Abstract 1163: Engineering Biomaterials For Vascular Differentiation And Regeneration

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Sharon Gerecht ◽  
Gordana Vunjak-Novakovic ◽  
Robert Langer
Keyword(s):  
Mammalian Cells ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Gene Expression Profiles ◽  
Vascular Differentiation ◽  
Human Escs ◽  
Differentiated Cells ◽  
And Migration

The goal of our studies was to utilize engineered biomaterials to understand the mechanisms underlying microenvironment regulatory cascades during vascular differentiation and regeneration. We found that the ECM polysaccharide hyaluronic acid (HA) is a developmentally relevant material for the growth of human embryonic stem cels (hESCs). Human ESCs encapsulated in HA hydrogels in conditioned media, maintained their undifferentiated state and could be further initiate vasculogenesis within the same system by supplementation with VEGF. In a continuous study we have developed a method for introducing pores into photocurable bioelastomer while maintaining mechanical properties. Biocompatibility studies demonstrated the ability to encapsulate, grow and differentiated cells in vitro , while subcutaneous transplantation revealed inflammatory response similar to other biocompatible materials. In addition, these In vivo experiments showed that the porous bioelastomer promotes ingrowth and integration with the host circulation, suggesting that porous bioelastomer may be utilized for tissue engineering applications. Mammalian cells respond to their substrates by complex changes in gene expression profiles, morphology, proliferation and migration. We report that the nanotopography of the substrate can be used to control various cellular responses of hESCs and human endothelial progenitor cell (hEPC). Poly(dimethylsiloxane) (PDMS) films were replica-molded on passivated silicon wafers to yield line-grating (600 nm ridges with 600 nm spacing and 600 ± 150 nm feature height), coated with fibronectin or collagen, and seeded with single-cell suspensions. These nanopattered PDMS substrates induced hESC alignment and elongation, mediated the organization of cytoskeletal components, and reduced proliferation. The addition of actin disrupting agents attenuated the alignment and proliferative effects of nanotopography. Human EPCs cultured on nanotopographic substrates elongated, and aligned with the structures, while their migration was also enhanced. Long-term cultures led to the formation of band-like structures of hEPCs as their proliferation was reduced, and the addition of matrigel led to the formation of ordered tube structures.

scholarly journals The MYEOV-MYC association promotes oncogenic miR-17/93-5p expression in pancreatic ductal adenocarcinoma

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Hongzhang Shen ◽  
Fuqiang Ye ◽  
Dongchao Xu ◽  
Liangliang Fang ◽  
Xiaofeng Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Ductal Adenocarcinoma ◽  
Invasion And Migration ◽  
And Migration

AbstractPancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy worldwide. As metastasis and malignant progression are primarily responsible for the poor clinical outcomes of PDAC, identifying key genes involved in these processes and the underlying molecular mechanisms of PDAC is vital. In this study, by analyzing TCGA PDAC data and matched GTEx data, we found that MYEOV expression is associated with poor survival in PDAC patients and higher in carcinoma tissues than in healthy tissues. Elevated levels of MYEOV led to enhanced cell proliferation, invasion and migration in vitro and in vivo. Transcriptome analysis results revealed that MYEOV mediates global alterations in gene expression profiles in PDAC cells. MiRNA-seq analysis showed that MYEOV regulates the expression levels of miR-17-5p and miR-93-5p, and its depletion resulted in reduced cell proliferation, invasion and migration, as observed in MYEOV-knockdown PDAC cells. These effects are likely due to the ability of MYEOV to regulate enrichment of the transcription factor MYC at the gene promoter regions of the two miRNAs. Furthermore, we identified a complex containing MYEOV and MYC in the nucleus, providing additional evidence for the association of MYEOV with MYC. Taken together, our results suggest that MYEOV promotes oncogenic miR-17/93-5p expression by associating with MYC, contributing to PDAC progression.

