrosine kinase inhibitors were subjected to western blot analysis to determine EGFR tyrosine kinase, GRB2‐SOS, Akt, JNK and STAT phoshorylation.

MT was significantly inhibited under EGFR tyrosine kinase inhibition. Similarly, coronary arteriolar MT was significantly reduced under GRB2‐SOS, JNK or STAT inhibitors. However, Akt inhibition had no effect on coronary arteriolar MT. Western blot analysis showed increased EGFR tyrosine kinase, GRB2‐SOS, Akt, JNK and STAT phosphorylation, which were inhibited under EGFR inhibition.

Conclusion

These novel findings suggest that EGFR dictates coronary arteriolar myogenic tone involves down stream signaling GRB2‐SOS, JNK and STAT, but not Akt.
Coronary Artery Endothelial Function and Inflammation in Hypercholesterolemic Pigs
Authors: Arce‐Esquivel Arturo A , Thorne Pamela K. , Turk James R. , Laughlin M. Harold
Published: June 2021

Inflammation is known to promote endothelial dysfunction and the development of cardiovascular disease. This study was designed to test the hypothesis that inflammation contributes to the development of endothelial dysfunction using a hereditary hypercholesterolemic (HH) swine model. We also tested the hypothesis that exercise training (ExT) decreases markers of inflammation in atherosclerotic lesions of HH coronary arteries. Fourteen HH pigs were randomly assigned into either exercise trained (EX) or sedentary groups (SED). ExT consisted of 16 weeks of treadmill running. At sacrifice coronary arteries (LAD, RCA) were harvested. In vitro assessment of endothelium‐dependent relaxation (EDR) was determined by administering increasing doses of bradykinin (BK) to RCA rings. Inflammation was quantified by immunohistochemistry using macrophage scavenger receptor A (SRA) and adipophilin antibodies. LAD and RCA sections were stained with Verhoeff's method for intima‐media thickness (IMT) assessments. Although BK‐induced relaxation tended to be greater in EX, EX did not significantly modify EDR in RCA rings (p=0.31). Average percentage for SRA, adipophilin staining and IMT were not significantly different in LADs or RCAs from EX and SED swine. We conclude that exercise training does not significantly decrease inflammatory marker expression in coronary arteries of this model of disease (NIH HL52940).
Hyperthyroidism enhances endothelium‐independent relaxation in rat aorta.
Authors: Carrillo‐Sepulveda Maria Alicia , Tostes Rita C , Webb R Clinton , Barreto‐Chaves Maria Luiza Morais
Published: June 2021

Thyroid hormone (T3) is known to promote vascular relaxation. However, the molecular mechanisms of T3 actions are incompletely characterized. Considering our previous study where T3 rapidly stimulated nitric oxide (NO) production by vascular smooth muscle cell (VSMC), we hypothesized that NO derived from smooth muscle represents an important component to mediate the vasodilators effects of T3. In vitro, chronic treatment of VSMCs for 24h with T3 stimulated NO production, and decreased expression of contractile proteins alpha actin, calponin as well as MLC phosphorylation levels. In vivo, aortas isolated from rats submitted to experimental hyperthyroidism (7ug/100g T3 for 14 days) presented decreased on expression of contractile apparatus proteins, as well as alterations in vascular reactivity. Functional assays demonstrated that relaxation responses to sodium nitroprusside (SNP) were greater in endothelium‐denuded aortic rings from T3‐treated rats and phenylephrine‐induced contraction was reduced. These results suggest that enhanced NO‐mediated vasodilation as well as decreased responses to constrictor stimuli contribute to vascular relaxation during hyperthyroidism. Financial support: NIH (USA), FAPESP (Brazil).
Rosiglitazone treatment down regulates AT1 receptor and collagen expression in cardiac myofibroblasts
Authors: Katwa Laxmansa C , Gould Amanda
Published: June 2021

Rosiglitazone, a Peroxisome Proliferator‐activated Receptor gamma (PPAR‐γ) agonist, is a commonly used anti‐diabetic drug. It is known that PPAR‐ γagonists play an important role in regulation of growth, differentiation and angiogenesis, including a decrease in myocardial infarction (MI). Our previous studies demonstrated that wound healing myofibroblasts (myoFb) isolated from the site of MI express angiotensin‐II (AngII) (Katwa et al J. Mol. Cell. Cardiol 29; 1375‐1386, 1997) and PPAR‐γ (Chintalgattu et al Cardiovasc Res 74:140‐150, 2007). Since these cells play a major role in tissue repair/remodeling at the site of MI; it is interesting to examine whether rosiglitazone treatment has any influence on type I collagen and AT‐1 receptor (AngII Type 1 receptor) expression in these cells. In the present study, we used primary cultures of adult rat cardiac myoFb isolated from the site of 4 week post‐MI and treated with (3 and 10 μM) rosiglitazone. Our results from both western blot and Real Time‐qPCR illustrated significant down regulation (p<0.05) of AT1 receptor expression at gene and protein levels. Rosiglitazone treatment also significantly decreased (p<0.05) the expression of type I collagen in cardiac myoFb, suggesting that rosiglitazone induced down regulation of type I collagen may be due to the decrease in the expression of AT‐1 receptors.
The role of local transcription and chromatin structure in establishing DNA replication origins
Authors: MacAlpine David , Gordan Raluca , MacAlpine Heather , DeNapoli Leyna , Ding Queying , Powell Sara , Hartemink Alex
Published: June 2021

