Felix C. Tanner

Drug-Eluting Stent and Coronary Thrombosis Biological Mechanisms and Clinical Implications

Although rare, stent thrombosis remains a severe complication after stent implantation owing to its high morbidity and mortality. Since the introduction of drug-eluting stents (DES), most interventional centers have noted stent thrombosis up to 3 years after implantation, a complication rarely seen with bare-metal stents. Some data from large registries and meta-analyses of randomized trials indicate a higher risk for DES thrombosis, whereas others suggest an absence of such a risk. Several factors are associated with an increased risk of stent thrombosis, including the procedure itself (stent malapposition and/or underexpansion, number of implanted stents, stent length, persistent slow coronary blood flow, and dissections), patient and lesion characteristics, stent design, and premature cessation of antiplatelet drugs. Drugs released from DES exert distinct biological effects, such as activation of signal transduction pathways and inhibition of cell proliferation. As a result, although primarily aimed at preventing vascular smooth muscle cell proliferation and migration (ie, key factors in the development of restenosis), they also impair reendothelialization, which leads to delayed arterial healing, and induce tissue factor expression, which results in a prothrombogenic environment. In the same way, polymers used to load these drugs have been associated with DES thrombosis. Finally, DES impair endothelial function of the coronary artery distal to the stent, which potentially promotes the risk of ischemia and coronary occlusion. Although several reports raise the possibility of a substantially higher risk of stent thrombosis in DES, evidence remains inconclusive; as a consequence, both large-scale and long-term clinical trials, as well as further mechanistic studies, are needed. The present review focuses on the pathophysiological mechanisms and pathological findings of stent thrombosis in DES.

Mechanisms underlying adverse effects of HDL on eNOS-activating pathways in patients with coronary artery disease

Therapies that raise levels of HDL, which is thought to exert atheroprotective effects via effects on endothelium, are being examined for the treatment or prevention of coronary artery disease (CAD). However, the endothelial effects of HDL are highly heterogeneous, and the impact of HDL of patients with CAD on the activation of endothelial eNOS and eNOS-dependent pathways is unknown. Here we have demonstrated that, in contrast to HDL from healthy subjects, HDL from patients with stable CAD or an acute coronary syndrome (HDLCAD) does not have endothelial antiinflammatory effects and does not stimulate endothelial repair because it fails to induce endothelial NO production. Mechanistically, this was because HDLCAD activated endothelial lectin-like oxidized LDL receptor 1 (LOX-1), triggering endothelial PKCβII activation, which in turn inhibited eNOS-activating pathways and eNOS-dependent NO production. We then identified reduced HDL-associated paraoxonase 1 (PON1) activity as one molecular mechanism leading to the generation of HDL with endothelial PKCβII-activating properties, at least in part due to increased formation of malondialdehyde in HDL. Taken together, our data indicate that in patients with CAD, HDL gains endothelial LOX-1- and thereby PKCβII-activating properties due to reduced HDL-associated PON1 activity, and that this leads to inhibition of eNOS-activation and the subsequent loss of the endothelial antiinflammatory and endothelial repair-stimulating effects of HDL.

Tissue Factor in Cardiovascular Diseases Molecular Mechanisms and Clinical Implications

Tissue factor (TF), formerly known as thromboplastin, is the key initiator of the coagulation cascade; it binds factor VIIa resulting in activation of factor IX and factor X, ultimately leading to fibrin formation. TF expression and activity can be induced in endothelial cells, vascular smooth muscle cells, and monocytes by various stimuli such as cytokines, growth factors, and biogenic amines. These mediators act through diverse signal transduction mechanisms including MAP kinases, PI3-kinase, and protein kinase C. Cellular TF is present in three pools as surface, encrypted, and intracellular protein. TF can also be detected in the bloodstream, referred to as circulating or blood-borne TF. Elevated levels of TF are observed in patients with cardiovascular risk factors such as hypertension, diabetes, dyslipidemia, and smoking as well as in those with acute coronary syndromes. TF may indeed be involved in the pathogenesis of atherosclerosis by promoting thrombus formation; in addition, it can induce migration and proliferation of vascular smooth muscle cells. As a consequence, therapeutic strategies have been developed to specifically interfere with the action of TF such as antibodies against TF, site-inactivated factor VIIa, or recombinant TF pathway inhibitor. Inhibition of TF action appears to be an attractive target for the treatment of cardiovascular diseases.

