Search PubMed⌕ Search

Biomedical subjects

Stephen L Hart

Publications and source records attributed to Stephen L Hart.

At least 19 recordsLinked to original sources
PubMed2006

Calcineurin regulates NFAT-dependent iNOS expression and protection of cardiomyocytes: co-operation with Src tyrosine kinase.

OBJECTIVE: To determine the role of calcineurin and Src tyrosine kinase in the regulation of inducible nitric oxide synthase (iNOS) expression and protection in cardiomyocytes. METHODS: iNOS expression was studied in isolated neonatal rat ventricular myocyte cultures in response to bacterial lipopolysaccharide (LPS) or following transfection with constitutively active calcineurin or Src and in hearts isolated from wild-type or calcineruin Abeta knockout mice. Cell injury in response to simulated ischemia-reperfusion was studied following overexpression of active calcineurin. Regulation of the iNOS gene promoter by calcineurin was studied using promoter-luciferase reporter and chromatin immunoprecipitation assays. RESULTS: Overexpression of constitutively active Src co-operated with [Ca2+]c elevation to induce iNOS expression, and LPS-induced iNOS expression was abrogated by pharmacological inhibition of calcineurin or tyrosine kinase. LPS also induced tyrosine kinase-dependent but calcineurin-independent phosphorylation of Src Tyr418. LPS induced myocardial iNOS expression in wild-type but not calcineurin Abeta knockout mice. Overexpression of constitutively active calcinuerin in isolated cardiomyocytes caused dephosphorylation and nuclear accumulation of the c1 isoform of nuclear factor of activated T-cells (NFATc1), induced strong iNOS expression, and induced NOS-dependent protection against simulated ischemia-reperfusion prior to cardiomyocyte hypertrophy. Co-transfection of a mouse iNOS promoter-luciferase reporter in combination with active calcineurin and wild-type or dominant negative Src confirmed that constitutive activation of calcineurin was sufficient for transactivation. Chromatin immunoprecipitation confirmed calcineurin-dependent in vivo binding of NFATc1 to consensus sites within the iNOS promoter. CONCLUSIONS: These results support a cardioprotective role for calcineurin mediated by NFAT-dependent induction of iNOS expression and co-operativity between calcineurin and Src.

Animals↗
PubMed2006

Analysis and optimization of the cationic lipid component of a lipid/peptide vector formulation for enhanced transfection in vitro and in vivo.

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K16GACRRETAWACG) and Lipofectin, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.

Animals↗
PubMed2006

Application to vascular adventitia of a nonviral vector for TIMP-1 gene therapy to prevent intimal hyperplasia.

Somatic gene transfer continues to have potential for the study and therapy of cardiovascular disease. We have developed a modular, self-assembling, nonviral system consisting of Lipofectin, integrin-targeting peptides, and plasmid DNA (LID) and we have applied this to a model of vascular injury, rat carotid angioplasty. Marker gene studies identified transfection of adventitial cells after vector delivery to that layer. Human tissue inhibitor of metalloproteinase-1 (hTIMP-1) was tested as a therapeutic gene product after direct application to the exposed adventitial layer. Vascular LID.hTIMP-1 transfection was confirmed by polymerase chain reaction and gene expression by immunohistochemistry at 7 days. Neointimal areas were 0.160 +/- 0.078 and 0.225 +/- 0.052 mm(2) for LID.hTIMP-1-transfected (n = 14) and LID.pCI-transfected (n = 12) vessels, respectively, at 14 days, and 0.116 +/- 0.068 mm(2) (n = 14) and 0.194 +/- 0.095 mm(2) (n = 14), respectively, at 28 days, representing a 29 and 40% reduction in neointimal hyperplasia at 14 and 28 days, respectively, after balloon dilatation. Neointima-to-media ratios were similarly reduced. In addition, expansile remodeling after balloon injury was inhibited at 14 days, the area within the external elastic lamina being 0.50 +/- 0.02 and 0.61 +/- 0.02 mm(2) in LID.hTIMP-1- and LID.pCI-transfected arteries, respectively (p < 0.0005). We have demonstrated an effective system of therapeutic gene transfer, particularly targeting the arterial adventitia, where transfer of genes involved in matrix remodeling and cell migration may be useful.

Animals↗
PubMed2005

The fractal structure of polycation-DNA complexes.

