Antioxidant effect of estrogen on cytomegalovirus-induced gene expression in coronary artery smooth muscle cells.

BACKGROUND: Pathogens infecting the arterial wall with resultant inflammation may contribute to atherogenesis. Human coronary artery smooth muscle cells (SMCs) infected with human cytomegalovirus (CMV) demonstrate a rapid increase in reactive oxygen species (ROSs), with activation of genes involved in viral replication and inflammation. Because estrogen appears to have antioxidant properties, we wished to determine whether this hormone attenuates SMC responses to CMV infection. METHODS AND RESULTS: Using confocal microscopy and an intracellular fluorescent dye activated by ROSs, we found that 17beta-estradiol (0.1 to 10 nmol/L) and its stereoisomer 17alpha-estradiol (which has low affinity for the estrogen receptor) dose-dependently inhibited ROS generation in CMV-infected SMCs. These effects were not blocked by the estrogen receptor inhibitor ICI 182,780. 3-Methoxyestrone, which lacks the phenolic hydroxyl group, did not interfere with ROS generation. We found that 17beta-estradiol and 17alpha-estradiol, but not 3-methoxyestrone, prevented binding of nuclear factor (NF)-kappaB to DNA. Furthermore, in SMCs transfected with the reporter constructs 3XkappaB-CAT, MIEP-CAT, or ICAM-CAT, cotransfection with a CMV-IE72 expression plasmid caused promoter and CAT activation. Treatment with 17beta-estradiol and 17alpha-estradiol, but not 3-methoxyestrone, inhibited CAT activity and, in CMV-infected SMCs, prevented IE72 and ICAM-1 protein expression and cytopathic effects. CONCLUSIONS: These findings indicate that estrogen molecules with an A-ring hydroxyl group have estrogen receptor-independent anti-CMV effects at physiological concentrations by inhibiting ROS generation, NF-kappaB activation, NF-kappaB-dependent transcription, and viral replication. To the extent that chronic infection of the vascular wall with CMV contributes to atherogenesis, these antioxidant actions of estrogen may be of therapeutic importance.

Competition for p300 regulates transcription by estrogen receptors and nuclear factor-kappaB in human coronary smooth muscle cells.

Previous studies suggest that estrogen may prevent expression of cell adhesion molecules implicated in vascular inflammation associated with atherosclerosis. We demonstrate the interaction and reciprocal interference of estrogen receptors (ERs) with p65, the nuclear factor-kappaB component, in smooth muscle cells that express ERalpha and ERss after exposure to 17ss-estradiol for 48 to 72 hours. ER and p65 do not associate directly, as shown by lack of coprecipitation, but instead compete for limiting amounts of p300, a close relative of the CREB-binding protein. Overexpressed p300 significantly reduced the inhibitory effect of ER on p65-dependent transcription as well as the inhibitory effect of p65 on ER-dependent transcription. These actions were ligand-dependent. The expression of both ER and nuclear factor-kappaB-dependent reporter genes was partially rescued from ER/p65 mutual inhibition by transient transfection of smooth muscle cells with a p300 expression vector. These actions of 17ss-estradiol may play an important role in the cytokine-induced expression of immune and inflammatory genes implicated in atherogenesis.

Cytomegalovirus gene regulation by reactive oxygen species. Agents in atherosclerosis.

Oxidative stress is implicated in the pathogenesis of atherosclerosis, and of viral infections caused by sendai virus, influenza and HIV. Vascular oxidative stress is due to inflammatory and immune responses of vascular cells, and to reperfusion after recanalization of blocked arteries. Because human cytomegalovirus (CMV) may contribute to atherogenesis by several mechanisms, and coronary artery smooth muscle cells (SMC) are permissive for the virus, we examined CMV interactions with SMC. Infection causes generation of intracellular reactive oxygen species (ROS) which activate NF-kappa B, a cellular transcription factor. NF-kappa B mediates expression of the CMV promoter and of genes involved in the immune and inflammatory responses. Antioxidants or aspirin inhibit ROS, NF-kappa B and CMV.

