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T Finkel

Publications and source records attributed to T Finkel.

At least 55 records · Page 3Linked to original sources
PubMed1996

Reactive oxygen species are downstream mediators of p53-dependent apoptosis.

Reactive oxygen species (ROS) have been implicated as potential modulators of apoptosis. Conversely, experiments under hypoxic conditions have suggested that apoptosis could occur in the absence of ROS. We sought to determine whether a central modulator of apoptosis, p53, regulates the levels of intracellular ROS and whether a rise in ROS levels is required for the induction of p53-dependent apoptosis. We transiently overexpressed wild-type p53, using adenoviral gene transfer, and identified cell types that were sensitive or resistant to p53-mediated apoptosis. Cells sensitive to p53-mediated apoptosis produced ROS concomitantly with p53 overexpression, whereas cells resistant to p53 failed to produce ROS. In sensitive cells, both ROS production and apoptosis were inhibited by antioxidant treatment. These results suggest that p53 acts to regulate the intracellular redox state and induces apoptosis by a pathway that is dependent on ROS production.

Adenoviruses, Human↗
PubMed1996

Regulation of reactive-oxygen-species generation in fibroblasts by Rac1.

In a variety of non-phagocytic cell types, there is a marked increase in intracellular levels of reactive oxygen species (ROS), including superoxide and H2O2, after ligand stimulation. We demonstrate that in NIH 3T3 cells transient expression of constitutively activated forms of the small GTP-binding proteins Ras or Rac1 leads to a significant increase in intracellular ROS. An increase in intracellular ROS is also demonstrated after growth factor [platelet-derived growth factor (PDGF) or epidermal growth factor (EGF)] or cytokine [tumour necrosis factor-alpha (TNF-alpha) or interleukin (IL)-1 beta] stimulation of NIH 3T3 cells. Expression of a dominant negative allele of Rac1 inhibits the rise in ROS seen after Ras expression or after stimulation by either growth factors or cytokines. These results provide the first demonstration of the pathway by which ligand stimulation of ROS occurs in non-phagocytic cells and suggest that the family of Ras-related small GTP-binding proteins may function as regulators of the intracellular redox state.

3T3 Cells↗
PubMed1996

Association between prior cytomegalovirus infection and the risk of restenosis after coronary atherectomy.

BACKGROUND: Restenosis occurs commonly after coronary angioplasty and atherectomy, but the causes of restenosis are poorly understood. Recently, it has been found that cytomegalovirus (CMV) DNA is present in restenotic lesions from atherectomy specimens. This and other evidence suggest that CMV may have a role in the process of restenosis. METHODS: We prospectively studied 75 consecutive patients undergoing directional coronary atherectomy for symptomatic coronary artery disease. Before atherectomy was performed, we measured blood levels of anti-CMV IgG antibodies to determine whether previous exposure to CMV increased the risk of restenosis, as determined by coronary angiography performed six months after atherectomy. RESULTS: After atherectomy, the mean (+/- SD) minimal luminal diameter of the target vessel was greater in the 49 patients who were seropositive for CMV than in the 26 patients who were seronegative (3.18 +/- 0.51 mm vs. 2.89 +/- 0.45 mm, P=0.01). After six months, however, the seropositive patients had a greater reduction in the luminal diameter (1.24 +/- 0.83 mm vs. 0.68 +/- 0.69 mm, P = 0.003), resulting in a significantly higher rate o restenosis in the seropositive patients (43 percent vs. 8 percent, P = 0.002). In a multivariable logistic-regression model, CMV seropositivity and the CMV titer were independently predictive of restenosis (odds ratios, 12.9 and 8.1, respectively). There was no evidence of acute infection, since the titer of anti-CMV IgG antibodies did not increase over time and tests for anti-CMV IgM antibodies were negative in all patients. CONCLUSIONS: Prior infection with CMV is strong independent risk factor for restenosis after coronary atherectomy. If confirmed, these findings may help identify patients at risk for restenosis.

Adult↗
PubMed1996

rac1 regulates a cytokine-stimulated, redox-dependent pathway necessary for NF-kappaB activation.

