Effect of Radiographic Contrast Media on Markers of Complement Activation and Apoptosis (Full title below)

Effect of Radiographic Contrast Media on Markers of Complement Activation and Apoptosis in Patients with Chronic Coronary Artery Disease Undergoing Coronary Angiography

________________________ ABSTRACT: The effects of radiographic contrast media on markers of complement activation and apoptosis in patients with chronic coronary artery disease (CAD) are unknown. The purpose of this study was to assess the comparative effects of ionic high-osmolar and non-ionic iso-osmolar radiographic contrast media on plasma markers of complement activation and apoptosis in patients with chronic CAD undergoing coronary angiography. Forty-four patients undergoing coronary angiography for chronic CAD were randomly assigned to receive the ionic high-osmolar radiographic contrast agent diatrizoate (Group A), or the non-ionic iso-osmolar contrast agent iodixanol (Group B) during angiography. Complement component 5 (C5a) and apoptotic markers sFas and sFasL were measured just prior to angiography and 1 hour after completion of angiography. Comparison of mean pre- and post-angiography plasma marker levels showed significantly greater increases in plasma levels in Group A than in Group B of C5a (29.30 ± 5.45 ng/ml for Group A and 0.47 ± 0.70 ng/ml for Group B (p Methods Patient selection. Consecutive patients with chronic stable angina pectoris and/or a positive exercise or pharmacologic stress test were referred for coronary angiography and were considered for inclusion in the study. Patients with acute coronary syndromes, subjects with coronary artery stenoses 1.0 mg/dl and patients with inflammatory diseases were excluded from the study. Protocol. Patients undergoing coronary angiography were randomly assigned to two groups. Group A received diatrizoate, a high-osmolar ionic radiographic contrast medium. Group B received iodixanol, a non-ionic iso-osmolar radiographic contrast medium. All patients underwent coronary angiography and left ventriculography using the Judkins technique. Left ventriculography was performed in the 30º right anterior oblique and 60º left anterior oblique view. Coronary stenoses were imaged in at least two orthogonal views. A normal arterial segment was identified immediately proximal and distal to the stenotic lesion. Measurements were conducted with an electronic caliper. In a similar fashion, minimal stenosis diameter was measured and its severity was expressed as a percentage reduction of normal diameter. Significant CAD was defined as at least a 50% reduction of minimal lumen diameter in at least one major coronary artery. Patients received diatrizoate (amidotrizoate meglucamine and amidotrizoate sodium) or iodixanol in accordance with the randomization sequence. All patients received unfractionated heparin intravenously during coronary angiography. Plasma levels of activated complement component 5 (C5a), as well as Fas (sFas) and Fas ligand (sFasL), soluble makers of apoptosis, were derived from peripheral blood samples drawn just prior to coronary angiography and 60 minutes after the final injection of the radiocontrast medium. Standard peripheral venipucture techniques were employed. Blood samples for determination of C5a were collected in pyrogen-free blood collection tables (Becton-Dickinson), with EDTA as the anticoagulant. The samples were centrifuged within 15 minutes of collection using a centrifuge with an integrated refrigeration system (at 4ºC, 3,000 g for 20 minutes). Plasma sample were maintained at -80ºC in multiple aliquots until analysis. Each aliquot was thawed and analyzed once; none were refrozen. C5a was measured using an enzyme-linked immuno-absorbent monoclonal antibody assay (BD Biosciences, San Diego, California), with a lower detection of 0.06 ng/ml. Cross-reactivity to C3a was Results Patient characteristics. The study population consisted of 44 patients: 22 in Group A, and 22 in Group B. Table 1 summarizes gender distribution, age, cardiovascular risk factors, distribution of CAD, volume of radiocontrast media used, as well as pre-angiographic plasma levels of markers, complement activation and apoptosis in both groups. Forty-one of 43 patients had coronary artery stenoses > 50%. There were no complications of coronary angiography except for the development of small hematomas (Discussion This study demonstrates a differential effect of ionic hyperosmolar and non-ionic iso-osmolar radiographic contrast media on plasma markers of complement activation and apoptosis in patients with chronic CAD undergoing coronary angiography. Specifically, the use of the ionic hyperosmolar agent (diatrizoate) was associated with significantly greater stimulation of serum C5a, sFas and sFasL levels than use of a non-ionic iso-osmolar agent (iodixanol). Among the factors of the complement cascade, C5a exhibits pro-inflammatory effects by binding to receptors of atherosclerotic lesions. C5a is one of the