Difference In Cellular Proliferation Research Articles

Pathogenic Roles of Cardiac Fibroblasts in Pediatric Dilated Cardiomyopathy.

Background Dilated cardiomyopathy (DCM) is a major cause of heart failure in children. Despite intensive genetic analyses, pathogenic gene variants have not been identified in most patients with DCM, which suggests that cardiomyocytes are not solely responsible for DCM. Cardiac fibroblasts (CFs) are the most abundant cell type in the heart. They have several roles in maintaining cardiac function; however, the pathological role of CFs in DCM remains unknown. Methods and Results Four primary cultured CF cell lines were established from pediatric patients with DCM and compared with 3 CF lines from healthy controls. There were no significant differences in cellular proliferation, adhesion, migration, apoptosis, or myofibroblast activation between DCM CFs compared with healthy CFs. Atomic force microscopy revealed that cellular stiffness, fluidity, and viscosity were not significantly changed in DCM CFs. However, when DCM CFs were cocultured with healthy cardiomyocytes, they deteriorated the contractile and diastolic functions of cardiomyocytes. RNA sequencing revealed markedly different comprehensive gene expression profiles in DCM CFs compared with healthy CFs. Several humoral factors and the extracellular matrix were significantly upregulated or downregulated in DCM CFs. The pathway analysis revealed that extracellular matrix receptor interactions, focal adhesion signaling, Hippo signaling, and transforming growth factor-β signaling pathways were significantly affected in DCM CFs. In contrast, single-cell RNA sequencing revealed that there was no specific subpopulation in the DCM CFs that contributed to the alterations in gene expression. Conclusions Although cellular physiological behavior was not altered in DCM CFs, they deteriorated the contractile and diastolic functions of healthy cardiomyocytes through humoral factors and direct cell-cell contact.

Cellular proliferation and apoptosis in Staphylococcus aureus–infected heifer mammary glands experiencing rapid mammary gland growth

Intramammary infections in nonlactating mammary glands are common and can occur during periods of rapid mammary epithelial cell (MEC) accumulation, which may ultimately reduce total MEC numbers. Reduced MEC numbers, resulting from impaired MEC proliferation and increased cellular apoptosis, are expected to reduce future milk yields. The objective of this study was to measure the degree of cellular proliferation and apoptosis in the epithelial and stromal compartment of uninfected and Staphylococcus aureus-infected mammary glands hormonally induced to grow rapidly. Nonpregnant heifers (n = 8) between 11 and 14 mo of age were administered supraphysiological injections of estradiol and progesterone for 14 d. One mammary gland of each heifer was randomly selected and infused with Staph. aureus (CHALL) while another mammary gland was designated as an uninfected control on d 8 of injections. Mammary tissues were collected on the last day of hormonal injections from center and edge parenchymal regions and subject to proliferation assessment via Ki-67 staining and apoptotic assessment via terminal deoxynucleotidyl transferase dUTP nick-end labeling. Differences in cellular proliferation between CHALL and uninfected control quarters were not apparent, but proliferation of MEC was marginally greater in edge parenchyma than in center parenchyma. Coincidently, CHALL quarters experienced a greater percentage of apoptotic MEC and lower percentage of stromal cells undergoing apoptosis than uninfected control quarters. This study also provides the first insight into the mechanisms that allow the mammary fat pad to be replaced by expanding mammary epithelium as edge parenchyma contained a greater percentage of apoptotic stromal cells than center parenchyma. When taken together, these data suggest that Staph. aureus intramammary infection impairs mammary epithelial growth through reductions in MEC number and by preventing its expansion into the mammary fat pad. These factors during periods of rapid mammary growth are expected to impair first lactation milk yield.

Downstream Branches of the Fibroblast Growth Factor Signaling Pathway Act Interdependently to Shape the Face

