Oxidative stress in atherogenesis and arterial thrombosis: the disconnect between cellular studies and clinical outcomes.

Atherosclerosis is a multifactorial disease for which the molecular etiology of many of the risk factors is still unknown. As no single genetic marker or test accurately predicts cardiovascular death, phenotyping for markers of inflammation may identify the individuals at risk for vascular diseases. Reactive oxygen species (ROS) are key mediators of signaling pathways that underlie vascular inflammation in atherogenesis, starting from the initiation of fatty streak development through lesion progression to ultimate plaque rupture. Various animal models of atherosclerosis support the notion that ROS released from NAD(P)H oxidases, xanthine oxidase, lipoxygenases, and enhanced ROS production from dysfunctional mitochondrial respiratory chain indeed have a causatory role in atherosclerosis and other vascular diseases. Human investigations also support the oxidative stress hypothesis of atherogenesis. This is further supported by the observed impairment of vascular function and enhanced atherogenesis in animal models that have deficiencies in antioxidant enzymes. The importance of oxidative stress in atherosclerosis is further emphasized because of its role as a unifying mechanism across many vascular diseases. The main contraindicator for the role oxidative stress plays in atherosclerosis is the lack of effectiveness of antioxidants in reducing primary endpoints of cardiovascular death and morbidity. However, this lack of effectiveness by itself does not negate the existence or causatory role of oxidative stress in vascular disease. Lack of proven markers of oxidative stress, which could help to identify a subset of population that can benefit from antioxidant supplementation, and the complexity and subcellular localization of redox reactions, are among the factors responsible for the mixed outcomes in the use of antioxidants for the prevention of cardiovascular diseases. To better understand the role of oxidative stress in vascular diseases, future studies should be aimed at using advances in mouse and human genetics to define oxidative stress phenotypes and link phenotype with genotype.

The no-reflow phenomenon in coronary arteries.

No reflow occurs when there is inadequate myocardial perfusion of a given segment of the coronary circulation without evidence of epicardial vessel obstruction. It is a rare but clinically significant condition associated with myocardial infarction and coronary interventions. Diagnosis is usually based on clinical signs of myocardial ischemia (symptoms and/or ECG changes) combined with coronary angiography. Management can be difficult and primarily consists of intracoronary administration of vasodilators. One interesting etiology is thromboembolism and this has become the focus for new potential treatments, including distal embolic protection devices.

Construction and characterization of a recombinant plasminogen activator composed of an anti-fibrin single-chain antibody and low-molecular-weight urokinase.

BACKGROUND: Targeting of plasminogen activators to the fibrin component of a thrombus by antibodies directed against human fibrin can enhance their thrombolytic potency and clot specificity. OBJECTIVES: To overcome the disadvantages of chemical conjugation, we investigated whether the recombinant fusion of a single-chain antibody and a plasminogen activator results in an active bifunctional molecule that might be useful as a therapeutic agent. METHODS: The cDNA of low-molecular-weight single-chain urokinase-type plasminogen activator, comprising amino acids Leu144-Leu411 (scuPA(LMW)), was cloned from human endothelial cells and fused to a single-chain antibody specific for the 7 N-terminal amino acids (beta(15-22)) in the beta-chain of human fibrin (scFv(59D8)). The fusion protein was purified using affinity chromatography with the beta(15-22)-peptide of human fibrin. RESULTS: Purified scFv(59D8)-scuPA(LMW) migrated as a 60-kDa band, which is consistent with a molecule composed of one scFv(59D8) and one scuPA(LMW) moiety. Both functions of the fusion molecule, fibrin-specific binding and plasminogen activation, were fully preserved. In human plasma clots, thrombolysis by scFv(59D8)-scuPA(LMW) is significantly faster and more potent compared with the clinically used urokinase. CONCLUSIONS: ScFv(59D8)-scuPA(LMW) constitutes a new recombinant chimeric plasminogen activator with a significantly enhanced thrombolytic potency and relative fibrin selectivity, that can be produced with modern methods at low cost, large quantities and reproducible activity in Escherichia coli.

