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In JoVE (1)
Other Publications (13)
- The Journal of Biological Chemistry
- Journal of Molecular Medicine (Berlin, Germany)
- The Journal of Biological Chemistry
- Nucleic Acids Research
- Nucleic Acids Research
- The International Journal of Biochemistry & Cell Biology
- Molecular Therapy : the Journal of the American Society of Gene Therapy
- Organic & Biomolecular Chemistry
- Biomedical Microdevices
- Advanced Materials (Deerfield Beach, Fla.)
- Colloids and Surfaces. B, Biointerfaces
- International Journal of Biomaterials
- Molecules (Basel, Switzerland)
Articles by Scott A. Irvine in JoVE
Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells
Ramya Bhuthalingam1, Pei Q. Lim1, Scott A. Irvine1, Subbu S. Venkatraman1
1School of Materials Science and Engineering, Nanyang Technological University
Other articles by Scott A. Irvine on PubMed
A Novel Role of Sp1 and Sp3 in the Interferon-gamma -mediated Suppression of Macrophage Lipoprotein Lipase Gene Transcription
The Journal of Biological Chemistry. Mar, 2002 | Pubmed ID: 11796707
The regulation of macrophage lipoprotein lipase by cytokines is of potentially crucial importance in the pathogenesis of atherosclerosis. We have shown previously that macrophage lipoprotein lipase expression is suppressed by interferon-gamma (IFN-gamma) at the transcriptional level. We investigated the regulatory sequence elements and the transcription factors that are involved in this response. We demonstrated that the -31/+187 sequence contains the minimal IFN-gamma-responsive elements. Electrophoretic mobility shift assays showed that the binding of proteins to two regions in the -31/+187 sequence was reduced dramatically when the cells were exposed to IFN-gamma. Both competition electrophoretic mobility shift assays and antibody supershift assays showed that the interacting proteins were composed of Sp1 and Sp3. Mutations of the Sp1/Sp3-binding sites in the minimal IFN-gamma-responsive elements abolished the IFN-gamma-mediated suppression of promoter activity, whereas multimers of the sequence were able to impart the response to a heterologous promoter. Western blot analysis showed that IFN-gamma reduced the steady state levels of Sp3 protein. In contrast, the cytokine decreased the DNA binding activity of Sp1 without affecting the protein levels. These studies therefore reveal a novel mechanism for IFN-gamma-mediated regulation of macrophage gene transcription.
Journal of Molecular Medicine (Berlin, Germany). Dec, 2002 | Pubmed ID: 12483461
Lipoprotein lipase (LPL) catalyses the hydrolysis of the triacylglycerol component of circulating chylomicrons and very low density lipoproteins, thereby providing non-esterified fatty acids and 2-monoacylglycerol for tissue utilisation. Research carried out over the past two decades have not only established a central role for LPL in the overall lipid metabolism and transport but have also identified additional, non-catalytic functions of the enzyme. Furthermore, abnormalities in LPL function have been found to be associated with a number of pathophysiological conditions, including atherosclerosis, chylomicronaemia, obesity, Alzheimer's disease, and dyslipidaemia associated with diabetes, insulin resistance, and infection. Advances have also been made in relating the various domains in the protein to different functions, and in understanding the mechanisms that are responsible for the changes in LPL expression seen in response to nutritional and other physiological changes, and during disease. This review summarises recent findings in relation to the structure, function, and regulation of LPL along with its important role in disease.
