Johannes Ruef

Induction of Rat Aortic Smooth Muscle Cell Growth by the Lipid Peroxidation Product 4-Hydroxy-2-Nonenal

BACKGROUND Atherosclerotic lesion formation is a complex process, in part mediated by inflammatory and oxidative mechanisms including lipid peroxidation. To further characterize the potential role of lipid peroxidation products in atherogenesis, we studied the effects of 4-hydroxy-2-nonenal (HNE) on rat aortic smooth muscle cell growth. METHODS AND RESULTS HNE, at concentrations of 1.0 and 2.5 micromol/L, significantly stimulated rat aortic smooth muscle cell growth as determined by cell counts, [3H]-thymidine uptake, and incorporation of bromo-deoxyuridine. To characterize the mechanism of HNE-induced mitogenesis, its effect on activation of intracellular growth signaling pathways was examined. Treatment with HNE resulted in activation of extracellular signal-regulated protein kinases ERK1 and ERK2, induction of c-fos and c-jun protein expression, and an increase in transcription factor AP-1 DNA binding activity. In addition, HNE induced expression of platelet-derived growth factor-AA (PDGF-AA) protein, and an anti-PDGF-AA antibody specifically inhibited HNE-mediated DNA synthesis, suggesting that growth factor induction may play a role in HNE-induced vascular smooth muscle cell growth. The role of redox-sensitive mechanisms in this process was further supported by the observation that HNE-induced DNA synthesis and AP-1 activation were inhibited by the antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate. CONCLUSIONS These data demonstrate that HNE, one of several important lipid peroxidation products, induces rat aortic smooth muscle cell growth through redox-sensitive mechanisms and growth factor expression. These observations are consistent with a role for lipid peroxidation products in vascular smooth muscle cell growth in atherogenesis.

Abciximab, Eptifibatide, and Tirofiban Exhibit Dose-dependent Potencies to Dissolve Platelet Aggregates

Platelet GPIIb/IIIa antagonists are not only used to prevent platelet aggregation, but also in combination with thrombolytic agents for the treatment of coronary thrombi. Recent data indicate a potential of abciximab alone to dissolve thrombi in vivo. We investigated the potential of abciximab, eptifibatide, and tirofiban to dissolve platelet aggregates in vitro. Adenosine diphosphate (ADP)-induced platelet aggregation could be reversed in a concentration-dependent manner by all three GPIIb/IIIa antagonists when added after the aggregation curve reached half-maximal aggregation. The concentrations chosen are comparable with in vivo plasma concentrations in clinical applications. Disaggregation reached a maximum degree of 72.4% using 0.5 &mgr;g/ml tirofiban, 91.5% using 3.75 &mgr;g/ml eptifibatide, and 48.4% using 50 &mgr;g/ml abciximab (P < 0.05, respectively). A potential fibrinolytic activity of the GPIIb/IIIa antagonists was ruled out by preincubation with aprotinin or by a plasma clot assay. A stable model Chinese hamster ovary (CHO) cell line expressing the activated form of GPIIb/IIIa was used to confirm the disaggregation capacity of GPIIb/IIIa antagonists found in platelets. Not only abciximab, but also eptifibatide and tirofiban have the potential to disaggregate newly formed platelet clusters in vitro. Because enzyme-dependent fibrinolysis does not appear to be involved, competitive removal of fibrinogen by the receptor antagonists is the most likely mechanism.

Differential Regulation of Protease Activated Receptor-1 and Tissue Plasminogen Activator Expression by Shear Stress in Vascular Smooth Muscle Cells

Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm2), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm2), mRNA expression was decreased (to 29% of stationary control; P<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control; P<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control; P<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface-associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca2+] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.

Flow Cytometric Monitoring of Glycoprotein IIb/IIIa Blockade and Platelet Function in Patients With Acute Myocardial Infarction Receiving Reteplase, Abciximab, and Ticlopidine Continuous Platelet Inhibition by the Combination of Abciximab and Ticlopidine

BackgroundImprovement of thrombolysis may be achieved by concomitant strong platelet inhibition. To monitor platelet function in patients with myocardial infarction (n=46) who were treated with the fibrinolytic agent reteplase, the glycoprotein (GP) IIb/IIIa blocker abciximab, and the ADP receptor antagonist ticlopidine, we developed a flow cytometric assay. Methods and ResultsBinding of abciximab to platelets was directly monitored as the percentage of platelets stained by a goat anti-mouse antibody. Blood drawn 10 minutes and 2 hours after the start of therapy with reteplase and abciximab and during the 12-hour infusion of abciximab demonstrated a maximal blockade of GP IIb/IIIa (10 minutes, 86.2±10.3%; 12 hours, 85.8±7.1%). Starting at 24 hours, abciximab binding gradually decreased (24 hours, 74.6±16.2%; 48 hours, 66.8±14.9%; 72 hours, 60.5±16.7%; 96 hours, 49.4±17.8%; 120 hours, 35.8±16.4%; and 144 hours, 29.9±15.3%). Binding of a chicken anti-fibrinogen antibody to platelets, indicating the level of functional blockade of GP IIb/IIIa, was inversely correlated with the binding of abciximab (r =−0.72, P <0.0001). In blood drawn at 10 minutes, platelet aggregation was maximally inhibited but recovered within 48 hours even if the majority of GP IIb/IIIa receptors were still blocked by abciximab. Reteplase did not influence abciximab binding and did not activate platelets, as measured by P-selectin expression, fibrinogen binding, and platelet aggregation. Platelet inhibition that was achieved during the first 24 hours by abciximab was directly maintained by additional treatment with ticlopidine. ConclusionsFlow cytometric monitoring of platelet function allows differentiation of the effects of reteplase, abciximab, and ticlopidine. The combination of abciximab and ticlopidine is an attractive therapeutic strategy that provides a fast and continuous platelet inhibition.