Cyclic strain increases protease-activated receptor-1 expression in vascular smooth muscle cells.

Cyclic strain regulates many vascular smooth muscle cell (VSMC) functions through changing gene expression. This study investigated the effects of cyclic strain on protease-activated receptor-1 (PAR-1) expression in VSMCs and the possible signaling pathways involved, on the basis of the hypothesis that cyclic strain would enhance PAR-1 expression, reflecting increased thrombin activity. Uniaxial cyclic strain (1 Hz, 20%) of cells cultured on elastic membranes induced a 2-fold increase in both PAR-1 mRNA and protein levels. Functional activity of PAR-1, as assessed by cell proliferation in response to thrombin, was also increased by cyclic strain. In addition, treatment of cells with antioxidants or an NADPH oxidase inhibitor blocked strain-induced PAR-1 expression. Preincubation of cells with protein kinase inhibitors (staurosporine or Ro 31-8220) enhanced strain-increased PAR-1 expression, whereas inhibitors of NO synthase, tyrosine kinase, and mitogen-activated protein kinases had no effect. Cyclic strain in the presence of basic fibroblast growth factor induced PAR-1 mRNA levels beyond the effect of cyclic strain alone, whereas no additive effect was observed between cyclic strain and platelet-derived growth factor-AB. Our findings that cyclic strain upregulates PAR-1 mRNA expression but that shear stress downregulates this gene in VSMCs provide an opportunity to elucidate signaling differences by which VSMCs respond to different mechanical forces.

p47phox is required for atherosclerotic lesion progression in ApoE(-/-) mice.

NADPH oxidase is upregulated in smooth muscle cells (SMCs) in response to growth factor stimulation, concomitant with increased reactive oxygen species (ROS) production. We investigated the role of ROS production by NADPH oxidase in SMC responses to growth factors and in atherosclerotic lesion formation in ApoE(-/-) mice. SMCs from wild-type, p47phox(-/-), and gp91phox(-/-) mice differed markedly with respect to growth factor responsiveness and ROS generation. p47phox(-/-) SMCs had diminished superoxide production and a decreased proliferative response to growth factors compared with wild-type cells, whereas the response of gp91phox(-/-) SMCs was indistinguishable from that of wild-type SMCs. The relevance of these in vitro observations was tested by measuring atherosclerotic lesion formation in genetically modified (wild-type, p47phox(-/-), ApoE(-/-), and ApoE(-/-)/p47phox(-/-)) mice. ApoE(-/-)/p47phox(-/-) mice had less total lesion area than ApoE(-/-) mice, regardless of whether mice were fed standard chow or a high-fat diet. Together, these studies provide convincing support for the hypothesis that superoxide generation in general, and NADPH oxidase in particular, have a requisite role in atherosclerotic lesion formation, and they provide a rationale for further studies to dissect the contributions of ROS to vascular lesion formation.

Randomized comparison of a strategy of predischarge coronary angiography versus exercise testing in low-risk patients in a chest pain unit: in-hospital and long-term outcomes.