The musculoskeletal tissues of the head develop from primordia, composed of neural crest cells, that evaginate and fuse to form the structures of the face. This occurs through a complex process controlled not only by regional differences in cellular proliferation, apoptosis, and differentiation, but also directed cellular migration and oriented cellular behavior. Disruption of normal development of the facial primordia can either prevent apposition and fusion of craniofacial primordia, leading to malformations such as cleft lip and/or palate. Previously, we found that stimulating signaling by fibroblast growth factor (FGF) in neural crest cells of chick embryos slowed proximodistal expansion of the facial primordia and widened the midface. Activation of the FGF pathway decreased cell proliferation in the frontonasal process (FNP) and disrupted cellular orientation, which were correlated with shape of the FNP. Blocking FGF signaling with a chemical inhibitor caused severe truncation of the FNP and decreased proliferation. Our previous results confirm that FGF signaling affects craniofacial morphogenesis, seemingly by altering multiple cellular functions. FGF receptors are receptor tyrosine kinases which utilize many common downstream signaling cascades that direct cellular proliferation, apoptosis, differentiation, migration, and oriented cell behaviors, but it is not clear which downstream pathway explains our previous results. In this study we used chemical inhibitors to block three pathways downstream of FGF receptor activation to determine their effect on facial morphology and cellular activity in the FNP of the chick embryo. Blocking MAPK, PLCγ, and PI3K pathways individually caused similar mild truncations of proximo‐distal growth in the FNP over 24 and 72 hours, and similar reductions in cellular proliferation. Cellular orientation was not significantly affected by any treatment. Blocking all three downstream pathways together caused a more severe proximo‐distal truncation of growth of the FNP, but not as severe as blocking Fgf signaling entirely. These results suggest that the downstream branches of the FGF pathway act inter‐dependently and in a non‐linear fashion to shape the face. Soon we will measure cellular orientation in the inhibitor cocktail treated FNPs and quantify the changes in FNP shape using three‐dimensional geometric morphometrics.

Augmenting Suture Tape Used in Rotator Cuff Surgery With Magnesium Increases in Vitro Cellular Adhesion of Human Subacromial Bursal Tissue

PurposeTo evaluate the effect of magnesium on cellular adhesion and proliferation of human subacromial bursal tissue (SBT), osteoblasts, and tenocytes on nonabsorbable suture tape commonly used in rotator cuff surgery.MethodsHuman SBT cells, primary human osteoblasts (HOBs), and primary human tenocytes were isolated from tissue samples and cultured in growth media. Commercially available collagen-coated nonabsorbable suture tape was cut into one-inch pieces, placed into 48-well culture dishes, sterilized under ultraviolet light, and treated with (+) or without (–) magnesium. For the (+) magnesium group, a one-time dose of 5 mM sterile magnesium chloride was added. Subsequently, cells were plated at a density of 20,000 cells/cm2. For each cell source (SBT, HOBs, tenocytes) cellular proliferation and adhesion assays on suture tape treated (+) or (–) magnesium were performed.ResultsSBT, HOBs, and tenocytes each demonstrated the ability to adhere and proliferate on suture tape. Augmenting suture tape with magnesium resulted in a significantly increased cellular adhesion of SBT compared with nonaugmented sutures (P = .001), whereas no significant differences were observed for HOBs (P = .081) and tenocytes (P = .907). Augmentation with magnesium demonstrated no significant difference in cellular proliferation of SBT (P = .856), HOBs (P = .672), and tenocytes (P = .251) compared with nonaugmented sutures.ConclusionsSBT, osteoblasts, and tenocytes each demonstrated the ability to adhere and proliferate on suture tape. In addition, augmenting the suture with magnesium resulted in a significantly increased cellular adhesion of SBT compared with nonaugmented sutures, whereas no significant differences were observed for osteoblasts and tenocytes. Further, magnesium did not impair the proliferative activity of SBT, osteoblasts, and tenocytes on suture tape used in rotator cuff surgery.Clinical RelevanceModifying the surface of the suture used for repair with application of magnesium may be an inexpensive and technically feasible option to improve the use of SBT for biologic augmentation of rotator cuff repair.

Vascular Smooth Muscle Cell Derived from IPS Cell of Moyamoya Disease - Comparative Characterization with Endothelial Cell Transcriptome