Interferon-gamma Stimulates the Expression of the Inducible CAMP Early Repressor in Macrophages Through the Activation of Casein Kinase 2. A Potentially Novel Pathway for Interferon-gamma-mediated Inhibition of Gene Transcription
The Journal of Biological Chemistry. May, 2003 | Pubmed ID: 12609974
Interferon-gamma (IFN-gamma) is a pleiotropic cytokine that modulates the immune function, cell proliferation, apoptosis, macrophage activation, and numerous other cellular responses. These biological actions of IFN-gamma are characterized by both the activation and the inhibition of gene transcription. Unfortunately, in contrast to gene activation, the mechanisms through which the cytokine suppresses gene transcription remain largely unclear. We show here for the first time that exposure of macrophages to IFN-gamma leads to a dramatic induction in the expression of the inducible cAMP early repressor (ICER), a potent inhibitor of gene transcription. In addition, a synergistic action of IFN-gamma and calcium in the activation of ICER expression was identified. The IFN-gamma-mediated activation of ICER expression was not blocked by H89, bisindoylmaleimide, SB202190, PD98059, W7, and AG490, which inhibit protein kinase A, protein kinase C, p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, calcium-calmodulin-dependent protein kinase, and Janus kinase-2, respectively. In contrast, apigenin, a selective casein kinase 2 (CK2) inhibitor, was found to inhibit response. Consistent with this finding, IFN-gamma stimulated CK2 activity and the level of phosphorylated cAMP response element-binding protein, which is known to induce ICER gene transcription, and this response was inhibited in the presence of apigenin. These studies, therefore, identify a previously uncharacterized pathway, involving the IFN-gamma-mediated stimulation of CK2 activity, activation of cAMP response element-binding protein, and increased production of ICER, which may then play an important role in the inhibition of macrophage gene transcription by this cytokine.
Interleukin-6 Represses the Transcription of the CCAAT/enhancer Binding Protein-alpha Gene in Hepatoma Cells by Inhibiting Its Ability to Autoactivate the Proximal Promoter Region
Nucleic Acids Research. Dec, 2003 | Pubmed ID: 14627805
The cytokine interleukin-6 (IL-6) plays key roles in the immune and inflammatory responses, acute-phase reaction and hematopoiesis. Such biological actions of IL-6 are characterised by both the activation and the inhibition of gene transcription. Unfortunately, in contrast to gene activation, the mechanism by which IL-6 suppresses transcription remains largely unclear. We have, therefore, investigated this aspect using the Xenopus laevis CCAAT/enhancer binding protein-alpha (C/EBPalpha) gene promoter as a model. We show by transient transfection assays of various promoter-luciferase DNA constructs into hepatoma cells that a C/EBP recognition sequence in the proximal promoter region is essential for the IL-6-mediated repression. Electrophoretic mobility shift assays showed that C/EBPalpha was the major protein that bound to this site and, consistent with its expression pattern, the binding was reduced when the cells were exposed to IL-6. Co-transfection assays revealed for the first time that the ability of C/EBPalpha, but not C/EBPbeta or Sp1, to transactivate the promoter was decreased dramatically when the cells were incubated with IL-6. These studies, therefore, identify a novel mechanism for IL-6-mediated repression of gene transcription that involves a reduction in C/EBPalpha-mediated activation.
A Critical Role for the Sp1-binding Sites in the Transforming Growth Factor-beta-mediated Inhibition of Lipoprotein Lipase Gene Expression in Macrophages
Nucleic Acids Research. 2005 | Pubmed ID: 15755745
Increasing evidence suggests that the cytokine transforming growth factor-beta (TGF-beta) inhibits the development of atherosclerosis. The lipoprotein lipase (LPL) enzyme expressed by macrophages has been implicated in the pathogenesis of atherosclerosis by stimulating the uptake of lipoprotein particles. Unfortunately, the action of TGF-beta on the expression of LPL in macrophages remains largely unclear. We show that TGF-beta inhibits LPL gene expression at the transcriptional level. Transient transfection assays reveal that the -31/+187 sequence contains the minimal TGF-beta-responsive elements. Electrophoretic mobility shift assays show that Sp1 and Sp3 interact with two regions in the -31/+187 sequence. Mutations of these Sp1/Sp3 sites abolish the TGF-beta-mediated suppression whereas multimers of the sequence impart the response to a heterologous promoter. TGF-beta has no effect on the binding or steady-state polypeptide levels of Sp1 and Sp3. These results, therefore, suggest a novel mechanism for the TGF-beta-mediated repression of LPL gene transcription that involves regulation of the action of Sp1 and Sp3.