scholarly journals Transcriptome analysis of gravitational effects on mouse skeletal muscles under microgravity and artificial 1 g onboard environment

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Risa Okada ◽  
Shin-ichiro Fujita ◽  
Riku Suzuki ◽  
Takuto Hayashi ◽  
Hirona Tsubouchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Muscle Mass ◽  
Muscle Atrophy ◽  
Transcriptome Analysis ◽  
Fiber Type ◽  
Expression Profiles ◽  
Space Station ◽  
Gene Expression Profiles

AbstractSpaceflight causes a decrease in skeletal muscle mass and strength. We set two murine experimental groups in orbit for 35 days aboard the International Space Station, under artificial earth-gravity (artificial 1 g; AG) and microgravity (μg; MG), to investigate whether artificial 1 g exposure prevents muscle atrophy at the molecular level. Our main findings indicated that AG onboard environment prevented changes under microgravity in soleus muscle not only in muscle mass and fiber type composition but also in the alteration of gene expression profiles. In particular, transcriptome analysis suggested that AG condition could prevent the alterations of some atrophy-related genes. We further screened novel candidate genes to reveal the muscle atrophy mechanism from these gene expression profiles. We suggest the potential role of Cacng1 in the atrophy of myotubes using in vitro and in vivo gene transductions. This critical project may accelerate the elucidation of muscle atrophy mechanisms.

scholarly journals Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. e66-e73 ◽  
Author(s):  
Chih-Wen Ni ◽  
Haiwei Qiu ◽  
Amir Rezvan ◽  
Kihwan Kwon ◽  
Douglas Nam ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Gene Expression Profiles ◽  
Disturbed Flow ◽  
A Genome ◽  
Pattern Comparison

Abstract Recently, we showed that disturbed flow caused by a partial ligation of mouse carotid artery rapidly induces atherosclerosis. Here, we identified mechanosensitive genes in vivo through a genome-wide microarray study using mouse endothelial RNAs isolated from the flow-disturbed left and the undisturbed right common carotid artery. We found 62 and 523 genes that changed significantly by 12 hours and 48 hours after ligation, respectively. The results were validated by quantitative polymerase chain reaction for 44 of 46 tested genes. This array study discovered numerous novel mechanosensitive genes, including Lmo4, klk10, and dhh, while confirming well-known ones, such as Klf2, eNOS, and BMP4. Four genes were further validated for protein, including LMO4, which showed higher expression in mouse aortic arch and in human coronary endothelium in an asymmetric pattern. Comparison of in vivo, ex vivo, and in vitro endothelial gene expression profiles indicates that numerous in vivo mechanosensitive genes appear to be lost or dysregulated during culture. Gene ontology analyses show that disturbed flow regulates genes involved in cell proliferation and morphology by 12 hours, followed by inflammatory and immune responses by 48 hours. Determining the functional importance of these novel mechanosensitive genes may provide important insights into understanding vascular biology and atherosclerosis.

scholarly journals Recapitulating tumour microenvironment in chitosan–gelatin three-dimensional scaffolds: an improved in vitro tumour model

2012 ◽  
Vol 9 (77) ◽  
pp. 3288-3302 ◽  
Author(s):  
Neha Arya ◽  
Viren Sardana ◽  
Meera Saxena ◽  
Annapoorni Rangarajan ◽  
Dhirendra S. Katti
Keyword(s):  
Cancer Progression ◽  
Expression Profiles ◽  
Three Dimensional ◽  
Gene Expression Profiles ◽  
Petri Dish ◽  
Tumour Microenvironment ◽  
Two Dimensional ◽  
Tumour Model