BackgroundMoyamoya disease (MMD) is an occlusive cerebrovascular disease, causing stroke in children and young adults with unknown etiology. The fundamental pathology is fibrocellular intimal thickening of cerebral arteries, in which vascular smooth muscle cells (VSMCs) are observed as one of the major cell types. Although the characteristics of circulating smooth muscle progenitor cells have been previously reported, the VSMCs are poorly characterized in MMD. We aimed to characterize VSMCs in MMD using induced pluripotent stem cell (iPSC)-technology. MethodsWe differentiated VSMCs from neural crest stem cells (NCSCs) using peripheral blood mononuclear cell-derived iPSCs and compared biological and transcriptome features under naïve culture conditions between three independent healthy control (HC) subjects and three MMD patients. VSMC transcriptome profiles were also compared to those of endothelial cells (ECs) differentiated from the same iPSCs. ResultsHomogeneous spindle-shaped cells differentiated from iPSCs exhibited smooth muscle cell marker expressions, including α-smooth muscle actin (αSMA, 82.3 ± 6.7% and 81.0 ± 6.7%); calponin (91.3 ± 2.1% and 90.9 ± 1.3%); myosin heavy chain-11 (MYH11, 96.9 ± 0.7% and 97.1 ± 0.3%) without significance of differences between the two groups. Real-time PCR showed few PECAM1 and CD34 gene expressions in both groups, indicating features of differentiated VSMCs. There were no significant differences in cellular proliferation (p = 0.45), migration (p = 0.60), and contractile abilities (p = 0.96) between the two groups. Transcriptome analysis demonstrated similar gene expression profiles of VSMCs in HC subjects and MMD patients with six differentially expressed genes (DEGs); while ECs showed a distinct transcriptome profile in MMD patients with 120 DEGs. The Wnt-signaling pathway was a significant pathway in VSMCs. ConclusionsThis is the first study that established VSMCs from NCSCs using MMD patient-derived iPSCs and demonstrated similar biological function and transcriptome profile of iPSC-derived VMSCs in MMD patients and HC subjects under naïve single culture condition. Comparative transcriptome features between iPSC-derived VSMCs and ECs, displaying distinct transcriptome in the ECs, suggested that pathological traits can be driven by naïve ECs predominantly and VSMCs may require specific environmental factors in MMD, which provides novel insight into the pathophysiology of MMD. Our iPSC derived VSMC model can contribute to further investigations of diagnostic and therapeutic target of MMD in addition to the current iPSC derived EC model.

Subacromial Bursa–Derived Cells Demonstrate High Proliferation Potential Regardless of Patient Demographics and Rotator Cuff Tear Characteristics

To investigate the influence of patient demographics and rotator cuff tear characteristics on the cellular proliferation potential of subacromial bursa-derived cells (SBDCs). Patients undergoing arthroscopic rotator cuff repair between December 2017 and February 2019 were considered for enrollment in the study. Basic demographic information as well as medical and surgical history were obtained for each patient. Subacromial bursa was harvested from over the rotator cuff tendon. Cellular proliferation was evaluated after 3 weeks of incubation by counting nucleated SBDCs. Fluorescence-activated cell sorting (FACS) analysis was performed to confirm the presence of mesenchymal stem cell (MSC) specific surface markers. Using preoperative radiographs and magnetic resonance imaging (MRI), acromiohumeral distance (AHD), severity of cuff tear arthropathy, and rotator cuff tear characteristics were evaluated. Seventy-three patients (mean age: 57.2 ± 8.5 years) were included in the study. There was no significant difference in cellular proliferation of SBDCs when evaluating the influence of age, sex, body mass index (BMI), smoking status, and presence of systemic comorbidities (p > .05, respectively). Similarly, there was no significant difference in cellular proliferation of SBDCs when looking at rotator cuff tear characteristics (size, tendon retraction, fatty infiltration, muscle atrophy), AHD, or severity of cuff tear arthropathy (p > .05). FACS analysis confirmed nucleated SBDCs to have a high positive rate of MSC specific surface markers. Subacromial bursa consistently demonstrated a high cellular proliferation potential regardless of patient demographics, rotator cuff tear characteristics, and severity of glenohumeral joint degeneration. These findings may alleviate concerns that subacromial bursa might lose cellular proliferation potential when being used for biologic augmentation in massive and degenerated rotator cuff tears, thus assisting in predicting tendon healing and facilitating surgical decision-making.

Dietary supplementation of micro-encapsulated sodium butyrate in healthy horses: effect on gut histology and immunohistochemistry parameters

BackgroundAs colic and intestinal disorders are a major concern in horses, the aim of the present study was to investigate the effect of dietary supplementation of butyrate, known to have a diverse array of beneficial effects on intestinal health. The effect of micro-encapsulated sodium butyrate supplementation on gut histology and immunohistochemistry parameters was studied in 14 healthy warmblood horses destined for slaughter in two separate periods. Horses were fed a low fiber - high starch diet, designed to induce subsequent starch overflow in the large intestine, aiming to create a mild challenge for large intestinal health. Treatment included supplementation with either micro-encapsulated sodium butyrate (Excential Butycoat®, Orffa, Werkendam, the Netherlands) or placebo (containing only coating material). The horses were fed for 20 consecutive days at a dosage of 0.4 g/kg BW (body weight). At day 21, the horses were slaughtered and intestinal samples were collected for determination of gut pH, villus length, crypt depth and area % of CD3+ and CD20+ cells.ResultsHorses on the butyrate supplemented diet had significantly reduced crypt depths in the right dorsal colon compared to placebo-fed horses (P < 0.001). However, a treatment x period interaction (P = 0.002) was discovered regarding this parameter, which could not be explained by the authors. Further investigation into the number of KI67+ cells in the RDC crypts did not reveal any significant differences between treatments (P = 0.650), indicating that the reduction in crypt depth in butyrate-fed horses could not be explained by a significant difference in cellular proliferation. Intestinal pH, villus length and expression of intestinal CD3+ and CD20+ cells were not significantly affected by treatment at any intestinal level.ConclusionsOur data indicate that supplementation of micro-encapsulated sodium butyrate to the equine diet did not influence gut histology (with the exception of a decrease found in the crypts of the RDC) or immunohistochemistry parameters in healthy horses. Further research is warranted to investigate the impact of butyrate supplementation in horses with intestinal disease.