The International Journal of Biochemistry & Cell Biology. Jan, 2006 | Pubmed ID: 16140560
Lipoprotein lipase expressed by the vasculature plays a key role in atherogenesis by enhancing the binding and uptake of lipoproteins and, thereby, leading to the formation of lipid-loaded foam cells. Hyperlipidemia also accelerates the progression of glomerular diseases and addition of exogenous lipoprotein lipase to mesangial cells has been shown to lead to an enhanced binding of lipoproteins to these cells. Despite such potential importance, the expression of endogenous lipoprotein lipase by cells of the glomeruli has, as yet, not been investigated. We show here for the first time that mesangial cells, but not epithelial cells, express lipoprotein lipase. The minimal lipoprotein lipase gene promoter was active in mesangial cells and inhibited by interferon-gamma, which is known to suppress its expression.
Receptor-targeted Nanocomplexes Optimized for Gene Transfer to Primary Vascular Cells and Explant Cultures of Rabbit Aorta
Molecular Therapy : the Journal of the American Society of Gene Therapy. Mar, 2008 | Pubmed ID: 18180778
We have developed new, synthetic vector formulations that display high efficiency of gene transfer to vascular cells and tissues. The formulations comprise cationic liposomes and cationic, receptor-targeting peptides that self assemble on mixing with plasmid DNA into receptor-targeted nanocomplexes (RTNs). One such RTN formulation was optimal for transfection of primary smooth muscle cells (LYD-1), while a second was optimal for transfection of rabbit aortic explants (LYD-2). In both RTNs, the peptide was a 16-lysine motif linked to the targeting sequence CYGLPHKFCG via a short spacer sequence. The major difference between LYD-1 and LYD-2 lay in the cationic lipid component, where LYD-1 contained ditetradecyl trimethyl ammonium (DTDTMA), an unsaturated, cationic lipid with a 14-carbon alkyl tail, whereas LYD-2 contained 2,3-dioleyloxypropyl-1-trimethyl ammonium chloride (DOTMA), a cationic lipid with an 18-carbon unsaturated alkyl tail. LYD-2 transfections of aortic explants were effective with incubations performed at room temperature for as little as 30 minutes, with either saline or glucose-based solutions. Transgene expression in the explants peaked at 5 days and persisted for 14 days. The kinetics of transfected gene expression, along with the efficacy of transfection with short incubation times, indicate that these new formulations may be useful tools in the development of molecular therapies for cardiovascular diseases.
Mono- and Dicationic Short PEG and Methylene Dioxyalkylglycerols for Use in Synthetic Gene Delivery Systems
Organic & Biomolecular Chemistry. Jul, 2008 | Pubmed ID: 18600277
A range of monocationic and dicationic dioxyalkylglycerol cytofectins have been synthesised possessing methylene and short n-ethylene glycol spacers. The monocationic compounds were found to be effective in transfections when formulated as lipopolyplexes with peptide and DNA components, in particular with shorter PEG head groups which may have less effect on peptide targeting in the ternary complex.
Biomedical Microdevices. Feb, 2015 | Pubmed ID: 25653062
Considerable interest has arisen in precision fabrication of cell bearing scaffolds and structures by free form fabrication. Gelatin is an ideal material for creating cell entrapping constructs, yet its application in free form fabrication remains challenging. We demonstrate the use of gelatin, crosslinked with microbial transglutaminase (mTgase), as a material to print cell bearing hydrogels for both 2-dimensional (2-D) precision patterns and 3-dimensional (3-D) constructs. The precision patterning was attained with 3 % gelatin and 2 % high molecular weight poly (ethylene oxide) (PEO) whereas 3-D constructs were obtained using a 5 % gelatin solution. These hydrogels, referred to as "bioinks" supported entrapped cell growth, allowing cell spreading and proliferation for both HEK293 cells and Human Umbilical Vein Endothelial Cells (HUVECs). These bioinks were shown to be dispensable by robotic precision, forming patterns and constructs that were insoluble and of suitable stiffness to endure post gelation handling. The two bioinks were further characterized for fabrication parameters and mechanical properties.