Owing to the reduced co-relationship between conventional flat Petri dish culture (two-dimensional) and the tumour microenvironment, there has been a shift towards three-dimensional culture systems that show an improved analogy to the same. In this work, an extracellular matrix (ECM)-mimicking three-dimensional scaffold based on chitosan and gelatin was fabricated and explored for its potential as a tumour model for lung cancer. It was demonstrated that the chitosan–gelatin (CG) scaffolds supported the formation of tumoroids that were similar to tumours grown in vivo for factors involved in tumour-cell–ECM interaction, invasion and metastasis, and response to anti-cancer drugs. On the other hand, the two-dimensional Petri dish surfaces did not demonstrate gene-expression profiles similar to tumours grown in vivo . Further, the three-dimensional CG scaffolds supported the formation of tumoroids, using other types of cancer cells such as breast, cervix and bone, indicating a possible wider potential for in vitro tumoroid generation. Overall, the results demonstrated that CG scaffolds can be an improved in vitro tool to study cancer progression and drug screening for solid tumours.

scholarly journals The Diverse Roles of the Global Transcriptional Regulator PhoP in the Lifecycle of Yersinia pestis

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1039
Author(s):  
Hana S. Fukuto ◽  
Gloria I. Viboud ◽  
Viveka Vadyvaloo
Keyword(s):  
Yersinia Pestis ◽  
Current Knowledge ◽  
Response Regulator ◽  
Expression Profiles ◽  
Critical Role ◽  
Gene Expression Profiles ◽  
Acanthamoeba Castellanii ◽  
Wide Range

Yersinia pestis, the causative agent of plague, has a complex infectious cycle that alternates between mammalian hosts (rodents and humans) and insect vectors (fleas). Consequently, it must adapt to a wide range of host environments to achieve successful propagation. Y. pestis PhoP is a response regulator of the PhoP/PhoQ two-component signal transduction system that plays a critical role in the pathogen’s adaptation to hostile conditions. PhoP is activated in response to various host-associated stress signals detected by the sensor kinase PhoQ and mediates changes in global gene expression profiles that lead to cellular responses. Y. pestis PhoP is required for resistance to antimicrobial peptides, as well as growth under low Mg2+ and other stress conditions, and controls a number of metabolic pathways, including an alternate carbon catabolism. Loss of phoP function in Y. pestis causes severe defects in survival inside mammalian macrophages and neutrophils in vitro, and a mild attenuation in murine plague models in vivo, suggesting its role in pathogenesis. A Y. pestisphoP mutant also exhibits reduced ability to form biofilm and to block fleas in vivo, indicating that the gene is also important for establishing a transmissible infection in this vector. Additionally, phoP promotes the survival of Y. pestis inside the soil-dwelling amoeba Acanthamoeba castellanii, a potential reservoir while the pathogen is quiescent. In this review, we summarize our current knowledge on the mechanisms of PhoP-mediated gene regulation in Y. pestis and examine the significance of the roles played by the PhoP regulon at each stage of the Y. pestis life cycle.

GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4108-4118 ◽  
Author(s):  
Naruyoshi Suwabe ◽  
Satoru Takahashi ◽  
Toru Nakano ◽  
Masayuki Yamamoto
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Erythroid Cells ◽  
Es Cell ◽  
Globin Mrna ◽  
Differentiated Cells ◽  
Vitro Analysis ◽  
Definitive Hematopoiesis

Abstract Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.

Gene Expression Profiles in Rat Liver Slices Exposed to Hepatocarcinogenic Enzyme Inducers, Peroxisome Proliferators, and 17α-Ethinylestradiol

2006 ◽  
Vol 25 (5) ◽  
pp. 379-395 ◽  
Author(s):  
Gisela Werle-Schneider ◽  
Andreas Wölfelschneider ◽  
Marie Charlotte von Brevern ◽  
Julia Scheel ◽  
Thorsten Storck ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rat Liver ◽  
Mode Of Action ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Peroxisome Proliferators ◽  
Liver Slices ◽  
Enzyme Inducers