We used static light scattering to obtain new measurements on the internal structure of aggregated non-viral gene-delivery particles in colloidal suspension. The vector particles are prepared by charge neutralization of plasmid DNA either by poly-L-lysine or by a Lipofectin/integrin-targeting peptide. We use established theories of the stability of colloidal particles and fractal concepts to explain the aggregation processes and demonstrate the existence of a new property (fractal dimension) of the aggregated vector particles. Aggregation is shown to produce particles with fractal dimensions in the range between 1.8 and 2.4; the former suggests a loose three-dimensional structure and the latter characterizes an aggregation process that leads to the formation of particles with tightly packed structures. We show that the fractal dimension of the vector particles is sensitive to changes in physicochemical conditions (ionic strength) of the buffer solution and propose that fractal dimension may provide a useful means of monitoring the physical state of non-viral delivery-vector particles during preparation and storage.

Cations↗
PubMed2005

Lipid carriers for gene therapy.

A wide variety of lipid molecules used as gene carriers has been reported and compared over the last twenty years. This review highlights a few examples of mechanistic analysis applied to the study of lipid carriers. The modular nature of the lipid structure offers itself up to a controlled, systematic analysis. Key to exploring structural variants is the understanding of the role each component and module plays in the formation of the lipoplex structure itself and their roles in the transfection pathway. Firstly, the lipid carrier must be able to package, and release into the cell its nucleic acid cargo. Uptake of lipoplexes into cells involves endocytic processes that lead inevitably to endosomal/liposomal degradation of the nucleic acid contents, unless lipid structures and designs are optimised to facilitate their release into the cytoplasm. Testing of possible endosomal escape mechanisms has led to improved lipid designs. For example, it was predicted that mixing of cationic lipids with shorter alkyl tails (<C18) from liposomes with the endosomal bilayer during the transfection process within the endosome should promote membrane fluidity, enhancing plasmid release and transfection efficiency. This hypothesis has been borne out numerous times. "Smart" molecules that respond to cellular cues such as endosomal pH have now been developed that appear to offer exciting opportunities for the future role of lipoplexes in clinical applications of gene therapy. The variability of cultured cell and model biological systems remains a challenge in designing improved lipids of general utility.

Alkylation↗
PubMed2004

Rate of transport of l-arginine is independent of the expression of inducible nitric oxide synthase in HEK 293 cells.

Expression of inducible nitric oxide synthase (iNOS) is generally accompanied by a parallel upregulation in l-arginine transport which is dependent, at least in part, on the synthesis of new carrier proteins. It is not clear however whether the induction of iNOS and its subsequent utilisation of l-arginine for NO synthesis contribute to the enhancement in l-arginine transport rates observed following induction of cells with pro-inflammatory mediators. To address this issue, we have transfected an iNOS construct in a pEGFP-N1 vector into HEK-293 cells and investigated the effects this has on l-arginine transport. The expression of iNOS through transfection resulted in the production of significant quantities of NO as detected by the standard Griess assay. Under these conditions, the transport of l-arginine was found to be unaltered, with rate of uptake being comparable in both transfected and non-transfected cells. Characterisation of the transporter(s) involved with uptake of l-arginine revealed features characteristic of the classical cationic amino acid transport system y(+). Further analysis of the expression profile of the cationic amino acid transporter (CAT) involved revealed the presence of transcripts for CAT-1 and CAT-2B. These data demonstrate that iNOS activity does not drive or enhance l-arginine transport despite the fact that HEK-293 cells transport l-arginine via the CATs, including CAT-2B which is thought to be critical for supply of substrate to iNOS.

Amino Acid Sequence↗
PubMed2004

Efficient transfection of non-proliferating human airway epithelial cells with a synthetic vector system.

BACKGROUND: The poor transfection efficiency of most synthetic vectors in non-dividing, airway epithelial cells is limiting the development of gene therapy for respiratory diseases such as cystic fibrosis. METHODS: Cultured airway epithelial cells, quiescent or replicating, were transfected with a lipid/peptide synthetic vector system and transfection efficiency assessed by luciferase assays or flow cytometry for green fluorescent protein expression. Tight junctions were disrupted by treatment with ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA). Cell division was inhibited by aphidicolin and assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation and assay by fluorescence microscopy or flow cytometry. In vivo transfection was performed by intratracheal instillation. RESULTS: Initial studies showed that this synthetic vector transfected 8% of non-dividing epithelial cells in culture compared with 45% of subconfluent, dividing cells. However, formulation of the vector with EGTA, which disrupts cellular tight junctions, induced a four-fold increase in the transfection efficiency of confluent, non-mitotic cells. The EGTA enhancement of transfection also correlated with a four-fold increase in vector binding to the cells, presumably via exposed basolateral receptors. Flow cytometry and double immunofluorescent staining for reporter gene expression and BrdU incorporation indicated efficient transfection of non-proliferating cells in a confluent monolayer by this vector. Furthermore, in vivo transfection of murine lung by intratracheal administration showed a six-fold enhancement of luciferase expression when lungs were pre-treated with EGTA. CONCLUSIONS: This study confirms the hypothesis that a lipid/peptide vector transfects non-dividing cells with high efficiency which contributes to its previously reported efficiency in transfecting bronchial epithelium, making it a good choice for gene transfer studies in the lung.