Oxidized low density lipoproteins inhibit relaxations of porcine coronary arteries. Role of scavenger receptor and endothelium-derived nitric oxide.

BACKGROUND We studied the effects of low density lipoprotein (LDL) on endothelium function. METHODS AND RESULTS Porcine epicardial and intramyocardial coronary arteries suspended in organ chambers for isometric tension recording were exposed to LDL for 2 hours and were then washed. In epicardial coronary arteries, oxidized LDL (30-300 micrograms/ml) but not native LDL or lysolecithin inhibited endothelium-dependent relaxations to serotonin, thrombin, and aggregating platelets (5,000-75,000/microliter). Endothelium-dependent relaxations to bradykinin and A23187 and endothelium-independent relaxations to SIN-1 were unaffected by oxidized LDL. In intramyocardial coronary arteries, oxidized LDL had no appreciable effect on relaxations to serotonin. The effect of oxidized LDL on the response to serotonin in epicardial coronary arteries was completely prevented by dextran sulfate (10 micrograms/ml). The inhibitory effect of oxidized LDL persisted in the presence of pertussis toxin. Similar to the lipoproteins, L-NG-monomethyl arginine (L-NMMA) reduced relaxations to serotonin but not to bradykinin in epicardial coronary arteries. In the presence of L-NMMA, oxidized LDL further reduced the response to serotonin. In arteries in which relaxations to serotonin were inhibited by oxidized LDL, L-arginine but not D-arginine induced a full relaxation. Pretreatment with L-arginine potentiated relaxations to serotonin in arteries exposed to oxidized LDL. CONCLUSIONS Thus, oxidized LDL activates the scavenger receptor on endothelial cells and inhibits the receptor-operated nitric oxide formation in epicardial but not in intramyocardial coronary arteries. The mechanism is not related to dysfunction of a Gi protein but is related to a reduced intracellular availability of L-arginine. The reduced nitric oxide formation at sites of early atherosclerotic lesions may favor platelet aggregation and vasospasm, both of which are known clinical events in patients with coronary artery disease.

Differential Effects of the Cyclin-Dependent Kinase Inhibitors p27Kip1, p21Cip1, and p16Ink4 on Vascular Smooth Muscle Cell Proliferation

BACKGROUND The cyclin-dependent kinase inhibitors (CKIs) have different patterns of expression in vascular diseases. The Kip/Cip CKIs, p27(Kip1) and p21(Cip1), are upregulated during arterial repair and negatively regulate the growth of vascular smooth muscle cells (VSMCs). In contrast, the Ink CKI, p16(Ink4), is not expressed in vascular lesions. We hypothesized that a variation in the inactivation of cdk2 and cdk4 during the G(1) phase of the cell cycle by p27(Kip1), p21(Cip1), and p16(Ink4) leads to different effects on VSMC growth in vitro and in vivo. METHODS AND RESULTS The expression of p27(Kip1) and p21(Cip1) in serum-stimulated VSMCs inactivated cdk2 and cdk4, leading to G(1) growth arrest. p16(Ink4) inhibited cdk4, but not cdk2, kinase activity, producing partial inhibition of VSMC growth in vitro. In an in vivo model of vascular injury, overexpression of p27(Kip1) reduced intimal VSMC proliferation by 52% (P<0.01) and the intima/media area ratio by 51% (P<0.005) after vascular injury and gene transfer to pig arteries, when compared with control arteries. p16(Ink4) was a weak inhibitor of intimal VSMC proliferation in injured arteries (P=NS), and it did not significantly reduce intima/media area ratios (P=NS), which is consistent with its minor effects on VSMC growth in vitro. CONCLUSIONS p27(Kip1) and p21(Cip1) are potent inhibitors of VSMC growth compared with p16(Ink4) because of their different molecular mechanisms of cyclin-dependent kinase inhibition in the G(1) phase of the cell cycle. These findings have important implications for our understanding of the pathophysiology of vascular proliferative diseases and for the development of molecular therapies.

