bluuebunny/crossref_metadata_2025 huggingface dataset:
Apo‐10′‐lycopenoic acid induces Nrf2‐mediated expression of phase II antioxidant genes and suppresses H2O2 induced oxidative damage in human bronchial epithelial cells
Authors: Lian Fuzhi , Wang Xiang‐Dong
Published: June 2021

Our previous study has demonstrated that apo‐10′‐lycopenoic acid (ALA), an enzymatic metabolite of lycopene, can suppress lung carcinogenesis in an animal model. However, the potential mechanism(s) underlying this protection is not well defined. It has been suggested that lycopene or its hydrophilic derivatives induce phase II detoxification and antioxidant enzymes by activating the transcription factor Nrf2 (nuclear factor E2‐related factor 2). In this study, we investigated whether purified ALA induces Nrf2‐mediated expression of phase II antioxidant genes and protects against H2O2‐induced oxidative damage. We found that ALA treatment significantly induced the expressions of several phase II enzymes (glutamate‐cysteine ligases, heme oxygenase‐1, and NAD(P)H : quinone oxidoreductase 1) and resulted in the nuclear accumulation of Nrf2 protein in a time‐ and dose‐dependent manner in BEAS‐2B human bronchial epithelial cells. Accordingly, ALA treatment increased total glutathione levels and suppressed endogenous reactive oxygen species generation in BEAS‐2B cells. Furthermore, pretreatment of ALA suppressed H2O2‐induced oxidative damage in BEAS‐2B cells as measured by lactate dehydrogenase releasing assay. In conclusion, these data suggest that ALA inhibits lung carcinogenesis by activating Nrf2 mediated antioxidant defense system. (Supported by NIH grant R01CA104932).
Evaluation of an online child body mass index calculator
Authors: Downs Elissa , Harnack Lisa
Published: June 2021

Most parents of overweight children are unable to accurately assess their child's weight status. In response, online tools have been developed to improve parents understanding of their child's weight. Most notably, the Center for Disease Control and Prevention (CDC) has developed an online Body Mass Index (BMI) calculator for children. We evaluated the effectiveness of this tool for improving weight perceptions of parents of preschool‐aged children. A sample of 124 parents of 2–5 year old children was recruited through an advertisement placed in a parent magazine distributed in Minnesota. Parents were given a preliminary survey which asked them to report how they would classify their child's weight. Next, they were asked to visit the CDC website. A follow‐up survey reassessed weight perceptions. After visiting the website 89% of parents correctly classified their child's weight, an increase from 79% during the pre‐survey (p<.0001). Among parents of preschoolers at‐risk for overweight or overweight (n=29), only 2 (7%) correctly classified their child initially. At follow‐up the proportion improved to 24%. Questions asked to assess parental understanding and acceptance of the BMI calculator provide insight into why parental perceptions did not improve more substantially. Results point to the need for additional evaluations and improvements to online BMI calculator tools such as the one provided by the CDC.
Cranberry consumption improves survival in the spontaneously hypertensive stroke‐prone rat
Authors: Scanlan Adam P , Shaughnessy Kevin S , Gottschall‐Pass Kathy T , Sweeney Marva I
Published: June 2021

Hypertension is a risk factor for stroke, a leading cause of death in North America. A reduction in systolic blood pressure of only 3 mm Hg reduces the incidence of cerebrovascular disease. Vascular integrity is altered in hypertension, characterized by arterial inflammation, oxidative stress and vasoconstriction. American cranberries (Vaccinium macrocarpon) contain polyphenols which have been shown to act as antioxidants and anti‐inflammatory agents in biological systems. We hypothesized that feeding cranberry‐enriched diets (CBD) to spontaneously hypertensive stroke prone rats (SHRSP) would reduce hypertension and incidence of stroke mortalities. Eight‐week‐old SHRSP and Wistar rats (n=10) were fed one of two diets: 3% cranberry + 4% NaCl or 4% NaCl, mixed into commercial chow. SHRSP eating CBD experienced a 10–20 mm Hg reduction in systolic blood pressure by week 6. This hypotensive effect may be due to a reduction in vasoconstriction as SHRSP eating CBD had significantly higher nitrite levels in their urine after 6 wks (p=0.034), suggestive of higher levels of NO release. SHRSP fed control diet had signs of oxidative stress, with elevated F2 isoprostane excretion in the urine. Feeding cranberry reduced urinary isoprostane excretion in SHRSP rats (p=0.01). Overall, SHRSP on CBD lived 40% longer than control rats (51.3 ± 3.8 days vs 70.5 ± 9.9 days, p= 0.039). These data suggest that cranberry supplementation reduces oxidative stress and vasoconstriction, resulting in lower blood pressure and increased longevity. Funded by NSERC & Atlantic Innovation Fund.
Long Term Effectiveness of A Dietary Portfolio of Cholesterol‐Lowering Foods in Hypercholesterolemic Subjects
Authors: Kendall Cyril WC , Nguyen Tri H , Fozdar Nishant , Faulkner Dorothea A , Marchie Augustine , Wong Julia MW , Josse Andrea R , Srichaikul Kristie K , Vidgen Edward , Holmes Candice , Josse Robert G , Leiter Lawrence A , Singer William , Jenkins David JA
Published: June 2021

Objective: To determine the long term effectiveness of advice to consume a dietary portfolio of cholesterol‐lowering foods on risk factors for heart disease.

Methods: Sixty‐six hyperlipidemic subjects were prescribed diets high in plant sterols (1.0 g/1000 kcal), soy protein (22.5 g/1000 kcal), viscous fibers (10 g/1000 kcal) and almonds (23 g/1000 kcal). Thirty subjects completed 3 years.

Results: LDL‐cholesterol (LDL‐C) was significantly reduced at years 1, 2, and 3 with mean reductions of 17.7%, 11.2%, and 10.1%, respectively (P<0.01, n=30). HDL‐cholesterol (HDL‐C) was significantly raised at years 1, 2, and 3 with mean increases of 3.9%, 3.1% and 4.5%, respectively (P<0.01, n=30). Changes in LDL‐C significantly correlated with compliance to the dietary portfolio. Subjects with greater than 75% compliance to the dietary portfolio achieved a mean 24% reduction in LDL‐C.