An investigation into the role of chronic Schistosoma mansoni infection on Human Papillomavirus (HPV) vaccine induced protective responses.

BackgroundSchistosoma mansoni is one of the most common helminth infections affecting a large population of people in sub-Saharan Africa. This helminth infection is known to cause immunomodulation which has affected the efficacy of a number of vaccines. This study examined whether a chronic schistosoma infection has an effect on the immunogenicity of HPV vaccine which is currently administered to girls and women aged 9 to 24. Little is known about the immune responses of the HPV vaccine in individuals with chronic schistosomiasis.MethodsThis study was carried out at the Institute of Primate Research (IPR) and involved an Olive baboon model. The experimental animals were randomly placed into three groups (n = 3–4); Two groups were infected with S. mansoni cercaria, and allowed to reach chronic stage (week 12 onwards), at week 13 and 14 post-infection, one group was treated with 80mg/kg of praziquantel (PZQ). Sixty four weeks post schistosoma infection, all groups received 2 doses of the Cervarix HPV vaccine a month apart. Specific immune responses to the HPV and parasite specific antigens were evaluated.ResultsAnimals with chronic S. mansoni infection elicited significantly reduced levels of HPV specific IgG antibodies 8 weeks after vaccination compared the PZQ treated and uninfected groups. There was no significant difference in cellular proliferation nor IL-4 and IFN-γ production in all groups.ConclusionChronic S. mansoni infection results in reduction of protective HPV specific IgG antibodies in a Nonhuman Primate model, suggesting a compromised effect of the vaccine. Treatment of schistosomiasis infection with PZQ prior to HPV vaccination, however, reversed this effect supporting anti-helminthic treatment before vaccination.

Abstract 5107: A novel MTA1 knock-in mouse model for the mechanistic and therapeutic studies of MTA1-driven prostate cancer

Abstract Metastasis-associated protein 1 is known to play role in prostate cancer progression and metastasis. Studies of MTA1-mediated prostate cancer tumorigenesis, progression and metastasis have been hampered by a lack of suitable experimental system. Here we describe a new conditional mouse model that shows strong MTA1 expression in the prostate. This model was generated through MTA1 transgene knocked into the mouse ROSA26 (R26) locus using CAG-LoxP-Stop-LoxP (LSL)-2HA-MTA1-T2A-GFP-pA construct (Applied StemCell,Inc, Menlo Park, CA). Briefly, after generation of a genetically modified founder mouse line Rosa26-3attP, resulted MTA1 transgene embryos were injected into foster mice to produce MTA1 founders, designated as R26-LSL-MTA1. To monitor tumor formation and development in live animals by using IVIS, we have crossbred R26-LSL-MTA1 with Pb-Cre+;Luc and generated transgenic lines that express MTA1 and Luc upon Cre-mediated removal of the LSL cassette, specifically in the prostate epithelium (Pb-Cre+;R26MTA1;R26Luc) (MTA1-tg). We confirmed prostate-specific expression of the MTA1 transgene on mRNA and protein levels. We evaluated prostate histology for differences in histology of control normal prostates (Pb-Cre-; MTA1-tg) and MTA1-tg mice at as early as 13-weeks of age. MTA1 overexpressing prostate showed signs of mPIN characterized by glandular structures composed of atypical proliferating luminal cells that fill out the lumen. We detected differences in cellular proliferation, as determined by increased PCNA and decreased p27 as well as Ki67 immunofluorescent staining in MTA1-tg prostate. Moreover, expression of several oncogenic MTA1-guided molecules was increased in the prostates of MTA1-tg mice. We have previously reported on the role of MTA1-mediated signaling in prostate tumor progression in the prostate-specific Pten heterozygous (Pb-Cre+; Pten+/f) and Pten null (Pb-Cre+; Ptenf/f ) mice. To determine the role of MTA1 overexpression in the context of PTEN loss, we generated MTA1-tg; Pten+/f and MTA1-tg; Ptenf/f mice to evaluate whether MTA1overexpression in the prostate promotes an earlier onset, greater total incidence, accelerated tumor growth rate and metastasis. We predict that MTA1 overexpression will cause higher activation of downstream pathways to promote cancer cell survival, which will result in increased incidence of invasive adenocarcinoma and probably lymph node metastasis. Mice will be sacrificed at different time points for prostate tissue and lymph nodes isolation and subsequent histopathological, immunochemical and molecular evaluation. Citation Format: Avinash Kumar, Swati Dhar, Nasir A. Butt, Pravin R. Phadatare, Kshiti Dholakia, Jogarao Vedula, Virginia Donovan, Anait S. Levenson. A novel MTA1 knock-in mouse model for the mechanistic and therapeutic studies of MTA1-driven prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5107.