In order to duplicate the genome within S‐phase, DNA replication must initiate at multiple start sites. We are using genomic approaches in the model organism Drosophila to elucidate how origins of replication are selected and regulated to maintain genomic stability. As part of the model organism ENCODE consortium we have characterized the average time of replication for all unique sequences, mapped early origins of replication and identified sites of preRC localization in multiple cell lines. We have found that ORC preferentially localizes to open chromatin near the transcription start site of active genes. ORC binding sites are enriched for the histone variant H3.3 which marks open and dynamic chromatin. The large number of ORC binding sites has provided us the opportunity to use machine learning algorithms to identify complex sequence elements that direct ORC localization. We have also found that the single X chromosome of male cell lines replicates significantly earlier than the autosomes, suggesting a link between DNA replication and dosage compensation. Funding from NIH grant HG004279.
Mitochondrial enzyme deficiency may underlie myopathic phenotype of female muscle‐specific LKB1‐KO mice
Authors: Mongillo Anthony D. , Malan Brandon B. , Kenney Steven G. , Brough Timise A. , Winder William W. , Thomson David M.
Published: June 2021

LKB1 and its target, AMPK, are regulators of muscle metabolism and play an important role in the control of mitochondrial biogenesis. Mitochondrial dysfunction leads to myopathic conditions in rodents and humans. We report here that the muscle‐specific knockout of LKB1 (KO) in female mice leads to a dysfunctional muscle phenotype and signs of congestive heart failure similar to other mitochondrial myopathies. At 9‐11 mo. of age, body weight (BW) was lower (p = 0.05) for KO vs control littermate (C) mice (21.6 ± 0.7 vs 26.2 ± 0.6 g). Absolute gastrocnemius (GAST) weight was lower for KO vs C mice (77.3 ± 2.9 vs 101.1 ± 3.7 mg), but not different as expressed relative to BW. Heart (HRT) weight relative to BW was greater in KO vs C mice (5.6 ± 0.3 vs 4.4 ± 0.1 mg). Cytochrome C (CytoC) and UCP‐2 levels were 40.1% and 59.3% lower, respectively, in KO vs C GAST muscles. CytoC, UCP‐2, UCP‐3, and cytoC oxidase‐1 (COX‐1) were all significantly lower in KO vs C HRT (33.8%, 50.8%, 49.5%, and 61.1% lower, respectively). Phosphorylation of CREB, one transcription factor controlling the expression of mitochondrial enzymes, was also decreased in HRT (40.1%) and GAST (35.6%) of KO vs C mice. Our findings suggest that impaired mitochondrial function may underlie the myopathic phenotype of LKB1‐deficient mice. This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR‐051928.
Unilateral denervation changes NRG/ErbB signaling in adult rat diaphragm muscle
Authors: Argadine Heather M , Hellyer Nathan J , Mantilla Carlos B , Zhan Wen‐Zhi , Sieck Gary C
Published: June 2021

Unilateral denervation (DNV) of rat diaphragm muscle (DIAm) increases protein synthesis and degradation, with net protein breakdown by 14 days after DNV. We hypothesized that neural influences such as the nerve‐derived trophic factor neuregulin (NRG‐1) are essential in maintaining DIAm protein balance. NRG activates receptor tyrosine kinases of the ErbB family, with the ErbB2‐ErbB3 co‐receptor showing highest affinity for NRG‐1 and strong phosphoinositol‐3‐kinase (PI3K) activation. Both protein synthesis (via Akt phosphorylation) and degradation (via the transcription factor FoxO3A) are regulated by PI3K activity. We used Western blot analyses to assess NRG‐1, ErbB2, ErbB3 and FoxO3A protein expression at 1, 3, 7, and 14 days after DNV. By 1 day after DNV, NRG‐1 expression decreased, whereas ErbB2 and ErbB3 expression increased. Consistent with reduced NRG/ErbB signaling, association of the PI3K regulatory subunit p85 with ErbB3 decreased at 14 days after DNV. FoxO3A expression increased at 7 and 14 days after DNV, in agreement with delayed net protein breakdown. Importantly, DIAm inactivity induced by spinal hemisection had no effect on ErbB2, ErbB3, or FoxO3A expression. Taken together, these results indicate that DNV‐induced skeletal muscle adaptations result from removal of a trophic influence such as NRG‐1 rather than inactivity itself.

