Edith Speir

Transforming Growth Factor Beta-1 in Acute Myocardial Infarction in Rats

TGF-beta 1 has been examined in the heart during myocardial infarction caused by ligation of the left coronary artery. Infarcted and uninfarcted myocardium have been compared by immunohistochemical staining of TGF-beta 1 and by Northern blot analysis of mRNA. Normal ventricular myocytes are strongly stained by an antibody to TGF-beta 1. Progressive loss of staining of these myocytes begins within 1 hr after coronary ligation. However, by 24-48 hr after ligation, intense staining of myocytes at the margin of infarcted areas is seen. Northern blots of infarcted myocardium 48 hr after ligation show a 3- to 4-fold increase in the principal 2.4 kb TGF-beta 1 mRNA; there is also a marked increase in a minor 1.9 kb transcript. In the same tissue samples, there is a 2-fold decrease in the mRNA for the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase. The results indicate a significant role for TGF-beta in the response of the heart to injury.

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).

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.

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.

Competition for p300 Regulates Transcription by Estrogen Receptors and Nuclear Factor-κB 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-&kgr;B component, in smooth muscle cells that express ER&agr; and ER&bgr; after exposure to 17&bgr;-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-&kgr;B–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 17&bgr;-estradiol may play an important role in the cytokine-induced expression of immune and inflammatory genes implicated in atherogenesis.

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

BackgroundWe 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 ResultsIn group 1 (n =6), after vasopressin administration coronary blood flow was decreased by 41% (p < 0.002) without changes in heart rate or aortic pressure, and left ventricular ejection fraction measured by radionuclide angiocardiography was decreased by 18% (p < 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 < 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 901% 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. ConclusionsThus, 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. (Circulation 1991;83:2111—2121)

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.

Antioxidant Effect of Estrogen on Cytomegalovirus-Induced Gene Expression in Coronary Artery Smooth Muscle Cells

BackgroundPathogens 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 ResultsUsing confocal microscopy and an intracellular fluorescent dye activated by ROSs, we found that 17&bgr;-estradiol (0.1 to 10 nmol/L) and its stereoisomer 17&agr;-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 17&bgr;-estradiol and 17&agr;-estradiol, but not 3-methoxyestrone, prevented binding of nuclear factor (NF)-&kgr;B to DNA. Furthermore, in SMCs transfected with the reporter constructs 3X&kgr;B-CAT, MIEP-CAT, or ICAM-CAT, cotransfection with a CMV-IE72 expression plasmid caused promoter and CAT activation. Treatment with 17&bgr;-estradiol and 17&agr;-estradiol, but not 3-methoxyestrone, inhibited CAT activity and, in CMV-infected SMCs, prevented IE72 and ICAM-1 protein expression and cytopathic effects. ConclusionsThese 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-&kgr;B activation, NF-&kgr;B–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.

Fibroblast growth factors are present in adult cardiac myocytes, in vivo

Summary We have previously shown that the adult heart contains mitogens immunologically identical to acidic and basic fibroblast growth factors. To determine whether these proteins are present in myocytes, we subjected lysates of freshly isolated myocytes to heparin-affinity chromatography. The 1.1 M – 3 M NaCl eluates stimulated incorporation of thymidine into DNA in quiescent Balb/c 3T3 fibroblasts, caused proliferation of vascular endothelial cells, and cross-reacted with antisera raised against acidic (1.1 M) and basic FGFs (1.5 M) by Western blotting and by RIA. These proteins may be involved in cellular differentiation and proliferation and may play an important role in regenerative and repair processes in the heart.

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.