bluuebunny/crossref_metadata_2025 huggingface dataset:
Mucosal protection by hypoxia‐inducible factor (HIF) prolyl hydroxylase inhibition
Authors: Keeley Simon , Furuta Glenn T , Colgan Sean P
Published: June 2021

A number of recent studies have implicated tissue hypoxia in both acute and chronic inflammatory diseases, particularly as they relate to mucosal surfaces involving epithelial cells. In this context, a protective role for the transcriptional regulator hypoxia‐inducible factor (HIF) has been demonstrated through conditional deletion of epithelial HIF‐1α in a murine model of colitis. Here, we hypothesized that pharmacologic activation of HIF would similarly provide a protective adaptation to murine colitic disease. For these purposes, we used a novel prolyl hydroxylase (PHD) inhibitor (FG‐4497) which readily stabilizes HIF‐1α and subsequently drives the expression downstream HIF target genes (e.g. EPO). Our results show that the FG‐4497‐mediated induction of HIF‐1α provides an overall beneficial influence on clinical symptoms (weight loss, colon length, tissue IFN and TNF) in murine TNBS colitis, most likely due to their barrier protective function and wound healing during severe tissue hypoxia at the site of inflammation. Taken together these findings emphasize the role of epithelial HIF‐1α during inflammatory diseases in the colon and may provide the basis for a therapeutic use of PHD inhibitors in inflammatory mucosal disease.
An essential role for STAT1 in KGF‐mediated protection from bleomycin‐induced pulmonary fibrosis
Authors: Yarlagadda Manohar Rama , Paglia Melissa , Huang Baocheng , Henry Adam , Morse Danielle , Shukla Manasi , Levy David , Ray Prabir
Published: June 2021

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by destruction of normal epithelial structure, proliferation of fibroblasts, deposition of connective‐tissue matrix proteins and varying degrees of inflammation. In a model of pulmonary fibrosis induced by the chemotherapeutic drug bleomycin, lung epithelial cell‐specific expression of keratinocyte growth factor (KGF) provided protection against lung fibrosis. In an effort to understand the mechanism underlying the protective effects of KGF, a microarray analysis revealed increased KGF‐induced expression of CXCL9 and CXCL10, chemokines whose expression was previously shown to be regulated by STAT1 and which have also been shown to protect against fibrosis in animal models. KGF treatment of epithelial cells in vitro resulted in STAT1 phosphorylation, providing a possible mechanism for the increase in expression of these STAT1‐dependent chemokine genes in vivo. In the bleomycin model, we have found that KGF is unable to protect from lung fibrosis in STAT1‐deficient mice. Our study shows that the epithelial cell‐specific growth factor KGF can protect from lung fibrosis at least in part by activating STAT1.
Rho kinase mediates Helicobacter pylori ‐induced tight junctional claudin‐4 disruption in human gastric epithelial cells
Authors: Lapointe Tamia K , OiConnor Pamela M , Ménard Daniel , Buret André G
Published: June 2021

Gastric epithelial barrier function is primarily maintained by tight junctions (TJ). Small Rho GTPases and Rho kinase (ROCK) are involved in TJ regulation. The ability of H. pylori (Hp) to subvert normal cellular pathways regulating TJ permeability may contribute to the development of gastric ulcers and adenocarcinomas. The mechanisms involved in Hp‐mediated junctional disruptions remain unclear.

AIM

The aim of this study was to assess the role of ROCK in Hp‐induced TJ disruption, using a human gastric epithelial (HGE‐20) cell line.

RESULTS

Immunostaining/Western blotting and activity assays showed cytosolic translocation of claudin‐4 and a 50% increase in ROCK activity in infected monolayers. ROCK inhibition reduced loss of claudin‐4 from the membrane. Treatment of HGE‐20 with conditioned media failed to induce the Hp‐mediated increase in ROCK activity. Caspase‐3 and Rho GTPases Rac‐1 and RhoA activity was significantly increased post infection, consistent with their involvement in the signaling events leading to ROCK activation and claudin‐4 redistribution.

CONCLUSION

These findings demonstrate that Hp activates ROCK and its upstream effectors caspase‐3 and Rho GTPases. The mechanism by which Hp disrupts gastric epithelial barrier structure involves ROCK‐dependent redistribution of claudin‐4, independently of epithelial or bacterial secreted factors. This work was funded by CIHR.
Statins and rapamycin act synergistically to enhance vascular endothelial cytoprotection
Authors: Mason Justin Charles , Hamdulay Shahir , Ali Faisal , Ali Nadira , Haskard Dorian O
Published: June 2021

Background: Heme oxygenase‐1 (HO‐1) and decay‐accelerating factor (DAF) are endothelial cytoprotective genes which limit vascular injury. Statins induce DAF, while rapamycin (Rap) upregulates HO‐1 expression. We asked whether statins and Rap act synergistically to enhance vasculoprotection.

Methods & Results: Treatment of EC with atorvastatin (AT) 0.5μM and Rap 1.25μM led to a synergistic increase in DAF expression 24‐72h post‐treatment (>200%, p<0.05). AT + Rap‐induced DAF expression was dependent upon gene transcription and de novo protein synthesis. Functional relevance was revealed by enhanced protection against complement‐mediated EC lysis, when compared to treatment with Rap or AT alone (35% v 75% and 55% lysis respectively, p<0.05), with protection lost in the presence of DAF inhibitory mAbs. Mevalonate inhibited synergy, confirming a role for HMG‐CoA reductase inhibition. Of note, AT + Rap increased HO‐1 enzyme activity, while HO‐1 antagonist ZnPPIX attenuated, and HO‐1 agonist CoPP enhanced, the synergistic response. Experiments, using iron chelator DFO and ferritin overexpression, suggested that depletion of intracellular free Fe2+ and increased ferritin, a consequence of HO‐1‐activation, was central to synergy. Finally, although synergy was reproduced by simvastatin and Rap, no synergy was seen between atorvastatin and cyclosporine, mycophenolate or methotrexate.