Viability, Proliferation, and Chondrogenesis of Equine Bone Marrow–Derived Mesenchymal Stromal Cells After Exposure to Varying Concentrations of Allogeneic Synovial Fluid In Vitro

Intraarticular administration of bone marrow–derived mesenchymal stromal cells (BMSCs) for treatment of acute or chronic joint pathology of the equine athlete has gained clinical interest. The objective of this study was to evaluate the effects of equine allogeneic synovial fluid on viability, proliferation, and chondrogenesis of BMSCs. Bone marrow was collected from eight horses aged 8–14 years old. Synovial fluid was collected from 25 horses aged 4–20 years old. Bone marrow–derived mesenchymal stem cells were cultured in medium supplemented with 25, 50, or 100% (vol/vol) synovial fluid for 72 hours. Viability and proliferation were measured after 24 and 72 hours. Bone marrow–derived mesenchymal stem cells were cultured in micromass for 28 days after which cell viability and glycosaminoglycan (GAG) content was quantified to assess chondrogenesis. A significant increase in viability was observed after 24 hours in culture with greater than 25% synovial fluid supplementation, but no difference in viability was observed after 72 hours of culture. A significant increase in cell number was observed in BMSCs exposed to 100% synovial fluid after 24 and 72 hours. However, no significant difference in cellular proliferation was detected between BMSCs cultured in 100% synovial fluid and control medium after 24 and 72 hours. Bone marrow–derived mesenchymal stem cells became elongated and aggregated into three-dimensional colonies during culture. Additionally, GAG production per viable BMSC in 100% synovial fluid was significantly increased compared to control medium. Supplementation or substitution of culture medium with synovial fluid was able to support BMSC viability, proliferation, and chondrogenesis in vitro.

Abstract 4168: Altering cisplatin sensitivity by manipulating the cellular metallome

Abstract The cellular metallome serves as an important reservoir to meet the biological needs of the cell, serving as enzyme co-factors and ensuring proper protein folding. Recent studies have suggested that cancer cells adapt their metallome in order to accommodate rapid cellular growth and metabolism pathways. Thus, altering the cancer metallome may yield new avenues to target cancer and change the responsiveness to chemotherapy. Here, we aim to characterize and understand how changes in the ovarian cancer metallome affect sensitivity to the chemotherapy drug cisplatin. Five ovarian cancer cell lines were used in experiments: A2780, A2780/CP70, SKOV3, OVCAR3, and CAOV3. The metallome was manipulated by treating cells with either zinc, copper, magnesium, calcium, iron, manganese, or the metal chelator tetrathiomolybdate. Cisplatin sensitivity after metal treatment was measured by WST-1 cell proliferation assays. Metallomes were quantitated by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) by measuring the following metals: zinc, copper, magnesium, calcium, molybdenum, manganese, iron, phosphorous, and sulfur. Cellular metal concentrations were normalized to sulfur levels. Changes in cisplatin sensitivity were evaluated by measuring cellular cisplatin uptake by ICP-MS. Treatment of ovarian cancer cells with various metals resulted in differences in cellular proliferation and metallome composition. The changes in the metallome were not singular in nature because altering one metal resulted in changes in multiple metals. Cisplatin sensitivity was additionally affected under different metal treatments. Preliminary data suggest that cisplatin uptake was altered as a mechanism to mediate the cellular response to cisplatin, however other mechanisms such as DNA repair might also play a role. In conclusion, we demonstrate that perturbations of the cancer metallome result in changes of various metals, cellular proliferation, and sensitivity to the chemotherapy drug cisplatin. The data lend further insight into how an individual’s metallomic status, a combination of nutritional and harmful metal exposure, might result in the development and progression of cancer and affect the response to chemotherapy. Citation Format: Lauren Amable, Eric Shide. Altering cisplatin sensitivity by manipulating the cellular metallome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4168. doi:10.1158/1538-7445.AM2017-4168