Egtazic Acid↗
PubMed2004

Severity of lung injury in cyclooxygenase-2-deficient mice is dependent on reduced prostaglandin E(2) production.

Levels of prostaglandin E(2) (PGE(2)), a potent inhibitor of fibroblast function, are decreased in the lungs of patients with pulmonary fibrosis, which has been shown to be because of limited expression of cyclooxygenase-2 (COX-2). To further investigate the relative importance of COX-2 and PGE(2) in the development of fibrosis we have used a selective COX-2 inhibitor and COX-2-deficient ((-/-) and (+/-)) mice in studies of bleomycin-induced lung fibrosis. We demonstrate in wild-type mice that bleomycin-induced lung PGE(2) production is predominantly COX-2 mediated. Furthermore, COX-2(+/-) mice show limited induction of PGE(2) and an enhanced fibrotic response with increased lung collagen content compared with wild-type mice after bleomycin injury (P < 0.001). In contrast, COX-2(-/-) mice show increased levels of lung PGE(2), compared with wild-type mice after injury (P < 0.05), because of compensatory up-regulation of COX-1, which appears to be associated with macrophage/monocytes but not fibroblasts derived from these mice. COX-2(-/-) mice show an enhanced and persistent inflammatory response to bleomycin, however the fibrotic response to injury was unaltered compared with wild-type animals. These data provide further direct evidence for the importance of up-regulating COX-2 and PGE(2) expression in protecting against the development of fibrosis after lung injury.

Animals↗
PubMed2004

Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display.

Human airway epithelial cell targeting peptides were identified by biopanning on 1HAEo-cells, a well characterised epithelial cell line. Bound phage were recovered after three rounds of binding, high stringency washing and elution, leading to the production of an enriched phage peptide population. DNA sequencing of 56 clones revealed 14 unique sequences. Subsequent binding analysis revealed that 13 of these peptides bound 1HAEo-cells with high affinity. Three peptides, SERSMNF, YGLPHKF and PSGAARA were represented at high frequency. Three clearly defined families of peptide were identified on the basis of sequence motifs including (R/K)SM, L(P/Q)HK and PSG(A/T)ARA. Two peptides, LPHKSMP and LQHKSMP contained two motifs. Further detailed sequence analysis by comparison of peptide sequences with the SWISSPROT protein database revealed that some of the peptides closely resembled the cell binding proteins of viral and bacterial pathogens including Herpes Simplex Virus, rotavirus, Mycoplasma pneumoniae and rhinovirus, the latter two being respiratory pathogens, as well as peptide YGLPHKF having similarity to a protein of unknown function from the respiratory pathogen Legionella pneumophila. Peptides were incorporated into gene delivery formulations with the cationic lipid Lipofectin and plasmid DNA and shown to confer a high degree of transfection efficiency and specificity in 1HAEo-cells. Improved transfection efficiency and specificity was also observed in human endothelial cells, fibroblasts and keratinocytes. Therefore, on the basis of clone frequency after biopanning, cell binding affinity, peptide sequence conservation and pathogenic similarity, we have identified 3 novel peptide families and 5 specific peptides that have the potential for gene transfer to respiratory epithelium in vivo as well as providing useful in vitro transfection reagents for primary human cell types of scientific and commercial interest.

Amino Acid Sequence↗
PubMed2003

Functional delivery of large genomic DNA to human cells with a peptide-lipid vector.