Conclusion: Treatment of EC with Rap and AT results in HO‐1‐dependent synergistic enhancement of DAF expression and protection against complement‐mediated injury. Combination therapies may provide the means to deliver immunosuppression and vasculoprotection for patients with systemic autoimmune diseases.
FGF mediates maintenance of vascular integrity in a Csk dependent manner
Authors: Murakami Masahiro , Zhuang Zhenwu , Moodie Karen , Stan Radu , Simons Michael
Published: June 2021

Background: Maintenance of vascular integrity is one of the most fundamental vascular functions; however, its regulation is not well understood.

Methods: To address the effect of FGF inhibition in adult vasculature, two mouse models were used: (1) endothelium‐specific transgenic expression of the dominant‐negative form of FGF‐R1 (FGF‐R1DN) under the tetracycline‐regulated Tie2 promoter and (2) adenovirus‐mediated systemic expression of soluble FGFR‐IgFc chimera proteins (Ad‐sFGFR). Results: Various in vivo studies demonstrated that inhibition of FGF signaling leads to: (1) disruption of EC adhesion and alignment and (2) increased vascular permeability in normal and ischemic tissues. In vitro studies confirmed that inhibition of FGF signaling increases EC monolayer permeability. This is accompanied with disappearance of VE‐cadherin and other junction proteins from cell‐cell contacts without changing total expression levels of these proteins or cell density/viability. Co‐immunoprecipitation experiments revealed disruption of FGF signaling led to decreased p120‐catenin and Csk association with VE‐cadherin. The absence of Csk in the VE‐cadherin complex can promote local Src activation which potentially leads to VE‐cadherin internalization and junction disassembly. In FGF‐R1DN EC, a VE‐cadherin mutant (Y685E) that constitutively binds Csk restored VE‐cadherin junction localization. This suggests that Csk recruitment to VE‐cadherin is critical for VE‐cadherin stabilization, and this process is regulated by FGF signaling.

Conclusion: Maintenance of vascular integrity is a dynamic process requiring ongoing FGF signaling.
The adhesion molecule ICAM‐2 regulates contact inhibition in endothelial cells
Authors: RANDI ANNA MARIA , Amsellem Valerie , Dryden Nicola , Birdsey Graeme , Haskard Dorian , Mason Justin Charles
Published: June 2021

Endothelial junctions play a key role in angiogenesis. ICAM‐2, constitutively expressed at endothelial junctions, mediates leukocyte recruitment and angiogenesis. The ICAM‐2 intracellular tail binds ERM proteins and α‐actinin. We generated an ICAM‐2 deficient line (δIC2) from mouse cardiac endothelial cells (EC), isolated from ICAM‐2 −/− mice by positive selection and immortalized with polyoma middle T. We also generated an over‐expressing line by infecting the δIC2 cells with a retroviral vector encoding for full‐length (FL) mouse ICAM‐2 (IC2FL), and two mutant ICAM‐2 lines, one lacking the intracellular tail (IC2δTL) and one where the ERM binding sites in the tail have been mutated (IC2δERM). δIC2 cells show defective in vitro angiogenesis on Matrigel, decreased cell migration and Rac activity; over‐expression of ICAM‐2 (IC2FL) reverses the phenotypes. Interestingly, IC2FL cells form a cobblestone monolayer at confluence and are contact‐inhibited whilst δIC2 cells grow and overlap, suggesting that ICAM‐2 is required for junction formation and contact‐dependent inhibition of cell growth. This involves signalling via ERMs, since mutants (IC2δTL) and (IC2δERM) also lack contact‐inhibition. Contact inhibition can be restored by over‐expressing constitutively active Rac (RacV12). Thus ICAM‐2 regulates EC growth and contact inhibition via signalling pathways involving ERMs and Rac.

Research supported by the British Heart Foundation and the EU FP6 Network MAIN.
Frizzled‐7 turnover at the plasma membrane is differentially regulated by non‐canonical and canonical Wnts and Wnt‐signaling components in endothelial cells.
Authors: Mao Catherine D , Barnett Corey D , Struewing Ian T
Published: June 2021

Endothelial cell (EC) migration is a key feature of vascular development, angiogenesis and tissue repair as well as of progression of numerous diseases. The Frizzled (Fz) receptors are cell polarity factors, which control morphogenesis through interaction with the Wnt ligands and activation of the beta‐catenin/TCF, planar cell polarity (PCP) and Ca2+/PKC pathways. We have recently identified a specific cleavage of the Fz7 receptors regulated by cell density and calpain1, a protease involved in the turnover of adhesion complexes. In EC, Fz7 localization at the plasma membrane was restricted to the leading edge of migrating cells and to early cell‐cell adhesion sites where it colocalized with VE‐cadherin and beta‐catenin. Interestingly, in a cell‐autonomous fashion, the non canonical Wnt11 and Wnt5A, and the Wnt‐signaling component dvl2 prevented Fz7 cleavage, while the canonical Wnt13A and stable beta‐catenin forms increased it. Dvl2 prevented also phosphorylation events taking place within Fz7 C‐terminus. Fz7 mutants displaying a decreased cleavage presented an altered phosphorylation pattern, an absence of dvl2 translocation at the plasma membrane and defect of beta‐catenin polarization during EC migration. Together our data show that Fz7 recapitulates a PCP pathway in EC regulated by cell‐cell adhesion and by non canonical Wnts. This work is supported by NIH‐HL68698 grant.
Household food security status before pregnancy as a risk factor for delivering a low birthweight infant
Authors: Hromi‐Fiedler Amber , Bermúdez‐Millán Angela , Chapman Donna , Segura‐Pérez Sofia , Damio Grace , Melgar‐Quiñonez Hugo , Pérez‐Escamilla Rafael
Published: June 2021