OBJECTIVES: This randomized trial compared a strategy of predischarge coronary angiography (CA) with exercise treadmill testing (ETT) in low-risk patients in the chest pain unit (CPU) to reduce repeat emergency department (ED) visits and to identify additional coronary artery disease (CAD). BACKGROUND: Patients with chest pain and normal electrocardiograms (ECGs) have a low likelihood of CAD and a favorable prognosis, but they often seek repeat evaluations in EDs. Remaining uncertainty regarding their symptoms and diagnosis may cause much of this recidivism. METHODS: A total of 248 patients with no ischemic ECG changes triaged to a CPU were randomized to CA (n = 123) or ETT (n = 125). All patients had a probability of myocardial infarction < or =7% according to the Goldman algorithm, no biochemical evidence of infarction, the ability to exercise and no previous documented CAD. Patients were followed up for > or =1 year and surveyed regarding their chest pain self-perception and utility of the index evaluation. RESULTS: Coronary angiography showed disease (> or =50% stenosis) in 19% and ETT was positive in 7% of the patients (p = 0.01). During follow-up (374+/-61 days), patients with a negative CA had fewer returns to the ED (10% vs. 30%, p = 0.0008) and hospital admissions (3% vs. 16%, p = 0.003), compared with patients with a negative/nondiagnostic ETT. The latter group was more likely to consider their pain as cardiac-related (15% vs. 7%), to be unsure about its etiology (38% vs. 26%) and to judge their evaluation as not useful (39% vs. 15%) (p < 0.01 for all comparisons). CONCLUSIONS: In low-risk patients in the CPU, a strategy of CA detects more CAD than ETT, reduces long-term ED and hospital utilization and yields better patient satisfaction and understanding of their condition.

PTP-epsilon, a tyrosine phosphatase expressed in endothelium, negatively regulates endothelial cell proliferation.

The vascular endothelium is a dynamic interface between the blood vessel and circulating factors and, as such, plays a critical role in vascular events like inflammation, angiogenesis, and hemostasis. Whereas specific protein tyrosine kinases have been identified in these processes, less is known about their protein tyrosine phosphatase (PTP) counterparts. We utilized a RT-PCR/differential hybridization assay to identify PTP-epsilon as a highly abundant endothelial cell PTP. PTP-epsilon mRNA expression is growth factor responsive, suggesting a role for this enzyme in endothelial cell proliferation. Overexpression of PTP-epsilon decreases proliferation by 60% in human umbilical vein endothelial cells (HUVEC) but not in smooth muscle cells or fibroblasts. In contrast, overexpression of PTP-epsilon (D284A), a catalytically inactive mutant, has no significant effect on HUVEC proliferation. These data provide the first functional characterization of PTP-epsilon in endothelial cells and identify a novel pathway that negatively regulates endothelial cell growth. Such a pathway may have important implications in vascular development and angiogenesis.

New tricks for old dogs: nonthrombotic effects of thrombin in vessel wall biology.

Thrombin is a serine protease that potently activates platelets and catalyzes the conversion of fibrinogen to fibrin. Thrombin also exerts direct effects on vascular cells, such as smooth muscle cells, via interactions with members of the protease-activated receptor family. Evidence in several animal models implicates thrombin-mediated signaling events in the response to injury that typifies vascular lesion formation in atherosclerosis and restenosis. In this review, we examine the activation of protease-activated receptors by thrombin, the downstream signaling events mediated by these receptors, and the physiological role of thrombin in vascular cells and vascular disease.

4-hydroxynonenal induces apoptosis, NF-kappaB-activation and formation of 8-isoprostane in vascular smooth muscle cells.

Oxidation of lipids is considered a key feature of atherogenesis. Lipid peroxidation products such as oxidized LDL or the bioactive aldehyde 4-hydroxynonenal (HNE) exert mitogenic effects on vascular smooth muscle cells (VSMC). These effects appear to be concentration-dependent since in addition to our previous reports on growth promotion at lower concentrations we here indicate induction of apoptosis in VSMC by 4-hydroxynonenal (HNE) at higher concentrations (100 micromol/L). In a line with HNE's previously documented effects on key mitogenic signaling elements, we also report on activation by this aldehyde of the redox-sensitive transcription factor NF-kappaB, a key regulator of apoptosis: HNE (1.0 micromol/L) induced DNA-binding of NF-kappaB in VSMC. The effect was inhibited by antioxidants, N-acetylcysteine and pyrrolidine dithio-carbamate. HNE caused phosphorylation but not degradation of the inhibitory subunit IkappaB-alpha. HNE itself acts as an oxidant as was investigated with measurements of 8-isoprostane which ranks among the most valuable available biomarkers of lipid peroxidation: HNE (1.0 micromol/L) increased 8-isoprostane levels in VSMC by 4.5-fold (p < 0.05). Compared to the controls, plasma samples from apoEnull mice exhibited elevated levels of 8-isoprostane (40 pg/mL, 3.2-fold increase) and the combined aldehydes HNE and malonaldehyde (1.5 micromol/L, 2.5-fold increase), (p < 0.05, resp). In addition, immunohistochemistry indicated the presence of HNE-protein adducts in atheroscerlotic lesions of apoEnull mice. Thus HNE is present in atherosclerotic tissue at concentrations that are bioactive in vitro. The data further indicate the involvement of the lipid peroxidation product HNE in atherogenesis.