Transcription profiling is used as an in vivo method for predicting the mode-of-action class of nongenotoxic carcinogens. To set up a reliable in vitro short-term test system DNA microarray technology was combined with rat liver slices. Seven compounds known to act as tumor promoters were selected, which included the enzyme inducers phenobarbital, α-hexachlorocyclohexane, and cyproterone acetate; the peroxisome proliferators WY-14,643, dehydroepiandrosterone, and ciprofibrate; and the hormone 17 α-ethinylestradiol. Rat liver slices were exposed to various concentrations of the compounds for 24 h. Toxicology-focused TOXaminer™ DNA microarrays containing approximately 1500 genes were used for generating gene expression profiles for each of the test compound. Hierarchical cluster analysis revealed that (i) gene expression profiles generated in rat liver slices in vitro were specific allowing classification of compounds with similar mode of action and (ii) expression profiles of rat liver slices exposed in vitro correlate with those induced after in vivo treatment (reported previously). Enzyme inducers and peroxisome proliferators formed two separate clusters, confirming that they act through different mechanisms. Expression profiles of the hormone 17 α-ethinylestradiol were not similar to any of the other compounds. In conclusion, gene expression profiles induced by compounds that act via similar mechanisms showed common effects on transcription upon treatment in vivo and in rat liver slices in vitro.

scholarly journals Transit-Amplifying Cells in the Fast Lane from Stem Cells towards Differentiation

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Emma Rangel-Huerta ◽  
Ernesto Maldonado
Keyword(s):  
Stem Cells ◽  
Mammalian Cells ◽  
Progenitor Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Differentiated Cells ◽  
Dividing Cells ◽  
Cycle Regulation ◽  
Potential Use ◽  
Skin Epidermis

Stem cells have a high potential to impact regenerative medicine. However, stem cells in adult tissues often proliferate at very slow rates. During development, stem cells may change first to a pluripotent and highly proliferative state, known as transit-amplifying cells. Recent advances in the identification and isolation of these undifferentiated and fast-dividing cells could bring new alternatives for cell-based transplants. The skin epidermis has been the target of necessary research about transit-amplifying cells; this work has mainly been performed in mammalian cells, but further work is being pursued in other vertebrate models, such as zebrafish. In this review, we present some insights about the molecular repertoire regulating the transition from stem cells to transit-amplifying cells or playing a role in the transitioning to fully differentiated cells, including gene expression profiles, cell cycle regulation, and cellular asymmetrical events. We also discuss the potential use of this knowledge in effective progenitor cell-based transplants in the treatment of skin injuries and chronic disease.

MLL-AF9 and MLL-ENL Induce Deregulation of Similar Downstream Candidate Genes: An Analysis across Experimental Protocols and Laboratories.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3372-3372
Author(s):  
Ashish R. Kumar ◽  
Robert K. Slany ◽  
Jay L. Hess ◽  
John H. Kersey
Keyword(s):  
Gene Expression ◽  
Fusion Protein ◽  
Fusion Gene ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Expression Systems ◽  
Rt Pcr ◽  
Conditional Expression