Little is know about the influence of household food security status on birth outcomes. Therefore, this study examined the relationship between household food security status and low birthweight among a sample of low income Latinas living in Hartford, Connecticut. Household food security status was assessed the year before pregnancy using a modified version of the U.S. Household Food Security Scale. Households were classified as food secure, mildly food insecure, moderately food insecure, or severely food insecure. Birthweight was dichotomized into low birthweight (< 2500 grams) and normal/high birthweight (> or = 2500 grams). Multivariate logistic regression evaluated risk factors for low birthweight. After adjusting for socioeconomic and demographic characteristics, moderate/severe household food insecurity before pregnancy (Odds Ratio=7.66; Confidence Interval=0.97−60.45; p=0.054), gaining less than the recommended weight during pregnancy (p=0.087), being U.S. born (p=0.033), and primiparity (p=0.090) were risk factors for delivering a low birthweight infant. These findings suggest household food insecurity status the year before pregnancy is an important determinant for delivering a low birthweight infant among Latinas. Funded by the Connecticut NIH EXPORT Center of Excellence for Eliminating Health Disparities among Latinos (NIH‐NCMHD Grant # P20MD0017650).
Role of Periostin in Maturation of the Mesenchymal Outflow Tract Septum
Authors: Conway Simon James , Snider Paige , Wang Jian
Published: June 2021

The critical requirement of the cardiac neural crest (CNC) cells during cardiovascular development is well documented, as are the severe and diverse congenital cardiovascular consequences associated with their removal and/or genetic manipulation. However, the actual role of the CNC in the outflow tract (OFT) itself and what role they play during mesenchymal cushion remodeling and generation of the mature heart remains unclear. Indeed, although the OFT cushions are uniquely colonized by invading CNCs (in contrast to the atrioventricular cushions), it is unclear what function (if any) the CNC play during remodeling and why OFT cushions (future aortic and pulmonary valves) differ from inlet atrioventricular cushions (future mitral and tricuspid valves). Our study of the transcriptional regulation of Periostin (a fasciclin‐family adhesion molecule that is continually expressed throughout cushion formation, remodeling and valvular homeostasis) identified a Periostin 3.9kb transcriptional regulatory module that drives in vivo restricted transient expression in subpopulation of CNC‐derived cells that colonize the distal OFT mesenchymal cushions. Given there are currently no OFT endocardially‐restricted promoters (or even a cushion‐specific promoter), we generated 3.9kb Periostin‐Cre transgenic mice. In combination with R26R reporter and R26‐diptheria toxin‐A genetic ablation mice, we have begun to analyze the role of the 3.9kb Periostin‐expressing OFT subpopulation. Lineage mapping and genetic cell ablation data will be presented. Combined these results indicate that we have identified a unique and powerful molecular tool with which to begin to understand/identify some of the master regulators that control how the OFT mesenchymal cushions are remodeled to give rise to mature heart and what are the molecular differences/origins between the OFT and atrioventricular cushions.
A new paradigm for teaching histology in Canada's first distributed medical school
Authors: Pinder Karen , Ford Jason , Ovalle William K.
Published: June 2021

To address the critical problem of inadequate physician supply in rural British Columbia, the University of BC launched an expanded and distributed medical program in 2005. Medical students engage in a common curriculum at 3 geographic sites across BC, in Vancouver, Prince George and Victoria. The distribution of the core Histology course required a thorough revision of our instructional methodology. We here report our progress and address the question “How can Histology be distributed to remote sites while maintaining the highest of educational standards?” Experience at UBC points to 3 specific challenges: (i) ensuring equitable student access to quality histological images, (ii) implementing a technological infrastructure that allows for real‐time teaching and interactivity across sites, and (iii) ensuring student access at all sites to Faculty expertise. High quality images (available through any internet connection) are provided within a new virtual slide box library of 300 light microscopic and 190 electron microscopic images. Our technological needs are met through a videoconference system that allows for live, interactive sharing of visual/audio materials across the 3 sites. This system also ensures student access to Faculty expertise during didactic teaching sessions. Student examination results and surveys demonstrate that the distribution of our Histology curriculum has been successful.
SLMAP overexpression in mouse heart remodels subcellular membranes involved in E‐C coupling
Authors: Nader Moni , Westendorp Bart , Salih Maysoon , Leenen Frans HH , Tuana Balwant S
Published: June 2021

SLMAP is a tail‐anchored membrane protein implicated in regulating the excitation‐contraction (E‐C) coupling. To define further its role in cardiac function we generated transgenic (Tg) mice with SLMAP1 expression driven by the α‐MHC promoter. Gross analysis indicated no changes in structure or function as a consequence of SLMAP1 expression although genes associated with cardiac remodeling such as (ANF) were up regulated. Immunohistochemical analysis revealed that SLMAP1 targets subcellular organelles and was associated with a moderate to severe vacuolization of membrane compartments in cardiomyocytes. The changes in membrane vacuolization appeared to be related to the level of SLMAP1 protein expressed as well as age of the animals with maximal membrane disruption noted at day 30–35. High resolution microscopy revealed the presence of large membranes vesicles filled with crystalline substance, consistent with dilated sarcoplasmic reticulum. The expression of proteins involved in regulating the E‐C coupling (RyR2, caveolin3, triadin) were down regulated whereas others (calsequestrin, calnexin, BiP) were up regulated. We concluded that SLMAP is involved in subcellular remodeling of E‐C coupling components and its regulated level in cardiomyocytes is critical to ensure a proper organization of subcellular compartments. (Supported by a grant from HSFO)
Attenuation of the post‐myocardial infarction stress response may promote improved survival and healing in the MRL mouse
Authors: Hunt Darlene , Campbell Patrick , Omens Jeffrey , McCulloch Andrew
Published: June 2021