Infectious agents in coronary artery disease: viral infection, aspirin, and gene expression in human coronary smooth muscle cells.

We and others have observational evidence that human cytomegalovirus (HCMV) may be a pathogen in human atherosclerosis and restenosis. We have experimental evidence that HCMV infects human coronary smooth muscle cells and initiates viral replication. Vascular cells generate reactive oxygen species in response to stress (such as infection or reperfusion) and this leads to increased transcription of atherosclerosis-related cellular and viral genes, and to reactivation of latent HCMV. Finally, we found that aspirin can attenuate this augmented gene transcription via direct and indirect effects.

Aspirin attenuates cytomegalovirus infectivity and gene expression mediated by cyclooxygenase-2 in coronary artery smooth muscle cells.

Human cytomegalovirus (CMV) infection of smooth muscle cells generates reactive oxygen species (ROS) and thereby activates nuclear factor kappaB (NFkappaB), which causes expression of viral and cellular genes involved in immune and inflammatory responses. These changes could account for the mounting evidence suggesting that CMV may contribute causally to restenosis and atherosclerosis. We found that CMV induces ROS, at least partly, through a cyclooxygenase-2 (COX-2)-dependent pathway. Moreover, the viral immediate-early (IE) gene products, IE72 and IE84, have the capacity to transactivate the COX-2 promoter. Aspirin and indomethacin, both cyclooxygenase inhibitors as well as direct ROS scavengers, reduce CMV-induced ROS, probably through both of these activities. Sodium salicylate also has antiviral effects as the result of its potent antioxidant properties. Furthermore, by reducing ROS, aspirin and sodium salicylate inhibit CMV-induced NFkappaB activation, the ability of IE72 to transactivate its promoter, CMV IE gene expression after infection of SMCs, and CMV replication in SMCs. This is the first time aspirin has been shown to have antiviral effects. Thus, it is possible that aspirin has previously unrecognized therapeutic effects in various clinical situations, such as in viral infections (when used as an antipyretic agent) and in atherosclerosis (when used as an antiplatelet agent).

Role of reactive oxygen intermediates in cytomegalovirus gene expression and in the response of human smooth muscle cells to viral infection.

Because cytomegalovirus (CMV) may contribute to restenosis and atherosclerosis and because smooth muscle cells (SMCs) are involved in these disease processes, we examined CMV-SMC interactions. Using confocal microscopy to identify a redox-sensitive fluorescent marker, we found that CMV infection of SMCs generates intracellular reactive oxygen intermediates (ROIs). CMV also activated nuclear factor kappa B (NF kappa B), a cellular transcription factor, as demonstrated by increased NF kappa B binding to DNA (electrophoretic mobility shift assay). Antioxidants inhibited activation, suggesting a role of ROIs in CMV-induced NF kappa B activation. By using antioxidants to assess the role of ROIs in modulating virally mediated effects, we also found that CMV-induced ROIs (1) are critical to the transactivation of the viral major immediate promoter (MIEP) by its immediate-early protein IE72 (determined by cotransfection of an IE72 expression vector and a reporter gene downstream from the MIEP) and (2) are necessary for IE72 expression (determined by immunocytochemistry) and viral replication (determined by viral titer assay on indicator cells) following CMV infection of SMCs. Because ROIs, through activation of NF kappa B, can also induce expression of cellular genes involved in immune and inflammatory responses, the ROI response to CMV infection may also represent a parallel survival mechanism that has evolved in the host cell to protect against viral infection. We conclude that CMV induces intracellular ROI generation within minutes after infection of SMCs and then uses these ROIs to facilitate its own gene expression and replication. Conversely, antioxidants inhibit CMV immediate-early gene expression and viral replication.

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.

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.

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.

In vitro effects of a recombinant toxin targeted to the fibroblast growth factor receptor on rat vascular smooth muscle and endothelial cells.