Social isolation increases cell proliferation in male and cell survival in female California mice (Peromyscus californicus)

Social environment has direct effects on an animal's behavior, physiology and neurobiology. In particular, adult neurogenesis is notably affected by a variety of social manipulations, including social isolation. We hypothesized that social isolation should have particularly acute effects on neurogenesis in a highly social (monogamous and bi-parental) species such as Peromyscus californicus, the California mouse. Adult male and female P.californicus mice were housed in isolation or in same-sex pairs for 4 or 24days. At the end of each period, either cell proliferation or cell survival was quantified with BrdU label and neuronal markers (either TuJ1 or NeuN). After 4days, isolated males had greater cellular proliferation in the dentate gyrus of the hippocampus (DG) than pair housed males. After 24days, isolate females demonstrated greater cell survival in the DG than paired females. Males demonstrated a similar, but non-significant pattern. No differences in cellular proliferation or cell survival were found in the subventricular zone (SVZ), or medial amygdala (MeA). These results add to the evidence which demonstrates that neurogenic responses to environmental conditions are not identical across species. These data may be critical in understanding the functional significance of neurogenesis as it relates to the interactions between social systems, social environment and the display of social behaviors.

Survival of adult generated hippocampal neurons is altered in circadian arrhythmic mice.

The subgranular zone of the hippocampal formation gives rise to new neurons that populate the dentate gyrus throughout life. Cells in the hippocampus exhibit rhythmic clock gene expression and the circadian clock is known to regulate the cycle of cell division in other areas of the body. These facts suggest that the circadian clock may regulate adult neurogenesis in the hippocampus as well. In the present study, neurogenesis in the hippocampal subgranular zone was examined in arrhythmic Bmal1 knockout (-KO) mice and their rhythmic heterozygous and wildtype littermates. Proliferation and survival of newly generated subgranular zone cells were examined using bromodeoxyuridine labelling, while pyknosis (a measure of cell death) and hippocampal volume were examined in cresyl violet stained sections. There was no significant difference in cellular proliferation between any of the groups, yet survival of proliferating cells, 6 weeks after the bromodeoxyuridine injection, was significantly greater in the BMAL1-KO animals. The number of pyknotic cells was significantly decreased in Bmal1-KO animals, yet hippocampal volume remained the same across genotypes. These findings suggest that while a functional circadian clock is not necessary for normal proliferation of neuronal precursor cells, the normal pruning of newly generated neurons in the hippocampus may require a functional circadian clock.

S131 Iron chelation reduces lung cancer proliferation in vitro

IntroductionThere is growing evidence that iron plays an important role within the lung cancer, the leading cause of cancer-related mortality worldwide. As a result of this, iron homeostasis has potential...

Dual Reporter Gene Imaging for Tracking Macrophage Migration Using the Human Sodium Iodide Symporter and an Enhanced Firefly Luciferase in a Murine Inflammation Model

The purpose of this study is to visualize the migration of reporter macrophages expressing both the human sodium iodide symporter (hNIS) and enhanced firefly luciferase (effluc) gene in mice with chemically induced inflammation. A macrophage cell line expressing both hNIS and effluc genes (Raw264.7/hNIS-effluc, herein referred to as a Raw264.7/NF) was established by cotransduction of two genes into a murine macrophage cell line (Raw264.7), and cell proliferation and phagocytic activity were compared between parental Raw264.7 and Raw264.7/NF cells. Both serial bioluminescence imaging (BLI) and small animal positron emission tomography (PET) imaging with I-124 were performed in inflammation-induced mice at various time points after intravenous injection of either Raw264.7 or Raw264.7/NF cells. There was no significant difference in cellular proliferation and phagocytic activity between parental Raw264.7 and Raw264.7/NF cells. Early distribution of Raw264.7/NF cells was successfully visualized in the lung and spleen by BLI, but not by I-124 PET imaging. BLI signals, but not PET signals, were observed from the inflammation site at day4 after the injection of Raw264.7/NF cells, and the signal intensity gradually increased until day8. In contrast, focal uptake of I-124 was first detected at the site of inflammation at postinjection day8, and signal intensity from the inflamed lesion was highest at that time point. While visualization of the inflamed lesion was possible by both BLI and PET imaging until day14, it was only possible by BLI until day21 after injection. Tracking of macrophage migration toward inflammation foci was successfully achieved in vivo from early time points by dual reporter gene imaging with a combination of nuclear and optical reporters. Multimodal reporter imaging of macrophages might successfully overcome the limitations of single reporter gene imaging in preclinical models of inflammation.