Introduction: MRL/MpJ (MRL) mice heal better following myocardial infarction (MI) than C57BL/6J (C57) controls, may regenerate cardiac muscle, and have improved long‐term survival, though the mechanisms are unclear. We compared acute post‐MI survival and healing differences between the two strains as well as the LG/J strain, the major genetic background of the MRL. Methods: MI was induced in adult male MRL, LG/J, and C57 mice by permanent ligation of the left coronary artery. Infarct size was measured after 1, 5, or 30 days. Microarray analysis was performed on RNA collected before and 1 or 5 days post‐MI. Survival was tracked through 70 days. Results: Survival was significantly greater in MRL than C57 mice, due to decreased LV rupture in MRLs during days 2–5 post‐infarct. LG/J mice also appear more susceptible to rupture. Infarct size on day 1 was not different between the MRL and C57 strains, but was significantly larger in C57 mice after 30 days. Preliminary results suggest infarcts of all 3 strains expand to a similar degree after 5 days. Microarray analysis indicates the expression of transcripts associated with induction of apoptosis, the immune response, and inhibition of cellular proliferation may be reduced in the MRL day 1 infarct compared to the C57. The relative attenuation of these processes may contribute to the MRL's survival advantage and promote improved healing. Support: NIH
Regulation of appendage regeneration in zebrafish
Authors: Poss Kenneth
Published: June 2021

Certain urodele amphibians and teleost fish display an elevated spectrum of organ regeneration with respect to mammals, with tissues like heart, retina, spinal cord, and appendages capable of impressive renewal after major injury. Zebrafish fins are complex structures containing bone, nerves, vasculature, connective tissue, and epidermis, and can achieve essentially perfect regeneration within 2 weeks of amputation. Here, we have utilized candidate gene approaches to dissect formation and function of the fin blastema, a proliferative mass of undifferentiated mesenchymal tissue that arises after ampuation and ultimately gives rise to the new structures of the regenerated appendage.
Chromosome dynamics during meiosis
Authors: Dernburg Abby F , Phillips Carolyn M , Sato Aya
Published: June 2021

Meiosis is the specialized cell division that enables organisms to carry out sexual reproduction. This process involves a series of tightly coordinated events in which chromosomes pair with their homologous partners, undergo crossover recombination, and eventually separate from each other. To accomplish this feat, chromosomes form de novo attachments to the nuclear envelope. These chromosome dynamics were first described in the early 20th century, but their functional significance was unknown. We have been investigating meiotic chromosome behavior in the nematode C. elegans. Genetic studies led us to identify an essential family of zinc finger proteins that recognize specific regions on each of the chromosomes These chromosome regions associate with a protein complex that bridges the nuclear envelope to engage with microtubules in the cytoplasm. Based on analysis of the resulting motion of the chromosomes, we hypothesize that these dynamics provide a kinetic proofreading mechanism that enables chromosomes to identify their proper partners.
HIV protease inhibitors block the zinc metalloproteinase ZMPSTE24 and lead to an accumulation of farnesyl‐prelamin A in cells.
Authors: Young Stephen G. , Coffinier Catherine , Hudon Sarah , Fong Loren , Hrycyna Christine
Published: June 2021

HIV protease inhibitors (HIV‐PIs) target the HIV aspartyl protease, which cleaves the HIV gag‐pol polyprotein into shorter proteins required for the production of new virions. HIV‐PIs are an important component of HIV treatment regimens but have been associated with lipodystrophy and other side effects. In both human and mouse fibroblasts, we showed that HIV‐PIs caused an accumulation of prelamin A. The prelamin A in HIV‐PI‐treated fibroblasts migrated more rapidly than nonfarnesylated prelamin A, comigrating with the farnesylated form of prelamin A that accumulates in ZMPSTE24‐deficient fibroblasts. The accumulation of farnesyl‐prelamin A in response to HIV‐PI treatment was exaggerated in fibroblasts heterozygous for Zmpste24 deficiency. HIV‐PIs inhibited the endoproteolytic processing of a GFP‐prelamin A fusion protein. The HIV‐PIs did not affect the farnesylation of HDJ‐2, nor did they inhibit protein farnesyltransferase in vitro. HIV‐PIs also did not inhibit the activities of the isoprenylcysteine carboxyl methyltransferase ICMT or the prenylprotein endoprotease RCE1 in vitro, but they did inhibit ZMPSTE24 (IC(50): lopinavir, 18.4 +/− 4.6 uM; tipranavir, 1.2 +/− 0.4 uM). We conclude that HIV‐PIs inhibit ZMPSTE24, leading to an accumulation of farnesyl‐prelamin A within cells. The inhibition of ZMPSTE24 by HIV‐PIs could play a role in the side effects of these drugs.
Three‐month effect of diabetes peer counseling on plasma lipids of Latinos enrolled in the DIALBEST trial: Preliminary findings.
Authors: Vega‐López Sonia , Calle Mariana C. , Fernandez Maria Luz , Chhabra Jyoti , D’Agostino Darrin , Samuel Grace K. , Segura‐Pérez Sofia , Damio Grace , Pérez‐Escamilla Rafael
Published: June 2021