The dominant mechanism responsible for restenosis after angioplasty is believed to be the activation of medial smooth muscle cells (SMCs), leading to their proliferation, migration to the subintima, and further proliferation. To develop novel strategies that might inhibit or prevent restenosis, we previously used a chimeric toxin composed of transforming growth factor-alpha (which targets the epidermal growth factor receptor) and mutated Pseudomonas exotoxin to preferentially recognize and kill rapidly proliferating, versus quiescent, vascular SMCs. We have recently cloned and expressed a recombinant gene encoding Pseudomonas exotoxin with a mutated (nonfunctional) cell recognition domain fused with the ligand acidic fibroblast growth factor, termed aFGF-PE66(4Glu)KDEL; thus, this recombinant toxin targets the fibroblast growth factor receptor. In the present study, we evaluated the relative effects of this chimeric toxin on quiescent versus rapidly proliferating vascular SMCs and also determined whether aFGF-PE66(4Glu)KDEL exerted different effects on SMCs versus endothelial cells. Rapidly proliferating SMCs (grown in 10% fetal bovine serum) were very sensitive to the cytotoxic effects of aFGF-PE66(4Glu)KDEL, whereas cytotoxicity was significantly less when the SMCs were in a quiescent state (grown in medium supplemented with 0.5% fetal bovine serum). The chimeric toxin was also significantly less cytotoxic against endothelial cells. Competition studies using excess acidic fibroblast growth factor indicated that the cytotoxic effects are specifically mediated by the fibroblast growth factor receptor. Thus, the present studies suggest a potentially expanded role of recombinant toxin therapy in restenosis: multiple receptors can be targeted, and cytotoxic effects, at least in vitro, can be preferentially directed to rapidly proliferating vascular SMCs, with relative sparing of vascular endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Elimination of smooth muscle cells in experimental restenosis: targeting of fibroblast growth factor receptors.

Factors in plasma and platelets do not fully account for the proliferation of smooth muscle cells in vascular injury, implying that additional factors are involved. Recently, we and others have observed that vascular injury regulates basic fibroblast growth factor, suggesting a further role for this pleiotropic factor. We report here that injury of rat arteries leads to an increase in fibroblast growth factor receptors in vascular smooth muscle cells. This up-regulation makes smooth muscle cells susceptible, in vitro and in vivo, to the lethal effects of a conjugate of basic fibroblast growth factor with the ribosome inactivator saporin. Saporin alone has no effect, whereas the conjugate kills proliferating, but not quiescent, smooth muscle cells in vitro. In vivo, one to three doses inhibit neointimal proliferation but have no apparent effect on the uninjured artery. Thus, the up-regulation of fibroblast growth factor receptors in vascular injury suggests new therapeutic possibilities for such refractory conditions as restenosis following balloon angioplasty.

Inhibition of smooth muscle cell proliferation by an antisense oligodeoxynucleotide targeting the messenger RNA encoding proliferating cell nuclear antigen.

BACKGROUND: The process by which normally quiescent vascular smooth muscle cells (SMCs) change into proliferating cells, which express and respond to multiple growth factors, plays a major role in restenosis after coronary angioplasty. We are attempting to inhibit SMC proliferation by interventions that inhibit specific factors involved in signal transduction pathways leading to cell division. To date, all studies taking this approach have attempted to block the effects of mitogens acting on the cell surface. In contrast, we have focused on a strategy that bypasses cell surface-mediated events by directly inhibiting the expression of proliferating cell nuclear antigen (PCNA), an intranuclear protein that functions in a final common pathway shared by diverse mitogen-induced signals. In the present investigation, we determined whether antisense oligodeoxynucleotides (ODNs) complementary to the messenger RNA of PCNA will inhibit PCNA expression and thereby reduce SMC proliferation. METHODS AND RESULTS: When antisense ODNs (15- or 18-mer), modified to inhibit their degradation, are introduced into the medium of rat aortic SMCs in concentrations ranging from 10 to 100 microM, the 18-mer ODN, in a concentration-related manner, decreases SMC growth (as assessed by cell counting) by more than 50%. This effect persists for at least 9 days. An ODN with the same nucleotides but a scrambled sequence has little effect. Western blots and immunocytochemistry indicate that the antisense ODN reduces expression of PCNA protein. CONCLUSIONS: Our results demonstrate that an antisense ODN directed at the messenger RNA of PCNA decreases expression of the PCNA gene product and reduces SMC proliferation. In addition, these results provide an important impetus to initiating in vivo studies to determine the feasibility of antisense strategies in the prevention of coronary restenosis.