Conclusions: A sustained long term benefit of the dietary portfolio was seen in hyperlipidemic subjects with lower LDL‐C and raised HDL‐C over a 3‐year period. The effectiveness of the dietary portfolio was greater with >20% LDL‐C reductions seen in subjects having over 75% compliance.

Funding: Canada Research Chair Endowment of the Federal Government of Canada; the Canadian Natural Sciences and Engineering Research Council of Canada; Loblaw Brands Limited; the Almond Board of California; Unilever Canada and Unilever Research & Development, Vlaardingen, The Netherlands; and Yves Veggie Cuisine.
Activation of protein kinase C disrupts epithelial apical junctions via ROCK‐dependent stimulation of myosin II contractility
Authors: Ivanov Andrei I , Samarin Stanislav N , Bachar Moshe , Parkos Charles A , Nusrat Asma
Published: June 2021

Activation of protein kinase C (PKC) destabilizes epithelial cell‐cell adhesions and therefore promotes cancer cell dissemination via unknown mechanism. The aim of this study was to investigate mechanisms underlying disassembly of epithelial tight junctions (TJ) and adherens junctions (AJ) induced by PKC activation. Exposure of HPAFII human pancreatic adenocarcinoma cell monolayers to either Octylindolactam V (OI) or phorbol ester caused a rapid (within 3–5 h) and orchestrated disassembly and internalization of AJ and TJ. Junctional disassembly was accompanied by translocation of the F‐actin motor myosin II to the lateral plasma membrane and by myosin II activation (phosphorylation). The OI‐induced disruption of AJ/TJ was significantly attenuated by either pharmacological inhibition of myosin II with blebbistatin or by siRNA‐mediated knockdown of the myosin IIA isoform. Furthermore, AJ/TJ disassembly was attenuated by inhibition of Rho‐associated kinase (ROCK) but was insensitive to blockage of MLCK, ERK, calmodulin or Rac‐1. These findings support a role of PKC in disassembly of epithelial junctions by stimulating ROCK‐dependent contractility of myosin II which in turn can contribute to cancer cell dissociation and metastasis. Supported by the Crohn's and Colitis Foundation of America (AII and SNS) and NIH (AN: DK59888 and CAP: DK61379).
Title: Analysis of differential hepatocyte nuclear factor 4 isoform expression during hepatic de‐differentiation and re‐differentiation in‐vitro
Authors: Bell Aaron W , Stoops John
Published: June 2021

Hepatocyte nuclear factor 4α is essential for liver development and maintenance of liver‐specific gene expression. HNF4α isoform expression is developmentally regulated. We have established a serum‐free hepatocyte culture system in which primary rat hepatocytes de‐differentiate and acquire a simplified morphologic phenotype resembling hepatoblasts. Addition of Matrigel, DMSO or phenobarbital to the cells induces re‐differentiation and restoration of hepatocyte‐specific gene expression and mature phenotype. Western blot and real‐time PCR analyses reveals that HNF4α isoform expression is directly correlated with hepatic differentiation status and mimics embryonic liver development. To determine the mechanism(s) of this differential regulation we examined the activation and/or inhibition of various kinase pathways with each of the differentiation‐inducing agents. Inhibition of TGFβRkinase or PI3Kinase with specific chemical inhibitors induces re‐expression of P1 isoforms and augments the re‐differentiation induced by DMSO, PB and Matrigel which also requires activation of the CaMKinase pathway. We propose that TGFβ□/Smad signaling and PI3Kinase are responsible for the expression of the P2‐derived isoforms and de‐differentiated phenotype and their inhibition coupled with activation of the CaMKinase pathway induces the P1‐derived HNF4α isoforms and the differentiated phenotype.
Regulation of ser‐45 mutated beta‐catenin in transgenic mouse livers
Authors: Thompson Michael David , Cieply Ben , Singh Sucha , Monga Satdarshan
Published: June 2021

Wnt/β‐catenin activation is frequently observed in hepatocellular carcinomas (HCC) due to multiple mechanisms. A common event is mutation at serine‐45 in β‐catenin rendering a stable protein. S45‐mutated β‐catenin was utilized to generate liver‐specific transgenic mice (Hep‐S45D‐Tg). Two independent lines were characterized and had a similar phenotype. Increased liver weight to body weight ratio from 10‐20% was observed in Hep‐S45D‐Tg (TG) relative to FVB wild‐type (WT). Increased levels of β‐catenin in the nucleus were observed at 1 month, which coincided with increased cyclin‐D1 and significantly higher Ki‐67‐positive hepatocytes. Interestingly, elevated β‐catenin was observed only at the hepatocyte membrane at 2 and 6 months with no change in proliferation but with an increase in average hepatocyte size. Decreased apoptosis was evident at 6 months only. No spontaneous tumors were observed in TG mice up to 12 months. DEN‐induced hepatocarcinogenesis was investigated in WT and TG mice. Females were protected up to 9 months. Male TG mice showed hepatic adenomas (HA) and HCC at 9 months, whereas WT mice showed HA only. Thus, TG mice demonstrated nuclear β‐catenin and activation that led to increased liver size at 1 month that was later regulated by sequestration of ‐catenin at the hepatocyte membrane. Also, TG mice showed higher incidence of HCC after DEN treatment.
Direct oxygenation and perfusion enhance viability and function of hepatocyte‐seeded scaffolds
Authors: Mbanu Chijioke A. , Matthew Howard W.T.
Published: June 2021