As part of the DIALBEST trial Latinos with type 2 diabetes enrolled in the “Amigos en Salud” (AES) program at Hartford Hospital are randomly assigned to either standard of care or an additional year long culturally‐tailored peer counseling (PC) service providing information and support on diabetes care and self management. Baseline (BL) and 3‐month plasma lipids have been measured in 56 subjects (44 females, 12 males; 32–75 y). BL total‐C was 183±43 mg/dL and 183±47 mg/dL for the control (n=27) and PC (n=29) groups, respectively and after 3 months decreased by 9% and 14% (time effect P=0.001). LDL‐C at BL was 100±34 mg/dL and 104±41 mg/dL for the control and PC groups, and after 3 months decreased by 10% and 20%, respectively (time effect P=0.007). HDL‐C decreased by 9% in the control group and only by 2% in the PC group. The LDL‐C/HDL‐C increased by 1% for the control group but decreased by 20% in the PC group (time × group interaction, 1‐tailed P=0.027). BL triglycerides were 139±68 mg/dL for the control and 158±85 mg/dL for the PC group and decreased by 6% and 17% respectively (time effect P=0.05). These preliminary results suggest that plasma lipids of Latino patients with type 2 diabetes enrolled in AES are being adequately controlled. The PC intervention is likely to further improve the LDL/HDL ratio. Funded by the Connecticut NIH EXPORT Center for Eliminating Health Disparities among Latinos (NIH‐NCMHD grant # P20MD001765).
Copper secretion from human breast epithelial cells is mediated by ATP7B and lactational hormones
Authors: Michalcsyk Agnes , Bastow Edward , Greenough Mark , Camakaris James , Linder Maria C , Mercer Julian F.B. , Ackland Leigh
Published: June 2021

Mammary epithelial cells that produce milk are unique in containing both of the Cu pumps, ATP7A and B, central to mammalian copper homeostasis but otherwise found individually. We have begun studies to clarify their roles in this gland using confocal microscopy. ATP7B was expressed in the luminal epithelial cells of human breast tissue, with a perinuclear location that shifted to a diffuse location with lactation. In the human mammary epithelial cell culture model (PMC42), ATP7B was also in the perinuclear region and redistributed to endosomes adjacent to, but not coincident with, the apical plasma membrane after treatment of the cultures with a lactational hormone regimen (3 days estrogen and progesterone then 3 days insulin, dexamethasone, prolactin). ATP7B was in a different subset of vesicles from those containing milk proteins and did not overlap with endosomes containing ATP7A. A physiological concentration of extracellular Cu was required for the redistribution effect, suggesting it was caused indirectly by increased intracellular Cu concentrations. Overexpression of ATP7B in the PMC42 cultures, grown as polarized monolayers, resulted in an enhanced efflux of Cu from the apical cell surface (determined with 64Cu). Our findings are consistent with the concept that ATP7B is important for the secretion of copper from the mammary gland into the milk. Supported by PHS Grant No. RO1 HD 46949.
Combination of aspirin and gamma‐tocopherol is superior to aspirin alone in protection of carrageenan‐induced inflammation in a rat model
Authors: Jiang Qing , Ames Bruce , Moreland Michelle
Published: June 2021

We examined whether combinations of aspirin and alpha‐tocopherol (aT) or gamma‐tocopherol (gT), two major forms of vitamin E, can alleviate aspirin‐caused stomach lesion and may be better anti‐inflammatories than aspirin alone. In carrageenan‐induced inflammation air‐pouch model, aspirin at 150mg/kg inhibited inflammation‐associated increase of prostaglanding E2 (PGE2) by 70% (P<0.02) at 6 h after initiation of inflammation, but at 18 h the inhibitory effect was vanished. The combination of aspirin and gT (30mg/kg) decreased PGE2 by 70% at 6h (P<0.02) and 40% at 18h (P<0.07). The combination, but not aspirin alone, resulted in a decrease in exudate volume (P < 0.05) and LDH increase, a tissue damage marker, in the exudate collected at inflammation site. gT reversed aspirin‐caused reduction of food intake and showed protection of aspirin‐induced lesion in the stomach. In contrast, combination of aspirin and alpha‐T did not show more beneficial effects than aspirin alone, but rather seemingly enhanced lesions in the stomach. Our study demonstrated that combination of gT and aspirin prolonged the anti‐inflammatory effect and attenuated aspirin‐caused adverse effect. combination may be useful in the treatment of chronic inflammatory conditions and cancer. (Supported by NIH R01AT001821)
Consumption of c9,t11–18:2 conjugated linoleic acid (CLA) does not influence somatic cell count, immune cell distribution, or bacterial content of human milk
Authors: Collier Natalie , McGuire Michelle K , Fox Larry W , Beerman Kathy A , Williams Janet , McGuire Mark A
Published: June 2021

Fatty acids have long been known to have biological roles beyond provision of energy. For example conjugated linoleic acid (CLA) isomers differentially influence a multitude of systems and responses including regulation of milk fat, modulation of carcinogenesis, partitioning of body fat, and mediation of immune function. Recent work from our laboratory suggests that the naturally‐occurring form of CLA (c9,t11–18:2) also decreases growth of Staphylococcus aureus, the organism thought to cause most infectious mastitis in breastfeeding women. We were therefore interested in whether milk‐borne S. aureus burden could be decreased by enhancing exposure of the mammary gland to this fatty acid. Healthy breastfeeding women (n = 16) were enrolled in this randomized, double‐blind, cross‐over, placebo‐controlled human intervention trial with two interventions: c9,t11–18:2 and placebo (sugar). Treatments were provided in the form of supplements; subjects consumed 4 supplements/d (~2 g daily) for 7 d with a 14 d washout period. Milk was collected on the last day of each period and analyzed for somatic cell count, distribution of immune cells, macronutrient content, and bacterial burden. ANOVA procedures were used to test for effect of treatment. Although CLA was enriched in milk, there was no effect of treatment on any of these variables. Funded in part by the United Dairymen of Idaho and NIH‐NRRI grant P20 RR15587.
Cholesterol provided by eggs and carbohydrate restriction distinctly modulate lipoprotein metabolism in adult men
Authors: Mutungi Gisella , Ratliff Joseph , Puglisi Michael , Volek Jeff S , Fernandez Maria Luz
Published: June 2021