From Soft Self-Healing Gels to Stiff Films in Suckerin-Based Materials Through Modulation of Crosslink Density and β-Sheet Content
Advanced Materials (Deerfield Beach, Fla.). Jul, 2015 | Pubmed ID: 26011516
Suckerins are block-copolymer-like structural proteins constituting the building blocks of the strong squid sucker-ring teeth. Here, recombinant suckerin-19 is processed into biomaterials spanning a wide range of elasticity, from very soft hydrogels to stiff films with elastic modulus in the gigapascal range. The elasticity is controlled by the interplay between the β-sheet content and induced di-tyrosine crosslinking.
Quantification of Aldehyde Terminated Heparin by SEC-MALLS-UV for the Surface Functionalization of Polycaprolactone Biomaterials
Colloids and Surfaces. B, Biointerfaces. Aug, 2015 | Pubmed ID: 26052108
A straight forward strategy of heparin surface grafting employs a terminal reactive-aldehyde group introduced through nitrous acid depolymerization. An advanced method that allows simultaneously monitoring of both heparin molar mass and monomer/aldehyde ratio by size exclusion chromatography, multi-angle laser light scattering and UV-absorbance (SEC-MALLS-UV) has been developed to improve upon heparin surface grafting. Advancements over older methods allow quantitative characterization by direct (aldehyde absorbance) and indirect (Schiff-based absorbance) evaluation of terminal functional aldehydes. The indirect quantitation of functional aldehydes through labeling with aniline (and the formation of a Schiff-base) allows independent quantitation of both polymer mass and terminal functional groups with the applicable UV mass extinction coefficients determined. The protocol was subsequently used to synthesize an optimized heparin-aldehyde that had minimal polydispersity (PDI<2) and high reaction yields (yield >60% by mass). The 8 kDa weight averaged molar mass heparin-aldehyde was then grafted on polycaprolactone (PCL), a common implant material. This optimized heparin-aldehyde retained its antithrombin activity, assessed in freshly drawn blood or surface immobilized on PCL films. Anticoagulant activity was equal to or better than the 24 kDa unmodified heparin it was fragmented from.
Smooth Muscle Cell Alignment and Phenotype Control by Melt Spun Polycaprolactone Fibers for Seeding of Tissue Engineered Blood Vessels
International Journal of Biomaterials. 2015 | Pubmed ID: 26413093
A method has been developed to induce and retain a contractile phenotype for vascular smooth muscle cells, as the first step towards the development of a biomimetic blood vessel construct with minimal compliance mismatch. Melt spun PCL fibers were deposited on a mandrel to form aligned fibers of 10 μm in diameter. The fibers were bonded into aligned arrangement through dip coating in chitosan solution. This formed a surface of parallel grooves, 10 μm deep by 10 μm across, presenting a surface layer of chitosan to promote cell surface interactions. The aligned fiber surface was used to culture cells present in the vascular wall, in particular fibroblasts and smooth muscle cells. This topography induced "surface guidance" over the orientation of the cells, which adopted an elongated spindle-like morphology, whereas cells on the unpatterned control surface did not show such orientation, assuming more rhomboid shapes. The preservation of VSMC contractile phenotype on the aligned scaffold was demonstrated by the retention of α-SMA expression after several days of culture. The effect was assessed on a prototype vascular graft prosthesis fabricated from polylactide caprolactone; VSMCs aligned longitudinally along a fiberless tube, whereas, for the aligned fiber coated tubes, the VSMCs aligned in the required circumferential orientation.
Molecules (Basel, Switzerland). Sep, 2016 | Pubmed ID: 27617991
The 3D bioprinting of stem cells directly into scaffolds offers great potential for the development of regenerative therapies; in particular for the fabrication of organ and tissue substitutes. For this to be achieved; the lineage fate of bioprinted stem cell must be controllable. Bioprinting can be neutral; allowing culture conditions to trigger differentiation or alternatively; the technique can be designed to be stimulatory. Such factors as the particular bioprinting technique; bioink polymers; polymer cross-linking mechanism; bioink additives; and mechanical properties are considered. In addition; it is discussed that the stimulation of stem cell differentiation by bioprinting may lead to the remodeling and modification of the scaffold over time matching the concept of 4D bioprinting. The ability to tune bioprinting properties as an approach to fabricate stem cell bearing scaffolds and to also harness the benefits of the cells multipotency is of considerable relevance to the field of biomaterials and bioengineering.