Progress in hepatic tissue engineering has been hindered by the challenge of adequately oxygenating high density hepatocyte cultures in 3‐D scaffolds. This effort evaluated the effects of direct scaffold oxygenation and variable perfusion rates on hepatocyte morphology, viability and function in densely seeded scaffolds. The fluid shear environment was assessed using computational fluid dynamics (CFD) modeling with pore and cell aggregate parameters taken from actual seeded scaffolds. Integral silicone tubing was used to facilitate gas exchange in chitosan scaffolds seeded with 80 million rat hepatocytes. Perfusion cultures and CFD simulations were run at flow rates of 0, 5, 10 and 15 ml/min. Cultures were evaluated at day seven. Increasing flow rates improved initial rates of albumin and urea secretion, and histological analysis showed increased cell aggregation and viability with higher flow rates. However at higher flows, synthesis rates declined more rapidly over the 7 days of culture. Viable hepatocyte aggregates were localized in areas of higher oxygen levels, notably in the vicinity of the integral oxygenation tubing. The results of this study indicate that direct oxygenation and higher perfusion rates facilitate hepatic function, but higher flows also pose a substantial risk of cellular damage due to fluid shear. Supported by Wayne State University.
Liver mesenchymal Thy‐1 cells: important player in oval cell mediated liver regeneration
Authors: Yovchev Mladen I , Zhang Jialin , Neufeld David , Grozdanov Petar , Dabeva Mariana D
Published: June 2021

Recently, we and others have reported that Thy1 antigen is expressed in rat liver myofibroblasts. To study further the properties and the function of these cells, we purified Thy‐1+ cells by MACS and FACS and analyzed their gene expression pattern using Affymetrix Rat expression array 230 2.0, RT‐PCR and IF microscopy. Thy‐1+ cells are highly activated in models of oval cell‐mediated liver regeneration, tightly surrounding the oval cell pseudoducts. Thy‐1+ cells appear, proliferate and disappear from the liver in parallel with oval cells. Thy‐1+ cells express mRNA for genes considered specific for stellate cells: desmin, v‐cam, gfap, pdgfrβ, integrin β3; for myofibroblasts: αsma, elastin, fibulin‐2, fibrilin‐1; for matrix remodeling proteins: mmp2, collagen I‐a1, ‐a2, III and IV; for mesenchymal stromal cells: CD54, CD73, CD105, CD166, SDF‐1; and for growth factors and cytokines that can stimulate oval cell growth: Il‐6, hgf, tweak. Co‐expression of Thy‐1 antigen with desmin, αSma and Pdgfrβ was confirmed by double IF microscopy. RT‐PCR analysis of isolated individual Thy‐1 expressing clones confirmed the heterogeneity of these cells. Conclusion: liver mesenchymal Thy‐1+ cells are the major supporting cells in the oval cells niche.

Supported by NIH grant R01 DK59321 (to M.D.D.)
Regulation of liver regeneration by T cell‐ derived lymphotoxin
Authors: Tumanov Alexei , Christiansen Peter , Koroleva Ekaterina , Anders Robert , Fu Yang‐Xin
Published: June 2021

The liver has a remarkable capacity to regenerate after injury or partial hepatectomy. The process of liver regeneration is tightly regulated by distinct signaling cascades involving components of innate immune system, cytokines and growth factors. However, the role of adaptive immune system in regulation of liver regeneration is not well defined. We found that mice deficient in T cells show a reduced capacity to regenerate after partial hepatectomy. We further demonstrate that surface Lymphotoxin (LT), a member of the TNF family of cytokines, provided by T cells is critical for liver regeneration. T cell‐specific LTβ deficient mice have increased liver damage and a reduced capacity to initiate DNA synthesis after partial hepatectomy. Transfer of splenocytes from wild type but not from LT deficient mice to lymphocyte deficient Rag mice improved liver regeneration. To develop a novel treatment to facilitate liver regeneration, we used an agonistic anti‐LTβR antibody which resulted in an increase in survival of Rag mice. Thus, our study revealed an unexpected role of lymphocyte‐restricted ligands in the control of liver regeneration.
Spontaneous cardiac hypertrophy results from the loss of Muscle Ring Finger 1 and 2
Authors: Rodriguez Jessica E , Li Luge , Lockyer Pamela , Patterson Cam , Willis Monte S
Published: June 2021

The muscle ring finger family of proteins (MuRF1‐3) are involved in the regulation of cardiac protein homeostasis. We recently described that MuRF1, like MuRF2, interacts with serum response factor, a transcription factor integral in regulating gene expression involved with cardiac growth. Since hearts from mice lacking either MuRF1 or MuRF2 are normal at baseline, we created MuRF1/MuRF2 double knock‐out mice to directly determine the possible redundancy of MuRF1 and MuRF2 in cardiac structure and function. When we crossed MuRF1/MuRF2 double heterozygous mice, we identified only 3 MuRF1/MuRF2 double null mice out of 231 at weaning, which was significantly less than the 14 (1/16) expected (χ2=0.0035). MuRF1/MuRF2 double knock‐out mice died prematurely at an average age of 2 weeks, with extraordinarily large hearts that took up most of the thoracic cavity. These hearts were notably hypertrophic with thickened anterior and posterior ventricular walls. Histologically, the hearts demonstrated increased cardiomyocyte size and extensive transmural fibrosis. These findings suggest overlapping roles of MuRF1 and MuRF2 in the regulation of cardiomyocyte size during development in vivo, consistent with their redundant interactions with proteins such as serum response factor and/or specific myofibrillar proteins they interact with.
High glucose alters tubular epithelial cell expression of molecules in the renin‐angiotensin axis
Authors: Kruss Szilvia , Danziger Robert , Kotlo Kumar , Setty Suman
Published: June 2021

Purpose: Aminopeptidase N (APN), a type II membrane ectoenzyme also known as CD13, is a major component of Glut‐4 containing vesicles. It has been postulated to regulate secretory function in general and glucose transport in particular. The present study was performed to determine the effect of glucose and insulin on the expression and activity of APN in renal epithelial cells.