The signal transduction pathway leading to the activation of the transcription factor NF-kappaB remains incompletely characterized. We demonstrate that in HeLa cells, transient expression of a constitutively active mutant of the small GTP-binding protein rac1 (V12rac1) leads to a significant increase in NF-kappaB transcriptional activity. In addition, expression of a dominant-negative rac1 mutant (N17rac1) inhibits basal and interleukin 1beta-stimulated NF-kappaB activity. Gel shift analysis using nuclear extract prepared from HeLa cells infected with a recombinant adenovirus encoding N17rac1 (Ad.N17racl) showed reduced levels of cytokine-stimulated DNA binding to a consensus NF-kappaB binding site. We demonstrate that rac proteins function downstream of ras proteins in the activation of NF-kappaB. In addition, V12rac1 stimulation of NF-kappaB activity is shown to be independent of the ability of rac proteins to activate the family of c-jun amino-terminal kinases. In an effort to further explore how rac proteins might regulate NF-kappaB activity, we demonstrate that expression of V12rac1 in HeLa cells or stimulation with cytokine results in a significant increase in intracellular reactive oxygen species (ROS). Treatment of cells with either of two chemically unrelated antioxidants inhibits the rise in ROS that occurs following V12rac1 expression as well as the ability of V12rac1 to stimulate NF-kappaB activity. These results suggest that in HeLa cells, rac1 regulates intracellular ROS production and that rac proteins function as part of a redox-dependent signal transduction pathway leading to NF-kappaB activation.

Cytokines↗
PubMed1996

Apoptosis in vascular disease: opportunities for genetic therapeutic intervention.

Apoptosis functions to efficiently eliminate normal cells no longer required in remodelling tissues, and abnormal cells exhibiting neoplastic phenotypes. Since injury is an important trigger to the development of vascular lesions, the balance of proliferation and apoptosis during the healing response, and aberrations of this process, may be important factors in lesion progression. The expression of genes that regulate this process, including c-myc and members of the bcl-2 and lce gene families, has been detected in the normal vasculature, and in atheromatous tissues. These observations suggest that the transfer of apoptosis-inducing genes into vascular lesions may be a feasible, non-inflammatory strategy to eliminate the cellular components of restenotic and atherosclerotic plaques.

Animals↗
PubMed1995

Requirement for generation of H2O2 for platelet-derived growth factor signal transduction.

Stimulation of rat vascular smooth muscle cells (VSMCs) by platelet-derived growth factor (PDGF) transiently increased the intracellular concentration of hydrogen peroxide (H2O2). This increase could be blunted by increasing the intracellular concentration of the scavenging enzyme catalase or by the chemical antioxidant N-acetylcysteine. The response of VSMCs to PDGF, which includes tyrosine phosphorylation, mitogen-activated protein kinase stimulation, DNA synthesis, and chemotaxis, was inhibited when the growth factor-stimulated rise in H2O2 concentration was blocked. These results suggest that H2O2 may act as a signal-transducing molecule, and they suggest a potential mechanism for the cardioprotective effects of antioxidants.

Acetylcysteine↗
PubMed1995

Gene therapy for vascular disease.

Highly efficient methods that are now available allow for the genetic modification of cells that compose the vessel wall. The application of these gene transfer systems are already providing information that has enhanced our understanding of a variety of cellular processes relating to vascular biology. Recent experimental results using this technology also hold promise for more powerful therapeutic options in the treatment of vascular disease. This review discusses the most commonly used methods of gene transfer and details some current applications of these methodologies in animal models of vascular disease.

Animals↗
PubMed1995

Interaction of human cytomegalovirus with p53: possible role in coronary restenosis.

Restenosis occurs in 25-50% of patients. Within 1-6 months after coronary angioplasty, excessive injury-induced smooth muscle cell (SMC) proliferation contributes to the development of restenosis; its causes remain unknown. The results of this study implicate human cytomegalovirus (HCMV) and HCMV-induced abnormalities in p53 function in the restenosis process. Almost 40% of restenosis lesions, obtained by atherectomy, demonstrated increased SMC p53 levels by p53 immunopositivity; sequencing revealed the p53 to be the wild type. A strong correlation was found between p53 immunopositivity and the presence of HCMV DNA. Moreover, the HCMV IE84 protein co-immunoprecipitates with p53, and p53 transcriptional capacity is reduced by IE84. Thus, HCMV may play a causal role in restenosis, which may be at least partly mediated by inhibiting p53 suppressor effects.