BACKGROUND: Nonviral gene transfer vectors have the potential to deliver much larger DNA constructs than current viral vectors but suffer from a low transfection efficiency. The LID vector, composed of Lipofectin (L), an integrin-targeting peptide (I) and DNA (D), is a highly efficient synthetic vector, both in vitro and in vivo, which may allow the transfer of genomic loci for gene therapy. METHODS: Transfection efficiencies were quantitated using the green fluorescent protein (GFP) reporter. Expression of a large genomic locus (NBS1 [Nijmegen breakage syndrome], encoding nibrin) was assessed by immunofluorescence. RESULTS: We report a systematic study of the parameters influencing delivery of BAC-based plasmids ranging in size from 12 to 242 kb using the LID vector. We showed 60% of cells were transfected with the smaller plasmids while plasmids up to 242 kb were consistently delivered to over 10% of cells. The number of transfected cells was related to number of plasmids in the transfection complex independent of plasmid size. Atomic force microscopy showed that LID particle size increased with plasmid size consistent with one plasmid molecule per particle. When LID vectors were used to deliver the NBS1 gene as a 143 kb construct to primary NBS cells, at least 57% of cells expressing GFP also expressed functional nibrin. CONCLUSIONS: We show that LID vectors represent a promising tool for the transfer of complete genomic loci.

Cell Cycle Proteins↗
PubMed2003

Enhancement of integrin-mediated transfection of haematopoietic cells with a synthetic vector system.

The aim of the present study was to develop and assess an integrin-targeted synthetic vector system for the transfection of haematopoietic cell lines and dendritic cells. The vector consists of a cationic liposome, Lipofectin (L), a peptide that both targets integrins and binds to DNA (I) and plasmid DNA (D). These components interact electrostatically to form the LID vector complex. Transfection conditions were optimized for the ratio of vector components, the amount of DNA and transfection incubation time. The kinetic analysis of transgene expression revealed a peak of activity at about 24 h, followed by a rapid decline over the next 48 h. Targeted gene delivery was demonstrated by comparing transfected luciferase reporter gene levels using LID complexes containing integrin-targeting peptide sequences with a control peptide. In addition, transfection levels of integrin-targeted LID complexes were significantly enhanced by treatment of cells with PMA, which was also shown to activate integrin receptors and enhance binding to fibronectin. Under optimized conditions transfection efficiencies of 19% for TF-1 cells, 28% for Jurkat cells and 10% for primary dendritic cells were achieved. The LID vector may thus find application for gene-transfer experiments in haematopoietic cell lines and for the development of genetic vaccines using transfected dendritic cells.

Cell Line↗
PubMed2003

Prediction of size distribution of lipid-peptide-DNA vector particles using Monte Carlo simulation techniques.

Concerns with insertional mutagenesis for retrovirus and immunogenicity for adenovirus have motivated research into development of non-viral vectors that can safely deliver desired gene constructs to target cells in tissues and organs. Many non-viral vectors suffer from unacceptably poor in vivo cell transfection and low transgene expression. Evidence suggests that cell transfection is linked to particle size - vector particles below about 200 nm are considered desirable. Experimental measurements indicate, however, that vector particles are susceptible to significant aggregation under most conditions of pH and ionic strength, including physiological conditions, although there are currently no means of predicting the kinetics of aggregation. The present paper addresses this challenge by presenting a mathematical framework based on the Monte Carlo simulation techniques for modelling the dynamics of aggregation. The approach is used to simulate the evolution of particle-size distribution for an integrin-targeting lipid-peptide-DNA vector system in buffers of different pH and ionic strength. The simulations required two input parameters, including the initial-size distribution of the particles and a fitting parameter (alpha). Comparison of simulations with experimental data showed that alpha was closely related to the zeta potential of the particles in the buffer medium, making simulations fully predictive. The modelling approach may be used in other vector systems.

DNA↗
PubMed2003

Biophysical characterization of an integrin-targeted non-viral vector.

BACKGROUND: The formulation of polycationic complexes containing plasmid DNA for optimal transfection in vitro and in vivo for DNA vaccination, gene therapy and other applications continues to be a major research goal. Here we present new data on the biophysical properties of an integrin-targeted plasmid DNA (LID) formulation. MATERIAL/METHODS: Two plasmids (D), pEGFP (4.7 kb) and pCI-luc (5.7 kb), were mixed with a synthetic a5b1 integrin-targeted peptide (I), [K]16 GACRRETAWACG, in the presence of a cationic liposome (L), Lipofectin, composed of DOTMA and DOPE to form LID complexes. The physical properties of the complexes were measured using a variety of techniques including dynamic light scattering and fluorescence methods. The in vitro gene delivery to neuroblastoma cells with LID complexes was also assessed. RESULTS: We demonstrate the effects of complex size and charge ratio on in vitro transfection of mouse (Neuro-2A) and human (IMR-32) neuroblastoma cells. We report a significant increase in the level of luciferase and green fluorescent protein expression when transfection is performed in buffers of physiological ionic strength and hypothesise that the enhancement in transfection is caused by an increase in the size of the complexes observed during mixing and maturation. CONCLUSIONS: Cell transfection is also shown to be dependent on complex size and charge ratio, with large complexes prepared at charge ratios above 4.0 demonstrating efficient transfection.

