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.

Neuropeptide-Y. A peptide found in human coronary arteries constricts primarily small coronary arteries to produce myocardial ischemia in dogs.

Neuropeptide-Y (NPY), a brain peptide, is located in the walls of human coronary arteries. This study assessed the effects of NPY on the coronary circulation in 40 chloralose-anesthetized, open-chest dogs. Intracoronary NPY (42 nmol over 5.2 min) caused a 39% reduction in coronary blood flow without changing heart rate or aortic pressure. To determine whether this vasoconstriction could produce ischemia, intramyocardial pH was measured in seven dogs (group I) and decreased from 7.45 +/- 0.06 to 7.37 +/- 0.06 pH units after NPY in the subendocardium (P less than 0.0002), and from 7.45 +/- 0.06 to 7.40 +/- 0.05 pH units (P less than 0.04) in the subepicardium of the infused zone. Left ventricular ejection fraction (LVEF), measured by radionuclide angiography, decreased from 0.52 +/- 0.08 to 0.42 +/- 0.12 U (n = 5, P less than 0.01) during NPY. NPY-induced vasoconstriction was also associated with ST-T wave changes on the electrocardiogram (ECG) in eight of nine other animals (group V). In another group of six dogs (group IV), the change in small vessel resistance accounted for 94% of the increase in total resistance, so that the primary vasoconstrictor effect of NPY was exerted on small coronary arteries. Thus, NPY, a peptide found in human coronary arteries, caused constriction of primarily small coronary arteries that was severe enough to produce myocardial ischemia as determined by ECG ST-T wave changes, and decreases in intramyocardial pH and LVEF in dogs.

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.

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.

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.

Role of cellular proteinases in acute myocardial infarction. I. Proteolysis in nonischemic and ischemic rat myocardium and the effects of antipain, leupeptin, pepstatin and chymostatin administered in vivo.

To test the hypothesis that cellular proteinases contribute to ischemic myocellular death, measurements were made of tyrosine release (an index of overall proteolysis) from incubated slices of nonischemic and ischemic myocardium obtained at various times after coronary artery occlusion in rats. Proteolysis failed to increase in ischemic myocardium throughout the first 24 hours of occlusion, when irreversible damage develops, indicating that cellular proteinases do not undergo generalized activation in this phase. These data represent the first assessment of myocardial proteolysis throughout the development of ischemic death, and suggest that cellular proteinases do not play a causal role in this process. However, the possibility remains that ischemia selectively accelerates the breakdown of vital proteins, a phenomenon that may not be detected by measuring overall proteolysis. To determine whether future studies on the effects of proteolytic inhibition on infarct size are feasible, the ability of the proteinase inhibitors antipain, leupeptin, pepstatin and chymostatin, given in vivo, to interfere with proteolysis in ischemic myocardium was also evaluated. Leupeptin (10 or 40 mg/kg) inhibited proteolysis in a dose-related fashion (-49 and -72%, respectively, p less than 0.001). Antipain (20 mg/kg) decreased protein breakdown by 60% (p less than 0.001). The combination of antipain (20 mg/kg), leupeptin (40 mg/kg) and pepstatin (5 mg/kg) suppressed proteolysis almost completely at both 15 minutes (-88%, p less than 0.001) and at 6 hours (-72%, p less than 0.05) of ischemia, that is, throughout the development of irreversible injury. These results demonstrate that whatever proteolysis is occurring during acute myocardial infarction is largely mediated by cathepsins A, B, D, L and H and by calcium-activated neutral protease (that is, the enzymes sensitive to the inhibitors used). Because antipain, leupeptin and pepstatin significantly suppress such proteolysis, these agents might be useful in further assessing any potential contribution of cellular proteinases to the production of ischemic myocellular death. In addition, this study provides a new experimental model that affords serial assessments of regional myocardial proteolysis during the evolution of myocardial infarction.

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.

A new device for the efficient pulverisation and extraction of myocardial biopsies for high energy phosphate analysis.

We developed a new device for processing frozen myocardial biopsies. Frozen samples of 20 to 50 mg were dropped into a 25 ml stainless steel centrifuge tube held in a custom-made aluminium container precooled in liquid nitrogen. A stainless steel pestle attached to a stainless steel disk was driven by a modified heavy-duty staple gun to pulverise the tissue rapidly at low temperatures. The tissue powder was extracted with 0.3N PCA at 0 degree C in the centrifuge tube which was then transferred to a Sorvall super-speed centrifuge. Values for adenosine triphosphate (ATP) were 5.6 +/- 0.7 mumol . g-1 wet weight (mean +/- SD). Creatine phosphate (CP) yield was 12.2 +/- 3 mumol . g-1 wet weight. The % recovery of an added internal standard for ATP was 86 +/- 18% and for CP 90 +/- 16% with the new method.