Methods: A heightened glucose milieu was created by culturing immortalized HK‐2 cells in 25 mM glucose as compared to the normal 5 mM glucose and a control for hyperosmolality (cells grown in 5 mM glucose with 20 mM mannitol)(OC). The model was validated by studying the altered growth and adhesion characteristics of the HK‐2 cells in the three conditions. Assays for APN activity and APN transcript abundance were performed on cells cultured in the three conditions. The effect of insulin and lisinopril on glucose‐induced changes APN activity was examined.

Results: APN transcript, protein, and activity were greater in cells treated with 25 mM vs 5 mM glucose and the same in cells treated with OC vs 5 mM glucose. Insulin reduced APN activity to that observed in low glucose (5mM) levels (p<0.05).

Conclusions: Glucose is capable of stimulating APN in renal epithelial cells. This stimulation was blocked by insulin, raising the possibilities that APN plays a role in glucose homeostasis in general and in the pathogenesis of diabetes specifically.
In Vivo Demonstration of Enhanced Oxidized Low Density Lipoprotein Uptake and Cholesterol Esterification by C‐Reactive Protein
Authors: Mohan Dasu Ravi , Devaraj Sridevi , Singh Uma , Yancey Patricia , Jialal Ishwarlal
Published: June 2021

CRP promotes oxidized low density lipoprotein (ox‐LDL) uptake in vitro. However, this effect has not been demonstrated in vivo. In this study, we investigated the effects of CRP on ox‐LDL uptake using the rat sterile air pouch model. Air pouches were made by injecting clean and sterile air into Wistar rats (n=5/group).Group 1 received purified CRP (25 μg/ml)/DiI‐ox‐LDL (50 μg/ml), Group 2 received DiI‐ox‐LDL/albumin (HuSA), and group 3 received 25 μg/ml HuSA. A second set of rats were pretreated with CD32, CD36, CD64 blocking antibodies, or Fucoidin, followed by ox‐LDL/CRP. Pouch macrophages were collected, and used for measuring ox‐LDL uptake by flow cytometry, stained with Oil‐Red O, and intracellular cholesterol accumulation by gas chromatography. CRP significantly increased ox‐LDL uptake in macrophages (9.5±0.8 to 25.7±2.2 MFI; P<0.05) and showed intense Oil‐Red O staining compared to group 2. CRP increased the cholesterol ester accumulation in group 1 rats compared to group 2 (2.8±0.5 to 1.3±0.4; P<0.05). The increased uptake of ox‐LDL by CRP was inhibited by CD32 (66%), CD64 (46%), CD36 (55%), Fucoidin (61%), CD32/CD36 (75%) compared to CRP/ox‐LDL (P<0.05). Our data provides convincing evidence that CRP significantly increases ox‐LDL uptake in rats, and this leads to accumulation of cholesterol esters, the hallmark of atherosclerosis.
The inhibition of insulin‐like growth factor 1 receptor pathway targets putative cancer stem cells in human malignant mesothelioma
Authors: Kim Yongbaek , Kai Kiyonori , Sills Robert C
Published: June 2021

Human malignant mesothelioma (HMM) is a fatal tumor with median survival time of 6–18 months despite of aggressive treatment schedule. Insulin‐like growth factor 1 receptor (IGF1R) pathway has known to be activated in various HMM cell lines. This study was performed to investigate the anti‐tumor effects and to elucidate an underlying mechanism of a chemical inhibitor of IGF1R pathway, AG1024, in HMM cell lines. The treatment of AG1024 decreased the cell proliferation in a dose‐dependent manner and also significantly enhanced the cytotoxicity of cisplatin which is a DNA binding anticancer drug. The inhibitory effects of AG1024 were largely dependent on the suppression of downstream pathways (PI3K and MAPK) and the changes in the expression of apoptosis‐related genes. Intriguingly, AG1024 treatment reduced the abundance of side population determined by flow cytometric analysis using Hoechst 33342 staining. The expression levels of stem‐cell related genes in the side population were altered by AG1024 treatment. The results of this study suggest that IGF1R pathway plays a crucial role in the maintenance of cancer stem cells in HMM, thus it could be a plausible target for cancer stem cell‐specific therapy to overcome chemoresistance of HMM.
Knock down of AQP1 in HMEC‐1 cells and WM115 cells changes the organisation of the cytoskeleton
Authors: Porta caterina AM La , Monzani Elena , Facchetti Floriana , perego Carla
Published: June 2021

Aquaporin 1 (AQP1) is specifically and strongly expressed in most microvasculature endothelial cells outside of the brain. Herein we have investigated the role of AQP1 in both microvascular endothelial (HMEC‐1) and in a human melanoma cell line (WM115). The knock down of AQP1 by RNA interference modified in both cells the organization of F‐actin as well as reduced the migration and invasive capacity significantly. In order to better understand the intracellular mechanisms elicited by AQP‐1, we have investigated the role of Lin‐7, a PDZ protein which is involved in the recruitment of receptors, adhesion molecules and signalling proteins and that can interact with cytoskeleton through beta‐catenin.

Our results showed that AQP1 co‐immunoprecipitated with Lin‐7 and the knock down of AQP‐1 decreased the level of expression of Lin‐7 and beta‐catenin. Interestingly, the inhibition of proteasome contrasted partially such a decrease.