Independent of ErbB1 gene copy number, EGF stimulates migration but is not associated with cell proliferation in non-small cell lung cancer

BackgroundLung cancer often exhibits molecular changes, such as the overexpression of the ErbB1 gene. ErbB1 encodes epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, involved mainly in cell proliferation and survival. EGFR overexpression has been associated with more aggressive disease, poor prognosis, low survival rate and low response to therapy. ErbB1 amplification and mutation are associated with tumor development and are implicated in ineffective treatment. The aim of the present study was to investigate whether the ErbB1 copy number affects EGFR expression, cell proliferation or cell migration by comparing two different cell lines.MethodsThe copies of ErbB1 gene was evaluated by FISH. Immunofluorescence and Western blotting were performed to determine location and expression of proteins mentioned in the present study. Proliferation was studied by flow cytometry and cell migration by wound healing assay and time lapse.ResultsWe investigated the activation and function of EGFR in the A549 and HK2 lung cancer cell lines, which contain 3 and 6 copies of ErbB1, respectively. The expression of EGFR was lower in the HK2 cell line. EGFR was activated after stimulation with EGF in both cell lines, but this activation did not promote differences in cellular proliferation when compared to control cells. Inhibiting EGFR with AG1478 did not modify cellular proliferation, confirming previous data. However, we observed morphological alterations, changes in microfilament organization and increased cell migration upon EGF stimulation. However, these effects did not seem to be consequence of an epithelial-mesenchymal transition.ConclusionEGFR expression did not appear to be associated to the ErbB1 gene copy number, and neither of these aspects appeared to affect cell proliferation. However, EGFR activation by EGF resulted in cell migration stimulation in both cell lines.

The effect of matrix metalloproteinase 2 and matrix metalloproteinase 2/9 deletion in experimental post-thrombotic vein wall remodeling

Vein wall fibrotic injury following deep venous thrombosis (VT) is associated with elevated matrix metalloproteinases (MMPs). Whether and by what mechanism MMP2 contributes to vein wall remodeling after VT is unknown. Stasis VT was produced by ligation of the inferior vena cava and tissue was harvested at 2, 8, and 21 days in MMP2 -/- and genetic wild type (WT) mice. Tissue analysis by immunohistochemistry, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and zymography was performed. Thrombus resolution was less at 8 days in MMP2 -/- compared with WT, evidenced by a 51% increase in VT size (P < .01), and threefold fewer von Willebrand's factor positive channels (P < .05). In MMP2 -/- mice, the main phenotypic fibrotic differences occurred at 8 days post-VT, with significantly less vein wall collagen content (P = .013), fourfold lower procollagen III gene expression (P < .01), but no difference in procollagen I compared with WT. Decreased inflammation in MMP2 -/- vein walls was suggested by ∼ threefold reduced TNFα and IL-1β at 2 days and 8 days post-VT (P < .05). A fourfold increase in vein wall monocytes (P = .03) with threefold decreased apoptosis (P < .05), but no difference in cellular proliferation at 8 days was found in MMP2 -/- compared with WT. As increased compensatory MMP9 activity was observed in the MMP2 -/-mice, MMP2/9 double null mice had thrombus induced with VT harvest at 8 days. Consistently, twofold larger VT, a threefold decrease in vein wall collagen, and a threefold increase in monocytes were found (all P < .05). Similar findings were observed in MMP9 -/- mice administered an exogenous MMP2 inhibitor. In stasis VT, deletion of MMP2 was associated with less midterm vein wall fibrosis and inflammation, despite an increase in monocytes. Consideration that VT resolution was impaired with MMP2 (and MMP2/9) deletion suggests direct inhibition will likely also require anticoagulant therapy.