Inflammatory Markers at the Site of Ruptured Plaque in Acute Myocardial Infarction Locally Increased Interleukin-6 and Serum Amyloid A but Decreased C-Reactive Protein

Background—Acute myocardial infarction (AMI) is associated with inflammation. However, it remains unclear whether it originates from the ruptured plaque or represents a systemic process. Methods and Results—In 42 patients with AMI, a balloon-based embolization protection device and aspiration catheter (PercuSurge) were used during acute coronary interventions. Samples from the site of the ruptured plaque were taken under distal balloon occlusion. Systemic samples were taken from the aorta. Sera, plaques, and thrombi were analyzed for inflammatory markers and lipoproteins. Systemic levels of C-reactive protein (CRP), interleukin-6 (IL-6), and serum amyloid A (SAA) in the aorta amounted to 3.0 mg/L, 5.0 ng/L, and 22.1 mg/L, respectively (interquartile ranges [IQRs], 1.1 to 7.4 mg/L, 5.0 to 6.5 ng/L, and 13.9 to 27.0 mg/L, respectively). In blood surrounding ruptured plaques, local levels of IL-6 (8.9 ng/L; IQR, 5.0 to 16.9 ng/L) and SAA (24.3 mg/L; IQR, 16.3 to 44.0 mg/L) were significantly higher, whereas CRP levels (2.5 mg/L; IQR, 0.9 to 7.7 mg/L) were decreased compared with the aorta (all P<0.0001). The coronary levels of IL-6 determined in vivo showed biological activity in vitro. Harvested thrombus contained CD68-positive monocytes expressing IL-6 and showed extracellularly and intracellularly positive staining for SAA, whereas CRP was found exclusively in the cytoplasm of phagocyting white blood cells. Conclusions—Coronary levels of IL-6 and SAA at the site of plaque rupture were increased relative to the systemic circulation, indicating local production of biologically active inflammatory mediators. In contrast, CRP was locally decreased, at least in part by uptake by the phagocyting cells, suggesting a systemic origin of the protein.

Genetic deletion of p66Shc adaptor protein prevents hyperglycemia-induced endothelial dysfunction and oxidative stress

Increased production of reactive oxygen species (ROS) and loss of endothelial NO bioavailability are key features of vascular disease in diabetes mellitus. The p66Shc adaptor protein controls cellular responses to oxidative stress. Mice lacking p66Shc (p66Shc−/−) have increased resistance to ROS and prolonged life span. The present work was designed to investigate hyperglycemia-associated changes in endothelial function in a model of insulin-dependent diabetes mellitus p66Shc−/− mouse. p66Shc−/− and wild-type (WT) mice were injected with citrate buffer (control) or made diabetic by an i.p. injection of 200 mg of streptozotocin per kg of body weight. Streptozotocin-treated p66Shc−/− and WT mice showed a similar increase in blood glucose. However, significant differences arose with respect to endothelial dysfunction and oxidative stress. WT diabetic mice displayed marked impairment of endothelium-dependent relaxations, increased peroxynitrite (ONOO−) generation, nitrotyrosine expression, and lipid peroxidation as measured in the aortic tissue. In contrast, p66Shc−/− diabetic mice did not develop these high-glucose-mediated abnormalities. Furthermore, protein expression of the antioxidant enzyme heme oxygenase 1 and endothelial NO synthase were up-regulated in p66Shc−/− but not in WT mice. We report that p66Shc−/− mice are resistant to hyperglycemia-induced, ROS-dependent endothelial dysfunction. These data suggest that p66Shc adaptor protein is part of a signal transduction pathway relevant to hyperglycemia vascular damage and, hence, may represent a novel therapeutic target against diabetic vascular complications.

Nitric Oxide Modulates Expression of Cell Cycle Regulatory Proteins A Cytostatic Strategy for Inhibition of Human Vascular Smooth Muscle Cell Proliferation