All together, our findings show, for the first time, that AQP1 triggers an intracellular mechanisms modulating and stabilizing the organization of cytoskeleton through Lin‐7/beta‐catenin interaction. A model for these functions of AQP1, at least for endothelial and melanoma cells, is presented.
Human monocytes and proinflammatory cytokines stimulate breast and prostate cancer cell invasion
Authors: Lindholm Paul Franklin , Lu Yi , Jovanovic Borko D , Sivapurapu Neela , Lee Chung
Published: June 2021

The objective of this study is to determine how monocyte/macrophages stimulate cancer cell growth and invasion. Co‐cultures with freshly isolated human monocytes or monocytoid cell lines stimulated invasion of several breast and prostate cancer cell lines. The co‐cultures produced high levels of IL‐6 and IL‐8 and increased MCP‐1 and Gro‐α chemokines above cancer cell or monocyte culture supernatants alone. Addition of purified recombinant interleukin‐8 and MCP‐1 proteins stimulated monocyte and cancer cell chemotaxis in Matrigel‐coated Transwell chambers. The co‐cultures also stimulated cancer cell NF‐κB reporter activity and nuclear localization. The co‐culture induced cancer cell invasion was blocked by biochemical inhibitors or dominant negative expression of IκBα or RhoA N19 mutants that inhibited NF‐κB activity. These studies indicate that human breast and prostate cancer cells cross‐communicate with monocytoid cells to stimulate cancer cell invasion in a NF‐κB dependent manner. This model has potential to identify treatments that block monocyte/macrophage‐induced cancer cell invasion and progression. Supported by Prostate S.P.O.RE. P50CA90386 and CDMRP DAMD‐17‐02‐1‐0162.
The Rho inhibitor p190RhoGAP controls VEGFR2 transcription and angiogenesis in vitro through antagonism between GATA2 and TFII‐I
Authors: Mammoto Akiko , Mammoto Tadanori , Ingber Donald
Published: June 2021

Angiogenesis can be controlled by mechanical interactions between cells and extracellular matrix (ECM) mediated by the Rho GTPase signaling pathway, as well as by the soluble angiogenic factor, vascular endothelial growth factor (VEGF) that stimulates capillary blood vessel development by binding VEGFR2 receptors on the endothelial cell surface. Here we show that the Rho inhibitor, p190RhoGAP, regulates angiogenesis by controlling VEGFR2 expression, and that it produces this effect by modulating cross antagonism between two transcription factors ‐ TFII‐I and GATA2 – that regulate VEGFR2 transcription. p190RhoGAP binds to both TFII‐I and GATA2, and sequesters them in the cytoplasm, thereby preventing VEGFR2 expression. TFII‐I decreases VEGFR2 promoter activity and expression as well as migration and capillary tube formation by human microvascular endothelial cells, whereas GATA2 produces the opposite effects, and antagonizes TFII‐I. Thus, the balance between TFII‐I and GATA2 appears to be critical for regulation of vascular network formation, and it appears to be controlled by p190RhoGAP that can sense both chemical and mechanical cues in the tissue microenvironment. (Supported by NIH)
Soluble epoxide hydrolase (Ephx2) is a susceptibility gene for heart failure in a rat model of human disease 3044
Authors: Hubner Norbert , Monti Jan , Fischer Judith , Paskas Svetlana , Heinig Matthias , Schulz Herbert , Goesele Claudia , Heuser Arnd , Fischer Robert , Schmidt Cosima , Schirdewan Alexander , Gross Volkmar , Hummel Oliver , Maatz Henrike , Patone Giannino , Saar Kathrin , Vingron Martin , Weldon Steven M. , Hammock Bruce D. , Rohde Klaus , Dietz Rainer , Cook Stuart A. , Schunck Wolf‐Hagen , Luft Friedrich C.
Published: June 2021

We aimed to idenify heart failure genes by using a rat model of the human disease. We performed invasive cardiac hemodynamic measurements in F2 crosses using spontaneously hypertensive heart failure rats (SHHF) and reference strains. We combined linkage analyses with genome wide expression profiling and identified Ephx2 as heart failure susceptibility gene in SHHF. We demonstrate that cis‐variation at Ephx2 co‐segregated with heart failure, increased transcript expression, protein expression, and enzyme activity, leading to a more rapid hydrolysis of cardioprotective epoxyeicosatrienoic acids. To confirm our results, we next tested the role of Ephx2 in heart failure by using knockout mice. We show that Ephx2 gene ablation protects from pressure overload‐induced heart failure and cardiac arrhythmias. We further demonstrate differential regulation of Ephx2 in human heart failure suggesting a cross‐species role for Ephx2 in this complex disease.
Synthesis and biological evaluation of soluble epoxide hydrolase (sEH) inhibitors: t‐AUCB and its derivatives
Authors: HWANG SUNG HEE , Tsai Hsing‐Ju , Morisseau Christophe , Liu Jun‐Yan , Hammock Bruce D.
Published: June 2021

Soluble epoxide hydrolase (sEH, EC ) is involved in the metabolism of endogenously derived fatty acid epoxides, such as arachidonic acid, linoleic acid, and other lipid epoxides. Epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase products of arachidonic acid, are known modulators of blood pressure and inflammation. A series of N,N′‐disubstituted ureas having a conformationally restricted cis‐ or trans‐1,4‐cyclohexane alpha to the urea were prepared and tested as sEH inhibitors. This series of compounds showed low nanomolar to picomolar activities against recombinant human sEH. Both isomers showed similar potencies, but the trans‐isomers were more metabolically stable in human hepatic microsomes. Furthermore, these new potent inhibitors show a greater metabolic stability in vivo than previously described sEH inhibitors. Among them, trans‐4‐[4‐(3‐Adamantan‐1‐yl‐ureido)‐cyclohexyloxy]‐benzoic acid (t‐AUCB, IC50 = 1.3 ± 0.05 nM) had excellent oral bioavailability (98%, n = 3) and blood area under the curve (AUC) in dogs and was effective in vivo to treat hypotension in lipopolysaccharide (LPS) challenged murine models. The synthesis, SAR, and PK properties of this series as well as the scale‐up process and efficacy of t‐AUCB along with PK profile of corresponding esters and salts of t‐AUCB also will be presented.