Acidic and basic fibroblast growth factors in adult rat heart myocytes. Localization, regulation in culture, and effects on DNA synthesis.

Basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF) are involved in the induction of embryonic mesoderm, angiogenesis, neuronal differentiation, and proliferation and survival of many cell types. In cardiac myocytes their roles are not well understood. Effects of fibroblast growth factors on reexpression of fetal actin genes have been reported. In freshly isolated adult rat cardiac myocytes, bFGF mRNA was not detectable by in situ hybridization, although the cells contained significant amounts of bFGF and aFGF as quantified by radioimmunoassays, mitogen assays with immunoneutralization, and Western blotting. After culturing, bFGF mRNA was detected (aFGF mRNA was not studied), and the cells contained 2.5-fold more bFGF and 60% more aFGF than freshly isolated cells. The FGFs were not found in conditioned medium. They were localized, especially in cultured cells, to the nucleus. Cultured myocytes bound fourfold more 125I-FGF than freshly isolated cells and expressed the fibroblast growth factor R-1 (flg) gene. The addition of bFGF or aFGF in serum-free medium to pure populations of myocytes (after 10 days in culture, at which time they are spread, beating, and multinucleated) led to increased thymidine incorporation. Expression of fibroblast growth factors and fibroblast growth factor receptors by adult cardiac myocytes that survive the shock and "dedifferentiation" of culturing may contribute to DNA synthesis and, by analogy, to other cell types, to regulation of ribosomal and actin genes, and to cell survival. These possibilities and their in vivo relevance will require further study.

Cytotoxic activity of chimeric proteins composed of acidic fibroblast growth factor and Pseudomonas exotoxin on a variety of cell types.

Chimeric proteins composed of acidic fibroblast growth factor (acidic FGF) and several forms of Pseudomonas exotoxin (PE) that cannot bind to the PE receptor have been produced in Escherichia coli by expressing chimeric genes in which DNA encoding acidic FGF is fused to various mutant forms of PE. These acidic FGF-PE fusion proteins were found to be cytotoxic to a variety of tumor cell lines including hepatocellular (PLC/PRF/5 and HEPG2), prostatic (LNCaP), colon (HT29), and breast (MCF-7) carcinomas at concentrations of 1-70 ng/ml. The cytotoxic effects of acidic FGF-PE were FGF-receptor specific as demonstrated by competition with excess acidic FGF and by showing that acidic FGF-PE bound to the FGF receptor with the same affinity as acidic FGF. Furthermore, the cell-killing activity of acidic FGF-PE was toxin-mediated, as an acidic FGF-PE mutant, which does not possess ADP-ribosylation activity, failed to kill cells. These findings demonstrate that acidic FGF-PE is a potent cytotoxic molecule that can be targeted to FGF receptor-bearing cells. Because acidic FGF is a potent angiogenic molecule, cytotoxic acidic FGF-PE chimeras may have utility as anti-angiogenic agents. These molecules could be helpful in determining the functional role of FGF receptors in cellular processes.

Coronary vasoconstriction induced by vasopressin. Production of myocardial ischemia in dogs by constriction of nondiseased small vessels.