Twenty eight men (BMI 26‐37 kg/m2) between 40‐70 y were recruited to evaluate the effects of carbohydrate restriction (CR, ~10% energy from carbohydrates) and dietary cholesterol (CHOL) on lipoprotein metabolism. Subjects were randomly allocated to the EGG group (640 additional mg CHOL/d) or the SUB group (0 mg additional CHOL/d) for 12 wk. Lipoprotein size and subclasses determined by use of NMR, apolipoproteins (apo) A‐I, A‐II, B, C‐II, C‐III, and E measured by antibody‐immobilized beads and Luminex MAP technology and lecithin cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein activities were measured both at baseline and post‐intervention. We observed a 45% decrease in plasma triglycerides for all subjects, which was associated with decreases in the large and medium VLDL subclasses (P < 0.001). In agreement with these observations, a decrease in apo B (P < 0.01) was also observed. In addition, CR resulted in a 133% increase in apo C‐II [lipoprotein lipase (LPL) activator] and a 65% decrease in apo C‐III, (LPL inhibitor) (P < 0.0001). The smaller HDL subclasses and apo A‐II levels were also decreased at 12 wk (P < 0.001). CHOL effects were mainly on LDL and HDL metabolism. Although an increase of the larger LDL subclass was observed for all subjects, the EGG group had a greater increase (P < 0.05). The EGG group also presented increases in HDL‐C, higher number of large HDL particles (P < 0.01) and an increased LCAT activity (P < 0.05) compared to the SUB group. These results indicate that CR favorably alters VLDL metabolism while CHOL provided by eggs favors the formation of less atherogenic lipoproteins, larger LDL and HDL.
Dynamic Impact of Income Growth on Diet Quality in China, 1989–2006: An Update
Authors: Du Shufa , Gordon‐Larsen Penny , Popkin Barry M
Published: June 2021

Earlier research covering 1989–1997 found adverse relationships between income and diet quality in China. This study uses data on 7718 subjects aged 20–45 years old from 7 waves of China Health and Nutrition Survey over a 17‐year period to explore income effects on diet quality in greater depth. Dietary intakes were measured using a combination of the weighing method and three consecutive 24‐h recalls and food groups and nutrients calculated. Detailed income data was used to characterize income elasticity. Multivariate longitudinal random‐effects models predicted (a) the probability of consuming any specific group of food and (b) the quantity of specific food group consumed as a function of income change over time, controlled for age, gender, education, urban residence, and major food prices. In the past decade, we did not see continued rapid decrease in grain consumption, but observed continued rapid increase in consumption of animal source foods (doubled in all income groups) and edible oil (tripled in low income and doubled in high income groups). Pork as % animal food decreased rapidly with high income. Significant shifts in income‐diet relationships in the period after 1997 were found and varied considerably by socioeconomic status. These income changes indicate an acceleration of the shift in the burden of less healthy diets (highly energy dense and obesogenic in the Chinese context) toward the poor.
JAM‐A regulates epithelial integrin expression and cell shape by activation of Rap1a and Rap1b through a complex with Afadin and PDZ‐GEFs
Authors: Severson Eric A , Parkos Charles A
Published: June 2021

Junctional adhesion molecule‐A (JAM‐A) is a tight junction protein implicated in the regulation of epithelial β1 integrin expression, cell shape and barrier function. We previously determined that this regulation requires both dimerization and PDZ binding motifs on different regions of JAM‐A. To gain further insight into JAM‐A mediated signaling in vitro, we downregulated expression of scaffolding proteins and found that loss of Afadin resulted in cellular changes similar to those observed with loss of JAM‐A. Immunofluorescence (IF) and immunoprecipitation (IP) studies revealed co‐association of Afadin with JAM‐A. Interestingly, the small GTPase Rap1 is known to interact with Afadin, regulate integrin function and have decreased activity after loss of JAM‐A. Downregulation of Rap1a resulted in decreased β1 integrin protein levels whereas loss of Rap1b resulted in altered cell shape. Since Rap1 activation is dependent on guanine nucleotide exchange factors (GEFs) we assessed expression of GEFs in vitro and identified PDZ‐GEF1 and PDZ‐GEF2 as major transcripts in colonic epithelia. IF and IP studies revealed co‐association of the PDZ‐GEFs with JAM‐A and loss of the PDZ‐GEFs resulted in diminished Rap1 activity. These results suggest that dimerized JAM‐A signals through a complex that may contain both Afadin and PDZ‐GEFs which, in turn, activate Rap1a and Rap1b to regulate β1 integrin levels and cell shape.

Supported by NIH DK‐61379
Renal Inflammation in Response to Cholesterol Feeding in the ApoE Deficient Mouse Detected In Vivo by Molecular Nanobeacons Targeted to VCAM‐1
Authors: Zhang Huiying , Kaneda Megan , Zhang Lei , Southworth Rick , Chen Junjie , Lanzan Gregory M , Wickline Samuel A
Published: June 2021

Atherosclerosis elicits kidney damage based on cholesterol‐macrophage response associated upregulated vascular cell adhesion molecule‐1 (VCAM‐1) expression. We employed a novel VCAM‐1 targeted fluorescent nanoparticles (NP) and immunochemistry to define the VCAM‐1 expression in apolipoprotein E deficient (ApoE−/−) mice kidney.

Methods: ApoE−/− mice fed on a high cholesterol diet for 35 weeks were injected with VCAM‐1 targeted NP (n=6). Control groups were comprised ApoE−/− littermates treated with non‐targeted NP (n=5), C57/BL6 mice treated with VCAM‐1 targeted (n=5) and non‐targeted (n=6) NP. Mice were sacrificed two hours after treatment to allow NP binding to vascular VCAM‐1 receptors. Frozen sections of mouse kidneys were prepared for fluorescent imaging. Immunostaining of VCAM‐1 was performed on three ApoE−/− and three C57/BL6 mice kidneys.