(supported in part by NIEHS grant ES02710, NIEHS Superfund grant P42 ES04699, and NIEHS Center grant P30 ES05707)
Pharmacokinetics of soluble epoxide hydrolase inhibitors in canine, rodent, feline, and primate species
Authors: Tsai Hsing‐Ju , Liu Jun Yan , Hwang Sung Hee , Kasagami Takeo , Kim In Hae , Jones Paul D. , Morisseau Christophe , Hammock Bruce D.
Published: June 2021

Soluble epoxide hydrolase inhibitors (sEHi) have been shown the therapeutic effects on hypertension, sepsis, and hypertrophy in different animal models. However, the inhibitors were hard to formulate which limits the administration methods, and their bioavailabilities were low leading to high dose treatments for observing some biological effects in vivo. Thus, compounds having improved physical properties and similar potency were recently synthesized. However, it is unknown the compounds perform consistent in vitro and in vivo among species. Herein, the results showed that the in vitro potency of some compounds were different among species, especially with dog and cat enzymes. Further, we also described the pharmacokinetic profiles in vivo of different animals. In summary, the inhibitors have different potency and pharmacokinetic profiles among species. With the information, sEHi will be invaluable tools to investigate the role of sEH and eicosanoids in biological systems. This work was supported in part by NIEHS Grant R37 ES02710, NIEHS SBRP Grant P42 ES04699, and NIEHS CEHS Grant P30 ES05707.
Molecular mechanisms involved in the regulation of the cytochrome P450 epoxygenase CYP2J5
Authors: Graves Joan P , Zhang Donghui , Maestro Miguel A , Servitja Joan M , King Lorraine M , Swope Deborah L , Korach Kenneth S , Ferrer Jorge , Zeldin Darryl C
Published: June 2021

CYP2J5 is expressed in kidney and liver, and metabolizes arachidonic acid to epoxyeicosatrienoic acids. Herein, we identify the molecular mechanisms responsible for regulation of this gene. The transcriptional start site of the Cyp2j5 gene was located and analysis of the proximal promoter region identified a putative HNF1alpha binding site and a putative androgen response element (ARE). Electrophoretic mobility shift assays (EMSAs) demonstrated binding of androgen receptor (AR) to the Cyp2j5 ARE. Chromatin immunoprecipitation assays with mouse kidney cells confirmed specific binding of AR to the Cyp2j5 ARE, and this was increased in cells treated with androgen compared to cells treated with vehicle. EMSAs also showed the binding of HNF1alpha to the putative Cyp2j5 binding site. Reporter assays with a Cyp2j5 promoter vector demonstrated activation when an HNF1alpha overexpression vector was co‐transfected but not when there was a mutation in the HNF1alpha site. Immunohistochemistry demonstrated a marked reduction in the expression of Cyp2j5 in livers but only minimal changes in kidneys of HNF1alpha knockout mice relative to wild type controls. These findings provide a molecular basis for the observed gender and tissue specific differences in the expression of the Cyp2j5 gene. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.
Importance of microsomal epoxide hydrolase (mEH) in toxicity, metabolism and formation of covalent protein adducts from the volatile air pollutant, naphthalene (NA): Comparison of mEH null and wild type (WT) mice
Authors: Krawiec David M , Sutherland Katherine , Morin Dexter , Thornton Jason , Buckpitt Alan , Van Winkle Laura
Published: June 2021

Human exposure to NA is due to combustion of both environmental and man‐made materials. Toxicity of NA is P450‐dependent, occurring in murine, but not rat, airways. However, the extent to which specific metabolic pathways (to epoxides, quinones and diol epoxides) contribute to cytotoxicity and the formation of covalent protein adducts is unknown. Upon inhalation exposure to NA (4h at 5–20 ppm) male mEH null animals had increased cytotoxicity in the bronchioles, but decreased susceptibility in the trachea and lobar bronchus as compared to WT mice, despite having a normal pattern of P450‐containing CC10 positive Clara cells. Formation rates of NA metabolites (conjugates plus diol) in the trachea were lower in EH null animals, but equivalent in the airways compared to WT. As expected, rates of diol formation were undetected/low in mEH null animals. Protein adducts detected after ip administration of NA were 2–4 fold higher in airway epithelium than elsewhere in the lung, and EH null animals showed significantly higher levels compared to WT. Bound liver adducts showed no differences between mEH and WT animals. These studies suggest that metabolites (1,2‐quinone, diol epoxide) arising via the diol pathway are not critical to NA toxicity or formation of covalent adducts in distal airway epithelium. This work also suggests that there are compensatory changes associated with disruption of mEH. NIEHS 04311, 12720 and 04699.
A high‐throughput fluorescence polarization assay for the determination of Kd for soluble epoxide hydrolase inhibitors
Authors: Ingraham Richard H. , Kroe Rachel R. , Morelock Maurice M. , Proudfoot John R. , Eldrup Anne B. , Canada Keith , Cardozo Mario , Grygon Christine A.
Published: June 2021

Soluble epoxide hydrolase (sEH) has been demonstrated to play a role in a number of physiological processes through its ability to convert epoxy fatty acids such as the highly active epoxyeicosatrienoic acids (EETs) into their less active vicinal diols. Because EETs exhibit potentially beneficial effects in various pathologies such as inflammation and hypertension, an sEH inhibitor could exert a therapeutic benefit by preventing their hydrolysis. We have developed a competitive binding fluorescence polarization screening assay for sEH inhibitors which utilizes a di‐alkylurea active site inhibitor linked to a rhodamine probe. The probe binds to various mammalian sEH enzymes with Kd values in the low nanomolar range. Inhibitor compounds were initially fit to yield IC50 values. However, using that simple fitting paradigm it was not possible to distinguish the affinities of more potent compounds. To address this, a sophisticated fitting algorithm was developed which enabled the use of more information from the binding curves and the calculation of Kd values for the inhibitors.
Analysis of the topology of the microsomal epoxide hydrolase on the cell surface with monoclonal antibodies with different epitope specificities
Authors: Duan Hongying , Yoshimura Kazunori , Kobayashi Nobuharu , Takagi Akira , Matsui Masanori , Ohno Satoshi , Sugiyama Kazuo , Morisseau Christophe , Hammock Bruce D. , Akatsuka Toshitaka
Published: June 2021