Early degradation of collagen after acute myocardial infarction in the rat.

After acute myocardial infarction (MI), proteolysis of necrotic myocardium is mediated by infiltrating inflammatory cells at the infarct margins. Collagen forms a structural fibroskeleton in healthy myocardium, and after MI this collagen may continue to provide significant tensile strength to the necrotic muscle wall. To determine whether collagen is also degraded (which might decrease infarct wall strength) and, if so, whether inflammatory cell proteases are implicated, hydroxyproline was measured from infarct zone and normal zone tissue from 24-hour infarcts produced in control rats and in rats made leukopenic (white blood cell count less than 300/mm3) by prior whole-body irradiation. Hydroxyproline was measured after precipitation of tissue homogenates with trichloroacetic acid to separate partially degraded collagen from larger collagen molecules that might retain structural importance. At 24 hours, there was significant (25%) collagen degradation in the infarct zone (p less than 0.01) in control rats but not in leukopenic rats. Tissue cell counts revealed a paucity of inflammatory cells in the infarct margins in leukopenic rats. Electron microscopic studies revealed greater preservation of collagen in the 24-hour-old infarcts of irradiated leukopenic rats compared with those of control rats. These results suggest that at 24 hours after experimental MI in the rat, there is significant collagen degradation mediated by inflammatory cell proteases.

Effect of ibuprofen on the healing phase of experimental myocardial infarction in the rat.

The healing phase of acute myocardial infarction (AMI) is initiated by proteolysis of necrotic myocardium, followed by infiltration of fibroblasts and deposition of collagen. To assess whether ibuprofen, a potent antiinflammatory agent, preserves existing collagen and enhances deposition of new collagen during infarct healing, biochemical and morphologic studies were made of experimentally induced myocardial infarcts in untreated rats and in rats treated with ibuprofen. All treated rats received 12.5 mg/kg of ibuprofen at 1, 6 and 18 hours after AMI. Group 1 rats underwent measurement of myocardial hydroxyproline (HP) content at 24 hours after AMI. Group 2 rats received ibuprofen, 12.5 mg/kg, twice a day for 2 additional days, with measurement of myocardial HP at 3 days (group 2a) or 21 days (group 2b) after AMI. Group 3 rats received ibuprofen, 12.5 mg/kg, twice a day for 6 additional days with measurement of HP content, or infarct size and degree of thinning at 21 days after AMI. Compared with untreated rats, ibuprofen-treated rats had significantly greater amounts of HP in the infarct at 24 hours (group 1, 8.9 +/- 2.2 nmol/mg dry weight vs untreated, 7.1 +/- 2.8 nmol/mg dry weight, p less than 0.04) and at 21 days (group 2b, 112 +/- 37 nmol/mg dry weight vs untreated, 91 +/- 39 nmol/mg dry weight, p less than 0.05, and group 3, 125 +/- 51 nmol/mg dry weight vs untreated, 91 +/- 39 nmol/mg dry weight, p less than 0.003). Substantial scar thinning was noted in all rats; no difference in scar thinning was noted between treated and untreated rats at 21 days after AMI.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Correlation of changes in cardiac calcium channels with hemodynamics in Syrian hamster cardiomyopathy and heart failure.

We compared hemodynamics with [3H]nitrendipine (calcium channel) binding to cardiac membranes from Bio 14.6 cardiomyopathic Syrian hamsters at 4 and 10 months with their F1B controls. A 50% increase in the number (Bmax) of nitrendipine binding sites (calcium channels) was seen only in the 4 month old myopathic vs controls (Bmax = 468 +/- 11 vs 309 +/- 10 fmol/mg prot with no change in affinity (KD) (KD = .65 +/- .12 vs .75 +/- .14 nM), while no differences in Bmax or KD were seen at 10 months (Bmax = 375 +/- 9 vs 362 +/- 7 fmol/mg prot/KD = .82 +/- .18 vs .89 +/- .17 nM) myopathic vs control respectively. Hemodynamic studies revealed no significant differences in cardiac output, cardiac index, stroke volume, heart rate, mean arterial pressure, peripheral resistance, body weight, heart weight at 4 months, but a significant decrease in peripheral resistance (1120 +/- 360 vs 2080 +/- 240) increase in body weight (118 +/- 2 vs 94 +/- 2 grams) and heart weight (97 +/- 5 vs 78 +/- 2 gms/100 gms body weight) in 10 month myopathic vs control animals. We conclude that the onset of cardiomyopathy at 4 months is associated with a selective increase in calcium channel binding sites and heart failure at 10 months is associated with a relative decrease in these sites.

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.

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.

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.

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.