Results: Relative area with VCAM‐1 expression was significantly higher in ApoE−/− kidneys than that of controls (Fig. A, p<0.05). Fluorescent imaging revealed that VCAM‐1 targeted NP bound specifically to the glomeruls of ApoE −/− mice kidney (Fig. B) but not to that of controls (images not shown).

Discussion: Upregulated VCAM‐1 in ApoE −/− mouse kidneys can be visualized with the targeted NP, suggesting VCAM‐1 targeted NP is a potential molecular imaging agent for early detection and monitoring of inflammation in renal disease.
Lack of Sex Differences in Muscle Stem Cell Transplantation for Cardiac Repair
Authors: Drowley Lauren , Okada Masaho , Payne Thomas , Botta Gregory , Oshima Hideki , Tobita Kimimasa , Keller Bradley , Huard Johnny
Published: June 2021

Cellular transplantation of stem cells is a potential method of repairing damaged myocardium. We identified a novel population of muscle‐derived stem cells (MDSCs) that can repair cardiac muscle. Sex differences have been seen in cardiovascular diseases and cellular therapy. Differences in regenerative capacity of male and female MDSCs have been observed in skeletal muscle and bone. In this study we compared male and female MDSCs to determine if there was a sex difference for cardiac repair. MDSCs were implanted into the hearts of both a cardiomyopathy mouse model and a myocardial infarction model. In the cardiomyopathy model, five populations of male and female cells were injected into both male and female mice. No differences were found with either host sex or cell sex in engraftment or blood vessels. For the injury model, cells were divided into 4 groups – high and low engrafting cells from both sexes, and injected into SCID mice after infarction to assess function at six weeks. No difference was seen in function between the groups, though all cell‐injected groups were significantly better than PBS controls. The infarcted hearts were stained for scar tissue, blood vessels, and engraftment, and no difference was seen between the cell groups. Though our lab has previously shown sex differences with MDSCs in skeletal muscle and bone, this does not appear to be the case in cardiac muscle. Since MDSCs seem to repair the heart through secretion of VEGF and chemoattracting host cells rather than directly regenerating the myocardium, this could explain why no difference was observed between male and female cells. This work was funded by grants to J. Huard from the MDA, the NIH (1U54AR050733‐01, HL 069368) and L. Drowley from the NIH (T32 EB001026‐05).
IFN‐γ production by neutrophils during S. pneumoniae pneumonia requires NADPH oxidase
Authors: Gomez John C. , Yamada Mitsuhiro , Rebert Nancy A. , Doerschuk Claire M.
Published: June 2021

Lung neutrophils produce interferon‐γ (IFN‐γ) in pneumonia induced by Streptococcus pneumoniae. The intracellular signals that control this process are not understood. Reactive oxygen species generated by NADPH oxidase can regulate intracellular pathways, including gene transcription in neutrophils. Our studies determined the role of NADPH oxidase in the production of IFN‐γ by neutrophils during pneumonia induced by S. pneumoniae. IFN‐γ production by neutrophils in the lungs of wild type (WT) and NADPH oxidase deficient (gp91phox −/−) mice 24 hours after intratracheal instillation of S. pneumoniae was determined by flow cytometry. The percentage of lung neutrophils that produced intracellular IFN‐γ was less in gp91phox −/− mice compared to WT mice (1.7 ± 0.1% vs. 23.8 ± 1.9% respectively, p<0.001). The total number of lung neutrophils was similar in WT and gp91phox −/− mice (19.7 ± 1.7 × 10 6 vs. 23.1 ± 3.5 × 106, respectively). Decreased IFN‐γ production in gp91phox −/− lung neutrophils was associated with elevated levels of activated JNK. Activation of p38 MAPK, ERK and NF‐κB was similar in WT and gp91phox−/− lung neutrophils. IFN‐γ production by neutrophils during S. pneumoniae pneumonia proceeds through signals that require NADPH oxidase and may contribute to host defense.

Supported by HL052466.
TG100–115, a PI3 Kinase γ/δ Inhibitor, Reduces Lung Inflammation and Airway Hyperresponsiveness in a Mouse Asthma Model
Authors: Racanelli‐Layton Adrienne Lynn , Eide Lisa , Jarmillo Claudia , Brown Jolene , Dellamary Luis , Martin Michael , Soll Richard , Doukas John
Published: June 2021

Rationale: The γ and δ isoforms of phosphoinositide 3‐kinase (PI3K) regulate immune/inflammatory responses by multiple cell types including Th2 T cells, mast cells, and eosinophils. We explored whether a dual PI3K γ/δ inhibitor could impact airway hyperresponsiveness (AHR) and inflammation.

Methods: Mice were sensitized with ovalbumin (OVA) on Day 0 and boosted on Day 5, followed by inhalation challenges on Days 12–13. TG100–115, a PI3K γ/δ inhibitor (IC50 = 83 and 235 nM, respectively), was delivered by inhalation on Days 11–14 for total daily doses of 100 mg/kg; dexamethasone (DEX) was delivered at 3 mg/kg i.p on Days 8–14. On Day 15, AHR was determined following aerosolized methacholine (MC) challenge, polymorphonuclear (PMN) cell counts were determined from bronchoalveolar lavage fluid (BALF), and lungs were processed for histology.

Results: Exposure to aerosolized TG100–115 resulted in sustained compound levels within the lung and airways with minimal plasma exposure. TG100–115 reduced both AHR and PMN accumulation vs. vehicle‐treated animals, to near maximal response of systemic DEX. Histology showed that both treatments reduced inflammation and mucin accumulation to near background levels.