Inhomogeneous background magnetic field in biological incubators is a potential confounder for experimental variability and reproducibility

This report shows that the background magnetic field in biological incubators can vary by orders of magnitude within and between incubators. These variations can be observed within the same incubator in locations that are centimeters apart from each other as well as between incubators that are identical and located in the same laboratory. Additionally, the values measured were frequently outside the range of magnitudes found naturally on the Earth's surface or ordinary habitation spaces. Exposure to such altered magnetic field environments has been experimentally shown to be sufficient to cause numerous effects in cell cultures. Examples of the effects reported span from differential generation of free radicals and heat shock proteins to differences in cellular proliferation, differentiation, and death. Although the effects are not well established and the molecular mechanism of action is currently under debate, these observations alone support the notion that the inhomogeneity of the background magnetic field in incubators is a potential confounding source of the variability and reproducibility for studies performed on cell cultures. In this regard, it is recommended that special measures be adopted to control the background magnetic fields in incubators when investigating the biological effects of exposure to magnetic fields of comparable characteristics as the ones measured in this study, or when studying small biological effects in general.

Abstract TP103: Brain Protection Effect By Exposure To Hydrogen Sulphide In Cerebral Infarct In Rat

Introduction: Hydrogen sulphide (H2S) can regulate oxygen consumption by competing with O2 in binding to cytochrome C oxydase. At micro molar concentrations H2S interferes with antinecrotic and antiapoptotic mechanisms, including up regulation of cytoprotective genes. We studied the effect of H2S exposure on lesion and functional recovery after permanent middle cerebral artery occlusion (pMCAO) in rats. Methods: Young adult male and female Sprague- Dawley rats distributed into three groups: 1- Sham (control for craniotomy); 2-Control: Surgery + permanent MCAO; 3- Treated: Surgery + permanent MCAO + inhalation of 40 ppM hydrogen sulphide. We analyzed functional outcome (Rogers modified scale), lesion volume by MRI at 24h and 14 day and by hematoxyline-eosin, cell death by TUNEL, repair markers implicated in brain plasticity (cellular proliferation by BrdU, GFAP (glial fibrillar acis protein), VEGF (vascular endothelial growth factor), Synaptophysine) by western-blot and immunofluorescence at 14 days. Rats were sacrificed at day 14th. Results: Treated animals showed a benefit in functional outcome both at 24h (p = 0,004445) and sacrifice day (p = 0,000942). We observed a decrease in infarct size at day 14th on MRI (p= 0,0383 ) and in Hematoxyline Eosine infarct areas (p= 0.026672 ). At 24 hours there was no statistical difference on MRI (p= 0,380644). TUNEL analysis showed a decrease in the expression of marked cells in the perinfarct zone of treated animals (p= 0,048877). We did not observe significant differences in cellular proliferation (BrdU), GFAP, VEGF and synapthophysin levels between control and treated groups. Conclusions: Exposure to hydrogen sulphide in rats with cerebral infarct was effective in functional recovery associated to brain protection mechanisms with reduction in size volume and cell death and may be associated with reperfusion increased but not in repair effects.

Increased Skin Papilloma Formation in Mice Lacking Glutathione Transferase GSTP

The glutathione S-transferase GSTP is overexpressed in many human cancers and chemotherapy-resistant cancer cells, where there is evidence that GSTP may have additional functions beyond its known catalytic role. On the basis of evidence that Gstp-deficient mice have a comparatively higher susceptibility to skin carcinogenesis, we investigated whether this phenotype reflected an alteration in carcinogen detoxification or not. For this study, Gstp(-/-) mice were interbred with Tg.AC mice that harbor initiating H-ras mutations in the skin. Gstp(-/-)/Tg.AC mice exposed to the proinflammatory phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) exhibited higher tumor incidence and multiplicity with a significant thickening of skin after treatment, illustrating hyperproliferative growth. Unexpectedly, we observed no difference in cellular proliferation or apoptosis or in markers of oxidative stress, although higher levels of the inflammatory marker nitrotyrosine were found in Gstp(-/-)/Tg.AC mice. Instead, gene set enrichment analysis of microarray expression data obtained from skin revealed a more proapoptotic and proinflammatory environment shortly after TPA treatment. Within 4 weeks of TPA treatment, Gstp(-/-)/Tg.AC mice displayed altered lipid/sterol metabolism and Wnt signaling along with aberrant processes of cytoskeletal control and epidermal morphogenesis at both early and late times. In extending the evidence that GSTP has a vital role in normal homeostatic control and cancer prevention, they also strongly encourage the emerging concept that GSTP is a major determinant of the proinflammatory character of the tumor microenvironment. This study shows that the GSTP plays a major role in carcinogenesis distinct from its role in detoxification and provides evidence that the enzyme is a key determinant of the proinflammatory tumor environment.