Aging, oxidative responses, and proliferative capacity in cultured mouse aortic smooth muscle cells.

The cellular mechanisms that contribute to the acceleration of atherosclerosis in aging populations are poorly understood, although it is hypothesized that changes in the proliferative capacity of vascular smooth muscle cells is contributory. We addressed the relationship among aging, generation of reactive oxygen species (ROS), and proliferation in primary culture smooth muscle cells (SMC) derived from the aortas of young (4 mo old) and aged (16 mo old) mice to understand the phenotypic modulation of these cells as aging occurs. SMC from aged mice had decreased proliferative capacity in response to alpha-thrombin stimulation, yet generated higher levels of ROS and had constitutively increased mitogen-activated protein kinase activity, in comparison with cells from younger mice. These effects may be explained by dysregulation of cell cycle-associated proteins such as cyclin D1 and p27Kip1 in SMC from aged mice. Increased ROS generation was associated with decreased endogenous antioxidant activity, increased lipid peroxidation, and mitochondrial DNA damage. Accrual of oxidant-induced damage and decreased proliferative capacity in SMC may explain, in part, the age-associated transition to plaque instability in humans with atherosclerosis.

Shear stress reduces protease activated receptor-1 expression in human endothelial cells.

Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

Thrombin regulates vascular smooth muscle cell growth and heat shock proteins via the JAK-STAT pathway.

The growth-stimulating effects of thrombin are mediated primarily via activation of a G protein-coupled receptor, PAR-1. Because PAR-1 has no intrinsic tyrosine kinase activity, yet requires tyrosine phosphorylation events to induce mitogenesis, we investigated the role of the Janus tyrosine kinases (JAKs) in thrombin-mediated signaling. JAK2 was activated rapidly in rat vascular smooth muscle cells (VSMC) treated with thrombin, and signal transducers and activators of transcription (STAT1 and STAT3) were phosphorylated and translocated to the nucleus in a JAK2-dependent manner. AG-490, a JAK2-specific inhibitor, and a dominant negative JAK2 mutant inhibited thrombin-induced ERK2 activity and VSMC proliferation suggesting that JAK2 is upstream of the Ras/Raf/MEK/ERK pathway. To elucidate the functional significance of JAK-STAT activation, we studied the effect of thrombin on heat shock protein (Hsp) expression, based upon the following: 1) reports that thrombin stimulates reactive oxygen species production in VSMC; 2) the putative role of Hsps in modulating cellular responses to reactive oxygen species; and 3) the presence of functional STAT1/3-binding sites in Hsp70 and Hsp90beta promoters. Indeed, thrombin up-regulated Hsp70 and Hsp90 protein expression via enhanced binding of STATs to cognate binding sites in the Hsp70 and Hsp90 promoters. Together, these results suggest that JAK-STAT pathway activation is necessary for thrombin-induced VSMC growth and Hsp gene expression.

Reactive oxygen species regulate heat-shock protein 70 via the JAK/STAT pathway.

Reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) activate intracellular signal transduction pathways implicated in the pathogenesis of cardiovascular disease. H(2)O(2) is a mitogen for rat vascular smooth muscle cells (VSMCs), and protein tyrosine phosphorylation is a critical event in VSMC mitogenesis. Therefore, we investigated whether the mitogenic effects of H(2)O(2), such as stimulation of extracellular signal-regulated kinase (ERK)2, are mediated via activation of cytoplasmic Janus tyrosine kinases (JAKs). JAK2 was activated rapidly in VSMCs treated with H(2)O(2), and signal transducers and activators of transcription (STAT) STAT1 and STAT3 were tyrosine-phosphorylated and translocated to the nucleus in a JAK2-dependent manner. Inhibition of JAK2 activity with AG-490 partially inhibited H(2)O(2)-induced ERK2 activity, suggesting that JAK2 is upstream of the Ras/Raf/mitogen-activated protein kinase-ERK/ERK mitogenic pathway. Because heat-shock proteins (HSPs) can protect cells from ROS, we investigated the effect of H(2)O(2) on HSP expression. H(2)O(2) stimulated HSP70 expression in a time-dependent manner, and AG-490 abolished H(2)O(2)-induced HSP70 expression. H(2)O(2) activated the HSP70 promoter via enhanced binding of STATs to cognate binding sites in the promoter. Regulation of chaperones such as HSP70 via activation of the JAK/STAT pathway suggests that in addition to its growth-promoting effects, this pathway may help VSMCs adapt to oxidative stress.

In situ hybridization.

In situ hybridization (ISH) is employed principally to detect mRNA within cells and tissues. ISH begins with the synthesis of a nucleic acid probe complementary in sequence to a cellular target. Through incorporation of a radioactive nucleotide into the probe, a cellular target may be visualized using autoradiography. A nonradioactive variation of this technique utilizing digoxigenin has been championed by some researchers, but our experience has been that radioactive probes produce more reliable and accurate results. Although not a difficult technique, ISH incorporates many steps, which must be performed with care and precision to obtain useful results.

Western blotting.

Western blotting is employed for the detection of proteins and other macromolecules immobilized on nitrocellulose membranes. This rapid and sensitive method enables the identification and quantification of a specific protein from cell lysates or a mixture of proteins. Following the resolution on denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE), the constituent polypeptides are transferred electrophoretically to a nitrocellulose membrane (1). The membrane is incubated in a solution containing primary antibody and the resultant antigen-antibody complex is detected by using appropriately labeled ligands. The most common method is based on the enzyme-linked immunodetection of antigen-specific antibodies using anti-IgG secondary antibodies conjugated with either horseradish peroxidase (HRP) or alkaline phosphatase (AP). Visualization of antibodyantigen complex is achieved through the use of an enhanced chemiluminiscent (ECL) method. It is possible to detect as little as 10-50 ng of protein with AP-conjugated secondary antibody and 0.5-1.0 ng of protein with HRP-conjugated secondary antibody using high affinity and high titer primary antibodies.

Angiotensin II induces gene transcription through cell-type-dependent effects on the nuclear factor-kappaB (NF-kappaB) transcription factor.

The vasopressor octapeptide, angiotensin II (Ang II), exerts homeostatic responses in cardiovascular tissues, including the heart, blood vessel wall, adrenal cortex and liver (a major source of circulating plasma proteins). One of the effects of Ang II is to induce expression of regulatory, structural and cytokine genes that play important roles in long-term control of blood pressure, vascular remodeling, cardiac hypertrophy and inflammation. The identification of nuclear signaling pathways and target transcription factors has provide important insight into cellular responses and the spectrum of genes controlled by Ang II. Here we will review how Ang II activates the transcription factors, Activator Protein 1 (AP-1), Signal Transducer and Activator of Transcription (STATs), and Nuclear Factor-kappaB (NF-kappaB). NF-kappaB is of particular interest because it is an important mediator of resynthesis of the Ang II precursor, angiotensinogen AGT. Through this positive feedback loop, long-term changes in the activity of the renin angiotensin system occur. Although NF-kappaB is ubiquitously expressed, surprisingly the mechanism for Ang II-inducible NF-kappaB regulation differs between aortic smooth muscle cells (VSMCs) and hepatocytes. In VSMC, Ang II induces nuclear translocation of cytoplasmic transactivatory NF-kappaB proteins through proteolysis of its inhibitor, IkappaB. By contrast, in hepatocytes, Ang II induces large nuclear isoforms of NF-kappaB1 to bind DNA through a mechanism independent of changes in IkappaB turnover. NF-kappaB activation depends upon the activity of DAG-sensitive PKC isoforms and ROS signaling pathway. These observations indicate that significant differences exist in Ang II signaling depending upon cell-type involved and suggest the possibility that tissue-selective modulation of Ang II effects is possible in the cardiovascular system.