We have reported the autoantibody response to microsomal epoxide hydrolase (mEH) in patients with hepatitis C and A (J. Autoimmunity 28:7, 2007). In another poster in this meeting, we demonstrated that RNA virus infection induced surface expression of mEH on hepatocyte cell lines, and made the cells more sensitive to a potent carcinogen DMBA. To analyze the topological changes of mEH in these conditions, we developed five groups of monoclonal antibodies with different epitope specificities as determined by ELISA with truncated mEH, western blotting and competitive binding assay. Three (type I to III) recognized the N‐terminus, one (type IV) recognized the C‐terminus, and one (type V) recognized only the conformational epitope. FACS analysis showed that all the epitopes except type IV were expressed on the surface of freshly isolated hepatocytes whereas none were detected on an immortalized hepatocyte cell line. When this cell line was infected with RNA viruses, only the type V was induced on the surface. A differentiated hepatocellular carcinoma (HCC) line, Huh7 also exhibited only the type V epitope on the surface, and its expression was enhanced by virus infection. An undifferentiated HCC line, HuH1 showed all the epitopes on the surface. These monoclonal antibodies seem to be useful to analyze the role of mEH in pathological conditions.
Microsomal epoxide hydrolase E404D mutation influences the turnover of epoxyeicosatrienoic acids
Authors: Burgener Julia , Marowsky Anne , Cronin Annette , Arand Michael
Published: June 2021

Traditionally, the microsomal epoxide hydrolase is thought of as a detoxifying enzyme as it hydrolyzes many carcinogenic epoxides to their corresponding dihydrodiols. Recent data by our group that substantiate earlier findings implicate that the mEH may also have a physiological role other than detoxification. The enzyme is capable of hydrolyzing epoxyeicosatrienoic acids (EETs), a class of signaling molecules known to regulate a wide variety of physiological functions, such as blood pressure, angiogenesis, inflammation and apoptosis. Thus, by metabolizing the EETs to the corresponding dihydrodiols, the mEH might also be implicated in the modulation of such processes.

The enzymatic reaction mechanism involves a nucleophilic attack followed by a hydrolysis via a water‐activating charge relay (H431 and E404). An artificial E404D mutant shows a faster metabolization rate compared to the wild type enzyme. This more efficient variant has, so far, not been found in the animal kingdom. We hypothesize that this may be connected to an altered EET metabolism by the E404D variant. So far, we have expressed recombinant wild type and E404D mEHs from rat, mouse and human and presently analyze their turnover rate with EETs using LC‐MS/MS.

We expect that our findings will elucidate some more details about the physiological role of mEH and may help to explain why in animals the less active version of mEH is established.
Soluble Epoxide Inhibition Differentially Modulates Flow Induced Vascular Remodeling in Spontaneously Hypertensive Stroke Prone Rats and Wistar Kyoto Rats
Authors: Simpkins Alexis N , Rudic R. Daniel , Roy Siddhartha , Hammock Bruce D. , Imig John D.
Published: June 2021

Although soluble epoxide hydrolase (SEH) inhibition restrains vascular smooth muscle cell (VSMC) proliferation in culture, SEH inhibition's affect on flow‐induced remodeling (FIR) is unknown. Here FIR was induced by left common carotid (LC) ligation in Wistar Kyoto (WKY) and spontaneously hypertensive stroke prone (SHRSP) rats. After 5 weeks of SEH inhibition via adamantanyl dodecanoic acid (AUDA) 2mg/day, FIR was assessed. Systolic blood pressure averaged 146±2mmHg in WKY (n=5) versus (vs) 194mmHg in SHRSP (n=4) rats and was not altered by AUDA. While, AUDA did not alter right common carotid (RC) structure in WKY, AUDA did reduce inward remodeling initiated by LC ligation in WKY, reducing wall to lumen ratio 25.9%, wall thickness 21.1 %, and medial area 19.5% vs control WKY (P<0.05). In contrast, in the SHRSP, AUDA enhanced outward remodeling of RC, increasing outer diameter (OD) 6.75%, wall thickness 6.8%, and medial area 17.1% vs control SHRSP (P<0.05). AUDA normalized inward remodeling in the ligated LC SHRSPs in comparison to control WKY, reducing lumen area 10.4% and OD 6.8% (P<0.05). Interestingly, this effect was due to cell proliferation as quantified by an increase in proliferating cell nuclear antigen (9.2 fold) and VSMC count (28%). These results suggest that SEH inhibition reduces inward remodeling in normotension and normalizes FIR in hypertension by modulating VSMC organization. (AHA and NIH)
Soluble epoxide hydrolase inhibition attenuates the development of renovascular hypertension in 2K1C rats.
Authors: Huskova Zuzana , Kopkan Libor , Walkowska Agnieszka , Vanourkova Zdenka , Hammock Bruce D. , Imig John D. , Cervenka Ludek
Published: June 2021

The present study tested the hypothesis that inhibition of soluble epoxide hydrolase (sEH), that increases the level of epoxyeicosatrienoic acids, would attenuate the development of renovascular hypertension in 2K1C rats. Renovascular hypertension was induced by placing a silver clip on the left renal artery in Wistar rats for 25 days. The sEH inhibitor, c‐AUCB was given in drinking water (0.5 mg/day) starting 48 hours before the renal artery clipping. Blood pressure (BP) was monitored by radiotelemetry in conscious animals during 30 days of experimental period. At the end of the experiment the ratio of left heart ventricular weight (mg) to tibial length (mm) (LVW/TL) was used for assessing cardiac hypertrophy.

After 30 days of experimental period, systolic BP was not different in sham operated rats treated with sEH inhibitor (106±2 to 104±3 mmHg; n=5) compared to sham control group (107±3 to 112±4 mmHg; n=4). However, the sEH inhibitor, c‐AUCB, attenuated the development of hypertension in treated 2K1C rats (110±2 to 138±3 mmHg, p<0.05; n=5) compared to untreated 2K1C (113±4 to 161±7 mmHg, p<0.05; n=6). Moreover, significantly higher LVW/TL ratio in untreated 2K1C compared to sham control group (26±1 vs. 20±1) was partially attenuated in 2K1C rats treated with sEH inhibitor (23±1, p<0.05).