Adult↗
PubMed1994

In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene.

Restenosis, a process characterized in part by excessive smooth muscle cell (SMC) proliferation in areas of vascular injury, occurs in up to 50% of patients undergoing balloon angioplasty. In an effort to develop a treatment strategy for restenosis, we constructed a replication-deficient recombinant adenovirus (AdMLP.HSTK) containing the herpes simplex virus thymidine kinase gene (HSV tk). This viral gene product phosphorylates the prodrug ganciclovir to form a nucleoside analog that inhibits DNA synthesis. Cultured primary rat SMCs infected with AdMLP.HSTK were completely growth-inhibited by incubation in ganciclovir-containing medium. In addition, when only a portion of the SMC population received the HSV tk transgene, an inhibitory effect on neighboring SMCs was evident. Evaluation of this strategy in vivo using a rat carotid balloon injury model demonstrated that local infection of injured arteries with AdMLP.-HSTK followed by 2 weeks of systemic ganciclovir treatment significantly (P < 0.01) reduced injury-induced SMC accumulation. In contrast, there was no suppression of injury-induced SMC accumulation in animals infected with AdMLP.HSTK but not receiving ganciclovir or in those animals infected with a control adenovirus and either treated or not treated with ganciclovir. These results demonstrate the potential utility of adenovirus-mediated gene transfer for treatment of restenosis after balloon injury.

Adenoviridae↗
PubMed1994

Ras proteins regulate multiple mitogenic pathways in A10 vascular smooth muscle cells.

We examined the role of ras proteins in mitogenesis and proliferation of A10 smooth muscle cells. An assay was developed in which a dominant negative ras gene could be transiently expressed in vascular smooth muscle cells. As opposed to cells transfected with a control plasmid, those transfected with a dominant negative ras expression plasmid exhibited a decrease in thymidine uptake in response to serum, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor and thrombin stimulation. In addition, expression of dominant negative ras blocked smooth muscle cell proliferation as assessed by the number of surviving colonies after transfection and neomycin selection. Our data is the first to show that ras is essential for smooth muscle cell proliferation and is a mediator in multiple smooth muscle cell mitogenic signalling pathways.

Animals↗
PubMed1994

Potential role of human cytomegalovirus and p53 interaction in coronary restenosis.

A subset of patients who have undergone coronary angioplasty develop restenosis, a vessel renarrowing characterized by excessive proliferation of smooth muscle cells (SMCs). Of 60 human restenosis lesions examined, 23 (38 percent) were found to have accumulated high amounts of the tumor suppressor protein p53, and this correlated with the presence of human cytomegalovirus (HCMV) in the lesions. SMCs grown from the lesions expressed HCMV protein IE84 and high amounts of p53. HCMV infection of cultured SMCs enhanced p53 accumulation, which correlated temporally with IE84 expression. IE84 also bound to p53 and abolished its ability to transcriptionally activate a reporter gene. Thus, HCMV, and IE84-mediated inhibition of p53 function, may contribute to the development of restenosis.

Adolescent↗
PubMed1994

Dynamic actin structures stabilized by profilin.

We describe the production and analysis of clonal cell lines in which we have overexpressed human profilin, a small ubiquitous actin monomer binding protein, to assess the role of profilin on actin function in vivo. The concentration of filamentous actin is increased in cells with higher profilin levels, and actin filament half-life measured in these cells is directly proportional to the steady-state profilin concentration. The distribution of actin filaments is altered by profilin overexpression. While parallel actin bundles crossing the cells are virtually absent in cells overexpressing profilin, the submembranous actin network of these cells is denser than in control cells. These results suggest that in vivo profilin regulates the stability, and thereby distribution, of specific dynamic actin structures.

Actins↗
PubMed1994

The basis of molecular strategies for treating coronary restenosis after angioplasty.