Indications for the presence of an atypical protease-activated receptor on rat platelets.

Activation of the protease-activated receptor (PAR)-1, one of four known PARs (PAR-1 to PAR-4), can be mimicked by thrombin receptor activating peptides (TRAPs) based on the PAR-1 tethered ligand. Interestingly, despite being activatable by thrombin, rodent platelets do not express PAR-1 and thus do not respond to PAR-1-derived TRAPs, indicating different activation mechanisms between human and rodent platelets. Using a rat platelet aggregation model, we determined that TRAPs based on the tethered ligand of PAR-1 fail to activate rat platelet aggregation at concentrations up to 1 mmol/l. In addition, TRAPs inhibit thrombin-mediated rat platelet aggregation, indicating the presence of a modified PAR-1 in this species. In order to determine characteristics of this putative receptor, we tested a panel of synthesized TRAPs based on the rat sequence (R) and human sequence (H) of the PAR-1 tethered ligand for their ability to inhibit thrombin-induced rat platelet aggregation. Peptides R1-9, R4-9, R4-10, and H4-10 inhibited rat platelet aggregation in response to alpha-thrombin [inhibitory concentration (IC) 50% 0.25-1.5 mmol/l]. None of these peptides blocked epinephrine-, collagen-, or arachidonic acid-induced platelet aggregation. Alanine substitution mapping of H4-10 indicated that both Leu4 and Arg5 are essential for inhibition. Inhibition of thrombin's catalytic activity required peptide concentrations tenfold higher than inhibition of platelet aggregation (IC50% 3-5 mmol/l). No prolongation of thrombin clotting time in response to TRAPs was detected at peptide concentrations up to 5 mmol/l. Our data suggest that (1) rat platelets express a PAR-1 subtype, (2) residues Leu4 and Arg5 of the tethered ligand peptide are required for binding to this new receptor, and (3) further analysis of peptide sequences might reveal a novel PAR-1 subtype.

Morphological patterns of death by myocytes in arrhythmogenic right ventricular dysplasia.

There are two forms of nuclear loss from eukaryotic cells: biochemical DNA degradation in apoptosis and nuclear extrusion from the cell body as seen in mammalian erythroblasts. In biopsies of right ventricular myocardium from 8 patients with arrhythmogenic right ventricular dysplasia (ARVD), we found not only a terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine triphosphate nick-end labeling (TUNEL)-positive nucleus in mononuclear myocytes, but also 1 or 2 TUNEL-positive nuclei in multinuclear myocytes. With electron microscopy, we found a nuclear dislocation to the cell periphery, followed by its extrusion into the extracellular space. Both the migration and extrusion of the nuclei of myocytes resemble the morphogenesis of human erythroblasts. Nuclear extrusion from myocytes may be another form of programmed cell death. In support of this possibility, we also found evidence of cytoplasmic degradation in right ventricular myocytes from our ARVD cases, a process similar to one often seen in developmental programmed cell death and differing from typical nuclear apoptosis. In our ARVD cases, we thus found several different patterns of cell death, all associated with initial preservation of the plasmalemma and avoidance of local inflammation. All these features may be different responses to common signals for selective non-necrotic (apoptotic) death of right ventricular myocytes.