These data demonstrate that sEH inhibition attenuates the development of renovascular hypertension.
A major ingredient of green tea rescues mice from lethal sepsis partly by inhibiting HMGB1
Authors: Wang Haichao , Li Wei , Ashok Mala , Zhu Cassie (Shu) , Sama Andrew
Published: June 2021

The pathogenesis of sepsis is mediated in part by bacterial endotoxin, which stimulates macrophages/monocytes to sequentially release early (e.g., TNF) and late (e.g., HMGB1) pro‐inflammatory cytokines. Our recent discovery of HMGB1 as a late mediator of lethal sepsis has prompted development of new experimental therapeutics. We previously reported that green tea brewed from the leaves of the plant, Camellia sinensis, is effective in inhibiting endotoxin‐induced HMGB1 release. Here we demonstrates that its major component, (−)‐epigallocatechin‐3‐gallate (EGCG) dose‐dependently abrogated HMGB1 release in macrophage/monocyte cultures, even when given 2–6 hours post LPS stimulation. Intraperitoneal administration of EGCG protected mice against lethal endotoxemia, and rescued mice from lethal sepsis even when the first dose was given 24 hours after cecal ligation and puncture. The therapeutic effects were partly attributable to: 1) attenuation of systemic accumulation of proinflammatory mediator (e.g., HMGB1) and surrogate marker (e.g., IL‐6 and KC) of lethal sepsis; and 2) suppression of HMGB1‐mediated inflammatory responses by preventing clustering of exogenous HMGB1 on macrophage cell surface. Taken together, these data suggest a novel mechanism by which the major green tea component, EGCG, protects against lethal endotoxemia and sepsis.
Self‐organization and pattern formation in calcifying vascular stem cells
Authors: Demer Linda L. , Yochelis Arik , Tintut Yin , Garfinkel Alan
Published: June 2021

We previously observed patterns of evenly spaced stripes of calcified cellular aggregates in cultures of calcifying vascular cells (CVC), bovine smooth muscle cells purified by dilutional cloning. We hypothesized that the patterns were formed by a reaction‐diffusion process driven by interaction of a known morphogen, bone morphogenetic protein‐2 (BMP‐2), with its inhibitor, matrix GLA protein (MGP). We used mathematical models based on the biochemical and physical properties of BMP‐2 (autocatalytic, chemoattractant, and osteogenic) and MGP (inhibitory of BMP‐2 and rapidly diffusing).

Computer simulations based on the math models predicted 2 experimental findings. Over 10–14 days, CVC cultures treated with exogenous MGP produced evenly spaced spots (nodules) rather than stripes, and CVC treated with warfarin (an inhibitor of MGP) produced stripes at double the density of untreated cultures. In addition, the models predicted conversion of diffuse cell density to focal nodules. In vivo, when CVC were implanted for 8 wks in subcutaneous diffusion chambers in hyperlipidemic mice, they produced tissue nodules resembling calcified atherosclerotic plaque.

Thus, calcified atherosclerotic lesions may arise from cellular aggregates formed by reaction‐diffusion processes. Harnessing this mechanism may ultimately allow clinical control of focal vs. diffuse vascular disease.
Role of epigenetic histone modifications during early embryonic stem cell differentiation
Authors: Fritsch Michael Kevin , Murdoch Fern , Singer Don , Lee Elliot , McCool Kevin , Alexanian Ruben , Oduro Akua
Published: June 2021

Early embryonic stem cell differentiation includes “exit” from the pluripotent state followed by “commitment” to a specific lineage. During both of these processes there are genome‐wide changes in gene transcription levels that are regulated, at least in part, by epigenetic changes in histone tail modifications. We have demonstrated both global and gene specific changes in histone tail modifications. We focused on the role of these marks as they regulate the earliest stages of differentiation in mouse ES cells.

Histone H3 phosphoacetylation (activating mark) is increased within the first 3 days of differentiation. Blocking general phosphorylation pathways markedly slowed exit from the pluripotent state. Blocking MSK1, the specific kinase responsible for phosphorylation on histone H3 still resulted in exit, but with diminished expression of early mesoderm markers. We also demonstrated a gene specific increase in acetylation (activating) of histones H3 and H4 and a global increase in acetylation of H4 as cells began lineage commitment. A pulse of the histone deacetylase inhibitor, TSA, maintained expression of markers of the undifferentiated state while also inducing expression of early mesoderm markers. TSA effects on later lineage commitment are being studied.

We propose that epigenetic modifications play an important role in early cell fate decisions.

This work was supported by NIH RO1 DK064243 from NIDDK.
Endodermal Sox17‐dependent pathways & cardiac specification
Authors: Liu Yu , Kaneda Ruri , Schneider Michael D
Published: June 2021

ES cells are unlimited in the potential to generate multiple cell types for cardiac repair; they also offer a faithful model of embryogenesis for dissecting the signal pathways governing cardiac development. Using lentiviral delivery of shRNA, we established the requirement for an endoderm‐expressed Sry‐box containing protein, Sox17, for cardiac myocyte differentiation in ES cells, through cell non‐autonomous mechanisms. Sox17 functions subsequent to mesoderm formation but prior to induction of Mesp1 and Mesp2, bHLH transcription factors that share an indispensable function in forming cardiac mesoderm. Sox17 induction is dependent on both the canonical Wnts and the Nodal/Activin pathways. The canonical Wnts‐beta‐catenin pathway, however, is chiefly responsible for the induction of mesoderm and is suppressed during cardiac specification; Sox17 binds and suppresses beta‐catenin‐dependent transcription, thus plays a key role in the process. The Nodal/Activin‐Cripto pathway is more specific in inducing Sox17 and subsequent endodermal cardiac‐inducing signals, including Hhex and Cerberus. Nodal/Activin‐induced Hhex and Cerberus are dependent on Sox17; both play important roles in cardiac development in ES cells. Further depicting the endoderm cardiac‐inducing signals may guide toward more efficient cardiac myocyte differentiation in ES cells.