Flavopiridol Inhibits Smooth Muscle Cell Proliferation In Vitro and Neointimal Formation In Vivo After Carotid Injury in the Rat

BACKGROUND Smooth muscle cell (SMC) proliferation is a critical component of neointimal formation in many models of vascular injury and in human lesions as well. Cell-cycle inhibition by gene transfer techniques can block SMC proliferation and lesion formation in animal models, although these methods are not yet applicable to the treatment of human disease. Flavopiridol is a recently identified, potent, orally available cyclin-dependent kinase inhibitor. METHODS AND RESULTS Using human aortic SMCs, we found that flavopiridol in concentrations as low as 75 nmol/L resulted in nearly complete inhibition of basic fibroblast growth factor-induced and thrombin-induced proliferation. At this dose, flavopiridol inhibited cyclin-dependent kinase activity, as measured by histone H1 phosphorylation, but had no effect on mitogen-activated protein kinase activation. Induction of the cell cycle-related proteins cyclin D1, proliferating cell nuclear antigen, and phosphorylated retinoblastoma protein was also blocked by flavopiridol. Flavopiridol had no effect on cellular viability. To test whether flavopiridol had a similar activity in vivo when administered orally, we examined neointimal formation in rat carotid arteries after balloon injury. Flavopiridol 5 mg/kg reduced neointimal area by 35% and 39% at 7 and 14 days, respectively, after injury. CONCLUSIONS Flavopiridol inhibits SMC growth in vitro and in vivo. Its oral availability and selectivity for cyclin-dependent kinases make it a potential therapeutic tool in the treatment of SMC-rich vascular lesions.

Cardioprotection by Local Heating: Improved Myocardial Salvage After Ischemia and Reperfusion

BACKGROUND Previous studies have shown that expression of the inducible 70-kD heat-shock protein (HSP72) by whole-body hyperthermia is associated with protection against ischemia-reperfusion injury. To develop techniques for regional elevation of heat-shock proteins that prevent extracardiac sequelae during whole-body hyperthermia, we sought to determine if local heating of the heart in vivo provides protection against ischemia-reperfusion injury in the rat. METHODS A thermal probe was used to locally heat rat hearts at two adjacent sites on the epicardial surface of the left ventricle. Rats were subjected to either 30 minutes of sham surgery (control; n = 10) or two local applications of the probe at 42.5 degrees to 43.5 degrees C for 15 minutes each (n = 9). After 4 hours, rats were subjected to 30 minutes of regional ischemia followed by 120 minutes of reperfusion. Hearts were removed and area at risk and infarct area were determined. RESULTS Localized heat stress resulted in a significant limitation of infarct size in heat-treated animals versus controls (mean +/- standard error of the mean infarct area/area at risk = 4.3% +/- 0.85 versus 19.2% +/- 3.4%; p < 0.005). Western blot experiments confirmed elevated HSP72 expression in left (heated) and right (nonheated) ventricular samples from treated animals (n = 6; left ventricular = 5.5-fold; right ventricular = 3.7-fold) compared with sham-operated controls. Controls treated with the probe at 37 degrees C (n = 4) showed no increases in HSP72. CONCLUSIONS Local heating of the heart is associated with elevated levels of HSP72 and improved myocardial salvage. The increase in expression of HSP72 is not limited to the heated region, but extends into nonheated regions of the heart as well. This may lead to the development of new techniques that improve methods of myocardial revascularization and heart transplantation procedures.

Fluid shear stress as a regulator of gene expression in vascular cells: possible correlations with diabetic abnormalities

Diabetes mellitus is associated with increased frequency, severity and more rapid progression of cardiovascular diseases. Metabolic perturbations from hyperglycemia result in disturbed endothelium-dependent relaxation, activation of coagulation pathways, depressed fibrinolysis, and other abnormalities in vascular homeostasis. Atherosclerosis is localized mainly at areas of geometric irregularity at which blood vessels branch, curve and change diameter, and where blood is subjected to sudden changes in velocity and/or direction of flow. Shear stress resulting from blood flow is a well known modulator of vascular cell function. This paper presents what is currently known regarding the molecular mechanisms responsible for signal transduction and gene regulation in vascular cells exposed to shear stress. Considering the importance of the hemodynamic environment of vascular cells might be vital to increasing our understanding of diabetes.