Conclusion: An aerosolized PI3K γ/δ inhibitor reduces asthma symptoms in a standard mouse model, and may represent a new anti‐inflammatory therapy for future clinical exploration.
The IkB family member Bcl‐3 regulates lung graft inflammation and injury
Authors: Gelman Andrew E , Kreisel Daniel , Sugimoto Seiichiro , Tietjens Jeremy
Published: June 2021

The IκB family member Bcl‐3 was recently shown to enhance the stability of p50 homodimers which in turn can inhibit the expression of NF‐κB dependent genes. However, the role of Bcl‐3 in solid organ injury is unknown. We asked if Bcl‐3 regulates ischemia‐reperfusion (I/R) injury in a mouse model of syngeneic orthotopic lung graft transplantation. B6→B6 and B6→Bcl‐3− /− transplants were compared for lung graft inflammation and function following cold ischemia and 24 hrs of reperfusion. Loss of Bcl‐3 expression resulted in exacerbated inflammatory mediator synthesis, pulmonary edema and poor graft function. However, I/R injury could be significantly averted in Bcl‐3− /− recipients by using an NF‐κB inhibitor indicating that Bcl‐3 controls graft inflammation through targeting NF‐κB activity. Also, we found 3‐fold more TNFα‐producing macrophages in the airways of Bcl‐3− /− recipients in comparison to B6 recipients. BrdU injection of Bcl‐3− /− recipients revealed that the elevated macrophage sequestration was not a result of altered monocyte chemotaxis but due to significantly higher numbers of dividing graft‐infiltrating monocytes and macrophages. Analysis of M‐CSF treated‐BrdU pulsed monocyte cultures also showed comparatively greater BrdU uptake and macrophage production from Bcl‐3− /− monocytes. Our data reveal that Bcl‐3 is a critical regulator of lung graft injury and inflammatory monocyte differentiation.
Oncogenic transcriptome of A/J lungs
Authors: Gohil Kishorchandra , Oommen Saji , Vasu Vihas T , Cross Carroll E , Witschi Hanspeter
Published: June 2021

The specific aim of this study was to identify gene‐networks that may cause lung oncogenesis in a mouse cancer model. A/J mice, in contrast to C57Bl6 (B6) have a high incidence of spontaneous lung tumors. To identify the oncogenic genes, RNA was isolated from lungs of A/J and B6 mice and analyzed with GeneChips for ~14,000 mRNAs. ~2000 differentially expressed genes could be divided in two major classes; overexpressed (~50%) or under expressed in A/J‐lungs compared to those in B6 lungs. Functional clustering identified ~20 distinct clusters, in each major class. These clusters included transcription factors, protein kinases, RNA processors, translation, mitochondrial, and immune response genes. Remarkably, these functional clusters were present in both, over expressed and under‐expressed gene‐classes. But the specific genes within a cluster were unique to each major class. Also noteworthy was the simultaneous over‐expression and under‐expression of protein kinases and, lack of protein phosphatase cluster in overexpressed gene‐class. In addition, a cluster of chromatin‐remodelling genes with repressor activities were differentially expressed between A/J and B6 strains. These transcriptomic analyses identify unique macro‐molecular networks that may contribute to spontaneous oncogenesis and offer targets for anti‐oncogenic interventions, possibly by RNA‐interference.
Matrix metalloproteinase‐7 controls pancreatic acinar cell transdifferentiation by activating the notch signaling pathway
Authors: Sawey Eric , Crawford Howard C.
Published: June 2021

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States, demonstrating the lowest five‐year survival rate of any cancer. An important observation made in cases of PDAC is the consistent presence of putative preneoplastic lesions, known as metaplastic ducts, which arise from a process known as acinar‐to‐ductal metaplasia. Molecular dissection of this process in vitro has shown that primary acinar cells, in response to EGF receptor ligands, can transdifferentiate into duct‐like epithelia in a Notch pathway dependent manner. Here we show that in vitro acinar transdifferentiation is dependent on Matrix Metalloproteinase‐7 (MMP‐7), a proteinase expressed in most metaplastic epithelia in vivo. MMP‐7 was found to be required for Notch activation, leading to dedifferentiation of acinar cells to a nestin‐positive transitional cell. We also showed that ADAM‐10, but not ADAM‐17, may be necessary for MMP‐7's effect on Notch activity. MMP‐7 activity was also found to be sufficient to induce the process, indicating that molecular signals capable of initiating MMP‐7 expression also have the potential to induce formation of metaplastic epithelia in the pancreas.
Genome‐wide Analysis of the HOXC6 Transcriptional Network in Prostate Cancer
Authors: Moreno Carlos S , Spyropolous Demetri D , Martin W. David , McCabe Colleen D
Published: June 2021

Homeobox (HOX) transcription factors are developmentally regulated genes that play crucial roles in tissue patterning. Homeobox C6 (HOXC6) is overexpressed in prostate cancers and correlated with cancer progression, but the downstream targets of HOXC6 are largely unknown. We have performed genome‐wide localization analysis to identify promoters bound by HOXC6 in prostate cancer cells. This analysis identified 468 reproducibly bound promoters whose associated genes are involved in functions such as cell proliferation and apoptosis. We have complemented these data with expression profiling of prostates from mice with homozygous disruption of the Hoxc6 gene to identify 31 direct regulatory target genes of HOXC6. We show that HOXC6 directly regulates expression of BMP7, FGFR2, IGFBP3, and PDGFRA in prostate cells and indirectly influences the Notch and Wnt signaling pathways in vivo. HOXC6 regulates genes with both oncogenic and tumor suppressor activities as well as several genes such as CD44 that are important for branching morphogenesis and metastasis to the bone microenvironment.