Supported by NIH grant AR51173.
Neuregulin‐1: a trophic factor for phrenic motoneurons
Authors: Issa Amine Najib , Mantilla Carlos B. , Zhan Whenzi , Fang Yun‐Hua , Sieck Gary C
Published: June 2021

Neuregulin (NRG) is a nerve derived trophic factor that regulates acetylcholine receptor expression during neuromuscular junction development. NRG also has important trophic effects in the adult central nervous system. We hypothesized that NRG is a trophic factor for phrenic motoneurons. In adult male Sprague‐Dawley rats, we examined NRG‐1 immunoreactivity in the vicinity of phrenic motoneurons. Retrogradely‐labeled phrenic motoneurons were imaged and reconstructed in 3D. NRG‐1 boutons were found mainly around the soma and primary dendrites. The co‐localization of NRG boutons and synaptic markers was determined using multicolor confocal microscopy and a custom‐developed application (Metamorph; Universal Imaging). We found that 94 ± 3% of NRG‐containing boutons resided on synapses, as determined by co‐localization with synaptophysin. In addition, 6 ± 3% of synapses on phrenic motoneurons (within 1 micrometer of soma and primary dendrite surface) expressed NRG. Overall, 13 ± 5 NRG boutons were present at the soma vs. 6 ± 3 at each primary dendrite of a motoneuron.Thus, 57% of NRG‐containing boutons were surrounding the motoneuron soma. In summary, NRG‐1 synaptic boutons are present at phrenic motoneurons in the adult, although its role is presently unknown.

Supported by NIH grant AR51173.
Diet‐induced obesity in rats decreases insulin sensitivity and baroreflex gain
Authors: Zhao Ding , Azar Afaf S. , Brooks Virginia L.
Published: June 2021

Baroreflex gain (BRG) is impaired in Zucker rats that are obese due to mutation of the leptin receptor. However, whether diet‐induced obesity also decreases BRG is unknown. To test this hypothesis, we measured BRG in conscious outbred SD male rats (~250 g) fed a moderately high fat diet (HFD; 33% kcal fat) for 4‐5 weeks; rats in the top tertile of weight gain were obesity prone (OP), whereas the lowest tertile were obesity resistant (OR). BRG was quantified using a 4 parameter sigmoidal fit of heart rate (HR) responses to slow ramp (3‐5 min) changes in arterial pressure (AP), induced by infusing nitroprusside and phenylephrine. To test if BRG impairment in OP rats is related to decreases in insulin sensitivity (IS), IS was also measured using the hyperinsulinemic‐euglycemic clamp method. OP rats weighed more than OR rats after 4 weeks of HFD (OP: 463±19 g, n=6; OR: 367±22 g, n=5; P<0.01). Neither AP (OP: 106±8 mmHg; OR: 95±4 mmHg) nor HR (OP: 373±27 bpm; OR: 369±3 bpm) differed significantly. BRG was reduced in OP rats (2.9±0.2 bpm/mmHg, OP; 4.0±0.3 bpm/mmHg, OR; P<0.05); however, other sigmoidal parameters were not different. Interestingly, OP rats also exhibited reduced IS (7.0±1.0 mg/kg ?min, OP; 22.6±1.9 mg/kg▸min, OR; P<0.01). In summary, BRG and IS decrease with diet‐induced obesity in rats. Because insulin enhances BRG, we speculate that the decreases in BRG and IS are functionally linked. Supported by AHA and NIH HL088552.
Neither gender nor the menstrual cycle affects sympathetic baroreflex sensitivity during orthostasis in humans
Authors: Fu Qi , Shibata Shigeki , VanGundy Tiffany B , Okazaki Kazunobu , Galbreath M Melyn , Levine Benjamin D
Published: June 2021

We tested the hypothesis that baroreflex control of muscle sympathetic nerve activity (MSNA) during orthostasis is impaired in women and can be affected by the menstrual cycle. MSNA and hemodynamics were measured supine and during a graded upright tilt (30º and 60º, 6 min each) in 11 young men and 11 women during the early follicular (EFP) and mid‐luteal phase (MLP) of the menstrual cycle. Sympathetic baroreflex sensitivity was quantified using the slope of the linear correlation between MSNA and diastolic pressure during spontaneous breathing. It was further assessed as the relation between MSNA and stroke index (SI), with SI derived from cardiac output (CO, C2H2 rebrathing), heart rate and body surface area. MSNA increased during tilting, and the responses were similar between genders and phases. Sympathetic baroreflex sensitivity did not differ between genders and phases supine [?280±151 (SD) units/beat/mmHg in men vs ?252±172 in EFP and ?272±131 in MLP in women, P=0.679 and 0.890], and at 30º (?648±429 vs ?610±262 and ?486±312, P=0.781 and 0.264) and 60º tilt (?792±447 vs ?831±305 and ?814±471, P=0.814 and 0.912); the sensitivity was not affected by the menstrual cycle. Changes in MSNA were associated with changes in SI, while the slope of the line was not different between genders and phases. These results suggest that neither gender nor the menstrual cycle affects sympathetic baroreflex sensitivity. Other factors rather than neural mechanisms contribute to gender differences in orthostatic tolerance in humans. 1