Excessive smooth muscle cell proliferation significantly contributes to restenosis, which occurs in 25% to 50% of patients within 6 months of coronary angioplasty. Because successful treatment will probably depend on our acquiring a comprehensive knowledge of the molecular and cellular mechanisms involved, this report reviews 1) information relevant to the molecular and cellular mechanisms responsible for the smooth muscle cell(s) response to vascular injury, and 2) several molecular-based therapeutic strategies currently being explored as possible approaches to the control of restenosis, including recombinant DNA technology to target delivery of cytotoxic molecules to proliferating smooth muscle cell(s), antisense strategies to inhibit expression of gene products necessary for cell proliferation and gene therapy.

Antisense Elements (Genetics)↗
PubMed1993

Detection and modulation in vivo of helix-loop-helix protein-protein interactions.

Studies are described that allow for the in vivo detection of helix-loop-helix (HLH) protein-protein interaction. The assay used requires HLH protein-protein interaction to reconstitute a functional GAL4 transcriptional activator, which in turn activates a reporter gene placed downstream of GAL4 DNA binding sequences. Using this assay, we are able to detect intracellular heterodimerization but not homodimerization of the MyoD, E12, and Id gene products. In addition, using this system we are unable to detect stable heterodimerization between MyoD and c-Jun. We also show that expression of activated rasH gene product does not inhibit and may stabilize HLH protein-protein interaction. This system may be of general utility in studying the modulation of transcription factor interactions.

Animals↗
PubMed1993

Efficient and selective adenovirus-mediated gene transfer into vascular neointima.

BACKGROUND: Previous attempts to target arterial smooth muscle cells (SMCs) for gene delivery using liposomal or retroviral methods were limited by low transfection efficiency. We therefore evaluated the efficiency of adenovirus-mediated gene delivery in cultured vascular SMCs and in an in vivo model of balloon injury-induced SMC cell proliferation. METHODS AND RESULTS: We used a recombinant adenovirus, Ad.RSV beta gal, which contained the beta-galactosidase (beta-gal) histochemical marker gene. For in vitro studies, rat aortic SMCs were incubated in media containing Ad.RSV beta gal for 5 to 120 minutes. The proportion of SMCs expressing the beta-gal gene product increased from 25% (5-minute exposure) to 80% (120-minute exposure). For in vivo studies, uninjured and injured rat carotid segments were incubated with 0.5 to 1.0 x 10(9) pfu Ad.RSV beta gal for 45 minutes. Uninjured arteries showed adenovirus-mediated gene transfer limited to the endothelium. Injured arteries were exposed to adenovirus 0, 3, 7, or 12 days after injury. In these segments, beta-gal expression was minimal with infection at 0 or 3 days after injury but marked when infection was delayed until 7 or 12 days after injury. Neointimal cells constituted the dominant target of adenovirus gene transfer, with efficiency of gene transfer ranging from 10% to > 75%. Medial SMCs, whether covered or uncovered by neointimal cells, were minimally infected. Infection with a control adenovirus vector showed no beta-gal staining. CONCLUSIONS: Recombinant adenovirus selectively targets neointimal cells with high-efficiency gene transfer. This suggests that adenovirus vectors should be useful in targeting cells for the delivery of genes whose products may be relevant to the treatment of restenosis.

Adenoviridae↗
PubMed1993

Efficient gene transfer into myocardium by direct injection of adenovirus vectors.

Previous studies have established that gene transfer into myocardial cells in vivo is detectable after direct injection of plasmid DNA. Recently, adenovirus vectors have been shown to provide an efficient method for gene transfer into a wide range of tissues. Therefore, this study sought to assess the efficiency and stability of adenovirus-mediated gene transfer into myocardium and to compare this method with that using plasmid-based gene transfer techniques. Adult rats underwent myocardial injection via a subdiaphragmatic approach. Gene transfer efficiency was compared using direct injection of an adenovirus vector encoding for the marker gene beta-galactosidase (beta-gal), a control adenovirus vector encoding for the cystic fibrosis transmembrane conductance regulator gene, a plasmid encoding for beta-gal, or a control plasmid. Hearts infected with an adenovirus vector containing the beta-gal gene showed significantly increased beta-gal enzymatic activity compared with hearts injected with beta-gal plasmid. Histological examination revealed that cardiac myocytes were the target of adenovirus-mediated gene transfer. A time course of gene expression showed that beta-gal enzymatic activity peaked during the first week following injection. Adenovirus vectors provide an efficient but transient method for in vivo gene expression in myocardium.

Adenoviridae↗