Animals↗
PubMed2002

Coleonema album: studies of the pharmacological action on smooth muscle in vitro and antimicrobial action of its essential oil.

The mode of action of the essential oil of Coleonema album, a South African species from the Cape region, was studied on smooth muscle in vitro using field-stimulated guinea-pig ileum. The oil produced an initial spasmogenic activity followed by spasmolysis, both actions being dose-dependent. The spasmolytic action was post-synaptic and not atropine-like and was unaffected by the adrenoceptor blockers phentolamine and propranolol. There was no evidence for the involvement of either cyclic guanosine monophosphate or cyclic adenosine monophosphate in the spasmolytic activity. The oil had some calcium channel blocking activity but this is not the primary explanation for its spasmolytic response. Antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Staphyloccocus aureus and Saccharomyces cerevisiae was very low and totally absent against Enterococcus hirae, suggesting that the oil has no worthwhile potential in this area. However, as the oil has a very disagreeable odour in its undiluted form, it could be a powerful insect repellent, as its local usage suggests, and this remains to be investigated.

Animals↗
PubMed2002

Effective gene transfer to solid tumors using different nonviral gene delivery techniques: electroporation, liposomes, and integrin-targeted vector.

In this study, we measured transfection efficiency in vitro and in vivo using the following nonviral approaches of gene delivery: injection of plasmid DNA, electroporation-assisted, liposome-enhanced, and integrin-targeted gene delivery, as well as the combination of these methods. Four histologically different tumor models were transfected with a plasmid encoding the green fluorescent protein (GFP) (B16 mouse melanoma, P22 rat carcinosarcoma, SaF mouse sarcoma, and T24 human bladder carcinoma) using adherent cells, dense cell suspensions, and solid tumors. Emphasis was placed on different electroporation conditions to optimise the duration and amplitude of the electric pulses, as well as on different DNA concentrations for effective gene delivery. In addition, transfection efficiency was correlated with cell density of the tumors. The major in vivo findings were: (a) electroporation-assisted gene delivery with plasmid DNA, employing long electric pulses with low amplitude, yielded significantly better GFP expression than short electric pulses with high amplitude; (b) electroporation combined with liposome-DNA complexes yielded the highest percentage of transfected tumor area in B16F1 tumor (6%); (c) transfection efficiency of electroporation-assisted plasmid DNA delivery was dependent on tumor type; (d) integrin-targeted vector, alone or combined with electroporation, was largely ineffective. In conclusion, our results demonstrate that some nonviral methods of gene delivery are feasible and efficient in transfecting solid tumors. Therefore, this makes nonviral methods attractive for further development.

Animals↗
PubMedDate not supplied

High efficiency transfection of porcine vascular cells in vitro with a synthetic vector system.

BACKGROUND: Gene therapy strategies for the treatment of vascular disease such as the prevention of post-angioplasty restenosis require efficient, non-toxic transfection of vascular cells. In vitro studies in these cells contribute to vector development for in vivo use and for the evaluation of genes with therapeutic potential. The aim of this project was to evaluate a novel synthetic vector consisting of a liposome (L), an integrin targeting peptide (I), and plasmid DNA (D), which combine to form the LID vector complex. METHODS: Cultures of porcine smooth muscle cells and endothelial cells were established and then transfected with the LID vector, using the reporter genes luciferase and green fluorescent protein and the metalloprotease inhibitor TIMP-1. RESULTS: The LID vector system transfected primary porcine vascular smooth muscle cells and porcine aortic endothelial cells with efficiency levels of 40% and 35%, respectively. By increasing the relative DNA concentration four-fold, incubation periods as short as 30 min achieved the same levels of luciferase transgene expression as 4 h incubations at lower DNA concentrations. The transfection did not affect cell viability as measured by their proliferative potential. Serum levels of up to 20% in the transfection medium had no adverse affect on the efficiency of transfer and gene expression in either cell type. Transfections with the cDNA for TIMP-1 produced protein levels that peaked at 130 ng/ml per 24 h and persisted for 14 days at 10 ng/ml per 24 h. CONCLUSION: This novel vector system has potential for studies involving gene transfer to cardiovascular cells in vitro and in vivo.

Amino Acid Sequence↗