Expression profiling has become an important tool for understanding gene deregulation in MLL-fusion leukemias. However, the results of gene profiling experiments are difficult to interpret when applied to leukemia cells because (i) leukemias arise in cells that differ greatly in their gene expression profiles, and (ii) leukemias most often require secondary genetic events in addition to the MLL fusion gene. Two principal model systems have been used to understand the direct effects of MLL-fusion genes. Knock-in models have the advantage of the fusion gene being under control of the physiologic promoter. On the other hand, conditional expression systems offer the ability to conduct short term experiments, permitting the analysis of direct effects on downstream genes. In the present combined-analysis, we used the Affymetrix U74Av2 oligonucleotide microarray to evaluate the effects of the MLL-fusion gene in vivo and in vitro respectively using two closely related MLL fusion genes - MLL-AF9 for knock-in and MLL-ENL for conditional expression. In the MLL-AF9 study, we compared gene expression profiles of bone marrow cells from MLL-AF9 knock-in mice (C57Bl/6, MLL-AF9+/−) to those of age-matched wild type mice (Kumar et. al. 2004, Blood). We used a t-test (p<0.05) to selected genes that showed significant changes in expression levels. In the MLL-ENL study, we transformed murine primary hematopoietic cells with a conditional MLL-ENL vector (MLL-ENL fused to the modified ligand-binding domain of the estrogen receptor) such that the fusion protein was active only in the presence of tamoxifen. We then studied the downstream effects of the fusion protein by comparing gene expression profiles of the cells in the presence and absence of tamoxifen. We used a pair-wise comparison analysis to select genes that showed a change in expression level of 1.5 fold or greater in at least two of three experiments (Zeisig et. al. 2004, Mol. Cell Biol.). Those genes that were up-regulated in both datasets were then compiled together. This list included Hoxa7, Hoxa9 and Meis1. The results for these 3 genes were confirmed by quantitative RT-PCR in both the MLL-AF9-knock-in and the MLL-ENL-conditional-expression systems. The remaining candidate genes in the common up-regulated gene set (not yet tested by quantitative RT-PCR) include protein kinases (Bmx, Mapk3, Prkcabp, Acvrl1, Cask), RAS-associated proteins (Rab7, Rab3b), signal transduction proteins (Notch1, Eat2, Shd, Fpr1), cell membrane proteins (Igsf4), chaperones (Hsp70.2), transcription factors (Isgf3g), proteins with unknown functions (Olfm1, Flot1), and hypothetical proteins. The results of the combined analysis demonstrate that these over-expressions are (i) a direct and sustained effect of the MLL-fusion protein, (ii) are independent of secondary events that might be involved in leukemogensis, and (iii) are independent of the two partner genes that participate in these fusions. The over-expression of a few genes in both the -in vitro and in vivo experimental systems makes these molecules very interesting for further studies, to understand the biology of MLL-fusion leukemias and for development of new therapeutic strategies.

scholarly journals Citrinin induces apoptosis via a mitochondria-dependent pathway and inhibition of survival signals in embryonic stem cells, and causes developmental injury in blastocysts

2007 ◽  
Vol 404 (2) ◽  
pp. 317-326 ◽  
Author(s):  
Wen-Hsiung Chan
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Mammalian Cells ◽  
Cell Injury ◽  
Reactive Oxygen Species Generation ◽  
Embryonic Stem ◽  
Subsequent Development ◽  
Cytochrome C Release

The mycotoxin CTN (citrinin), a natural contaminant in foodstuffs and animal feeds, has cytotoxic and genotoxic effects on various mammalian cells. CTN is known to cause cell injury, including apoptosis, but the precise regulatory mechanisms of CTN action, particularly in stem cells and embryos, are currently unclear. In the present paper, I report that CTN has cytotoxic effects on mouse embryonic stem cells and blastocysts, and is associated with defects in their subsequent development, both in vitro and in vivo. Experiments in embryonic stem cells (ESC-B5) showed that CTN induces apoptosis via ROS (reactive oxygen species) generation, increased Bax/Bcl-2 ratio, loss of MMP (mitochondrial membrane potential), induction of cytochrome c release, and activation of caspase 3. In this model, CTN triggers cell death via inactivation of the HSP90 [a 90 kDa isoform of the HSP (heat-shock protein) family proteins]/multichaperone complex and subsequent degradation of Ras and Raf-1, further inhibiting anti-apoptotic processes, such as the Ras→ERK (extracellular-signal-regulated kinase) signal transduction pathway. In addition, CTN causes early developmental injury in mouse ESCs and blastocysts in vitro. Lastly, using an in vivo mouse model, I show that consumption of drinking water containing 10 μM CTN results in blastocyst apoptosis and early embryonic developmental injury. Collectively, these findings show for the first time that CTN induces ROS and mitochondria-dependent apoptotic processes, inhibits Ras→ERK survival signalling via inactivation of the HSP90/multichaperone complex, and causes developmental injury in vivo.

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