BACKGROUND: We studied the effect of intracoronary administration of arginine-8-vasopressin on blood flow in nondiseased coronary arteries and determined whether this vasoconstriction was severe enough to produce ischemia in 30 dogs. METHODS AND RESULTS: In group 1 (n = 6), after vasopressin administration coronary blood flow was decreased by 41% (p less than 0.002) without changes in heart rate or aortic pressure, and left ventricular ejection fraction measured by radionuclide angiocardiography was decreased by 18% (p less than 0.0005). In group 2 (n = 6), ischemia was confirmed by measurement of transmural pH changes. Administration of vasopressin decreased subendocardial pH of the infused zone from 7.40 +/- 0.03 to 7.31 +/- 0.07 (p less than 0.01). The subendocardial pH of the zone not infused with vasopressin did not change. To overcome the intrinsic regulation of blood flow, operating primarily in small coronary arteries, we hypothesized that vasopressin must increase resistance primarily in large rather than small coronary arteries. After intracoronary infusion in group 3 (n = 6), however, most (94%) of the increase in resistance during vasopressin administration was explained by an increase of resistance in small coronary arteries. In group 4 (n = 9), vasopressin decreased coronary blood flow by 50% and decreased local shortening by 90% at a time when systemic hemodynamics were unchanged. Coronary constriction induced by vasopressin, or the recovery from it, also was not altered by cyclooxygenase blockade. CONCLUSIONS: Thus, vasopressin produces myocardial ischemia by constricting small, nondiseased coronary arteries severely enough to overcome the competition from normal coronary regulation, and this ischemic event is not mediated by prostaglandin products.

Culture-induced increase in acidic and basic fibroblast growth factor activities and their association with the nuclei of vascular endothelial and smooth muscle cells.

The activity of acidic and basic fibroblast growth factor-like mitogens (aFGF, bFGF) extracted from cultured bovine aortic endothelial (BAEC) and rat aortic smooth muscle cells (SMC) was compared with that of freshly isolated cells from the same tissues. Extracts of subendothelial extracellular matrix (ECM) and cell lysates of cultured BAEC contained 4-fold more bFGF-like activity than the extracts of fresh cells. ECM and cell lysates of SMC yielded 10-fold more bFGF-like activity than the fresh cell lysates. We consistently find aFGF-like activity in both cell types. In the case of BAEC, cultured cells and ECM contained 3-fold more aFGF-like activity when compared with freshly isolated cells, whereas in cultured SMC, aFGF-like activity in cell and ECM extracts was 8-fold higher than in fresh cell extracts. The mitogens extracted from cell lysates and from the ECM are closely related to aFGF or bFGF by the criteria that they bind to heparin-sepharose and elute at 1.1 M (aFGF) or 1.5 M (bFGF) NaCl, have molecular weights of about 18,000, and react with anti-aFGF (1.1 M), or anti-bFGF (1.5 M) antibodies when analyzed by Western blots and by radioimmunoassay specific for aFGF and bFGF. This mitogenic activity is inhibited by neutralizing antibodies to aFGF and bFGF. In addition, the column fractions are potent mitogens for Balb/c 3T3 fibroblasts. Acidic and basic FGF-like mitogenic activity could also be extracted from the cell nuclei. The subcellular localization of both FGFs was visualized in both nuclei and cytoplasm with immunoperoxidase. Compared with primary SMC, secondary SMC had an increased capacity to bind 125IaFGF to high affinity receptors, while binding to freshly isolated BAEC and SMC was negligible. We conclude that FGFs are present at low levels in freshly isolated cells and that propagation in cell culture provides a stimulus for production of these mitogens.

Isolation, characterization, and localization of heparin-binding growth factors in the heart.

Acidic and basic fibroblast growth factors (aFGF and bFGF) are angiogenic polypeptide mitogens for cells of mesodermal and neuroectodermal origin. In this report we describe the purification from several normal human hearts (including a very fresh, nonischemic sample) of heparin-binding, acid-, heat- and trypsin-sensitive 14-18-kD peptides that crossreact with antisera against aFGF and bFGF. Further evidence includes (a) prevention of mitogenicity by protamine and by anti-bFGF, (b) displacement of 125I-bFGF from cell membranes, and (c) stimulation of capillary endothelial cell migration. Specific immunohistochemistry localized bFGF to endothelial cells and, surprisingly, to cardiac myocytes, with almost no immunoreactivity in smooth muscle cells. These peptides may function in cardiac embryogenesis, hypertrophy, atherogenesis, angiogenesis, and wound healing, and may also have endocrine, neurotropic, or vasomotor functions.