BACKGROUND We examined the effect of NO on the proliferation and cell cycle regulation of human aortic vascular smooth muscle cells (VSMCs). METHODS AND RESULTS The NO donor diethylenetriamineNONOate (10(-5) to 10(-3) mol/L) inhibited proliferation in response to 10% fetal calf serum (FCS) and 100 ng/mL platelet-derived growth factor-BB in a concentration-dependent manner. This effect was not observed with disintegrated diethylenetriamineNONOate or with the parent compound, diethylenetriamine. Adenoviral transfection of endothelial NO synthase (NOS) inhibited proliferation in response to FCS, which was prevented with N(G)-nitro-L-arginine methyl ester. NOS overexpression did not inhibit proliferation in response to platelet-derived growth factor, although the transfection efficiency and protein expression were similar to those of FCS-stimulated cells. Nitrate release was selectively enhanced from FCS-treated cells, indicating that NOS was activated by FCS only. NO caused G(1) cell cycle arrest. Cytotoxicity was determined with trypan blue exclusion, and apoptosis was assessed with DNA fragmentation. Cyclin-dependent kinase 2 expression level, threonine phosphorylation, and kinase activity were inhibited. Cyclin A expression was blunted, whereas cyclin E remained unchanged. p21 expression was induced, and p27 remained unaltered. The effect on cyclin A and p21 started within 6 hours and preceded the changes in cell cycle distribution. Proliferation in response to 10% FCS was barely inhibited with 8-bromo-cGMP (10(-3) mol/L) but was blunted with both forskolin and 8-bromo-cAMP. Proliferation in response to 2% FCS was inhibited with 8-bromo-cGMP, but it did not mimic the cell cycle effects of NO. CONCLUSIONS NO inhibits VSMC proliferation by specifically changing the expression and activity of cell cycle regulatory proteins, which may occur independent of cGMP. Adenoviral overexpression of endothelial NOS represents a cytostatic strategy for gene therapy of vascular disease.

Connective Tissue Growth Factor Induces Apoptosis in Human Breast Cancer Cell Line MCF-7

Connective tissue growth factor (CTGF) is a member of an emerging CCN gene family that is implicated in various diseases associated with fibro-proliferative disorder including scleroderma and atherosclerosis. The function of CTGF in human cancer is largely unknown. We now show that CTGF induces apoptosis in the human breast cancer cell line MCF-7. CTGF mRNA was completely absent in MCF-7 but strongly induced by treatment with transforming growth factor β (TGF-β). TGF-β by itself induced apoptosis in MCF-7, and this effect was reversed by co-treatment with CTGF antisense oligonucleotide. Overexpression of CTGF gene in transiently transfected MCF-7 cells significantly augmented apoptosis. Moreover, recombinant CTGF protein significantly enhanced apoptosis in MCF-7 cells as evaluated by DNA fragmentation, Tdt-mediated dUTP biotin nick end-labeling staining, flow cytometry analysis, and nuclear staining using Hoechst 33258. Finally, recombinant CTGF showed no effect on Bax protein expression but significantly reduced Bcl2 protein expression. Taken together, these results suggest that CTGF is a major inducer of apoptosis in the human breast cancer cell line MCF-7 and that TGF-β-induced apoptosis in MCF-7 cells is mediated, in part, by CTGF.

Rapamycin, but Not FK-506, Increases Endothelial Tissue Factor Expression. Implications for Drug-Eluting Stent Design

Background—Drugs released from stents affect the biology of vascular cells. We examined the effect of rapamycin and FK-506 on tissue factor (TF) expression in human aortic endothelial cells (HAECs) and vascular smooth muscle cells (HAVSMCs). Methods and Results—Rapamycin enhanced thrombin- and tumor necrosis factor (TNF)-α–induced endothelial TF expression in a concentration-dependent manner. The maximal increase was 2.5-fold more pronounced than that by thrombin or TNF-α alone and was paralleled by a 1.4-fold higher TF surface activity compared with thrombin alone. Rapamycin by itself increased basal TF levels by 40%. In HAVSMCs, rapamycin did not affect thrombin- or TNF-α–induced TF expression. In contrast to rapamycin, FK-506 did not enhance thrombin- or TNF-α–induced endothelial TF expression. Thrombin induced a transient dephosphorylation of the mammalian target of rapamycin downstream target p70S6 kinase. Rapamycin completely abrogated p70S6 kinase phosphorylation, but FK-506 did not. FK-506 antagonized the effect of rapamycin on thrombin-induced TF expression. Rapamycin did not alter the pattern of p38, extracellular signal–regulated kinase, or c-Jun NH2-terminal kinase phosphorylation. Real-time polymerase chain reaction analysis revealed that rapamycin had no influence on thrombin-induced TF mRNA levels for up to 2 hours but led to an additional increase after 3 and 5 hours. Conclusions—Rapamycin, but not FK-506, enhances TF expression in HAECs but not in HAVSMCs. This effect requires binding to FK binding protein-12, is mediated through inhibition of the mammalian target of rapamycin, and partly occurs at the posttranscriptional level. These findings may be clinically relevant for patients receiving drug-eluting stents, particularly when antithrombotic drugs are withdrawn or ineffective, and may open novel perspectives for the design of such stents.