most potent chemotactic factors for monocytes, mast cells and T lymphocytes. It enhances the release of tumor necrosis factor alpha and interleukin-1 from macrophages as well was the generation of reactive oxygen species.7 Our findings concerning C5a are consistent with previous studies reporting the induction of pro-inflammatory responses by radiographic contrast media. A study by Laskey et al reported a similar differential effect of ionic and non-ionic radiographic contrast media on a variety of inflammatory markers (interleukin-6, tumor necrosis factor alpha-1 and tumor necrosis factor alpha-2).8 Neutrophil degranulation resulting in the release of neutrophil myeloperoxidase has also been reported in response to infusion of radiographic contrast media.9 Activation of the complement pathways in response to radiographic contrast media has been demonstrated previously.5,10,11 However, to our knowledge, this is the first report of a differential effect of high- and iso-osmolar radiographic contrast media on plasma inflammatory and apoptotic markers in patients with chronic CAD undergoing coronary angiography. This is a particularly relevant clinical finding since plasma levels of C5a have been shown to be related to cardiovascular risk independent of other inflammatory markers (hs-CRP, serum amyloid A, fibrinogen), thus adding to their predictive value.7 Fas (APO-1 or CD95) is a cell-surface receptor that transduces apoptotic signals from Fas ligand (FasL).12 It is a member of the tumor necrosis factor receptor superfamily (TNFRSF). It shares a cytoplasmic motif with TNF RI, referred to as the “death domain,” which binds cytoplasmic signaling molecules to trigger the cytoplasmic apoptotic signal.12,13 FasL, the physiological agonist for Fas, is also a transmembrane protein12,13 with homology to the tumor necrosis factor family in its extracellular domain. Mice with mutations in either Fas or FasL exhibit accumulation of activated lymphocytes and classical autoimmune symptoms, suggesting that a major function of Fas-mediated apoptosis is the elimination of activated immune cells from the peripheral circulation.14 Stimulation of sFas and sFasL may have cardiovascular implications. It has been shown that serum sFasL concentration is elevated with atherosclerosis and inflammatory disease in patients with hypertension.15 Moreover, the sFas/sFasL system is upregulated in patients with myocardial dysfunction in proportion to the severity of heart failure.16 Given these meaningful links of this signaling pathway to cardiovascular disease, it is of particular interest that in our study, high- and iso-osmolar radiographic contrast media appeared to exert a differential effect on the levels of sFas and sFasL when used for coronary angiography in patients with CAD. Little information exists concerning the release kinetics and metabolism of C5a, sFas and sFasL in cardiac tissue. The differential effect of diatrizoate and iodixanol on plasma levels of C5a, sFas and sFasL suggests that their generation cannot be explained by release kinetics alone. Study limitations. One limitation of the present study is the fact that the interventional cardiologists performing coronary angiography were not blinded as to the type of radiographic contrast medium used. However, the personnel involved in the handling of blood samples, laboratory measurements, angiographic analysis and data processing were blinded to patient data and type of radiographic contrast medium used. Whether elevation of plasma levels of C5a, sFas and sFasL persisted beyond 1 hour after infusion of radiographic contrast media is uncertain, as no measurements were made after this time. This study was designed to assess the comparative effects of high- and iso-osmolar radiographic contrast mediation plasma markers of complement activation and apoptosis before and after coronary angiography in patients with chronic CAD. Whether the observed differences will translate into differential short- or long-term clinical outcomes is uncertain and is beyond the scope of this investigation. Conclusion Use of the ionic hyperosmolar radiographic contrast agent diatrizoate was associated with significantly greater stimulation of plasma C5a, sFas and sFasL levels than use of the non-ionic iso-osmolar contrast agent iodixanol in patients with chronic CAD undergoing coronary angiography. The results suggest that ionic hyperosmolar radiographic contrast media produce a more robust inflammatory and apoptotic milieu than non-ionic osmolar radiographic contrast media. ___________________________ From the Department of Cardiology, Athens General Hospital, Athens, Greece and the *Division of Cardiology, University of Missouri-Columbia School of Medicine, Columbia, Missouri. The authors report no financial relationships or conflicts of interest regarding the content herein. Manuscript submitted March 2, 2009, provisional acceptance given March 12, 2009, and final version accepted March 30, 2009. Address for correspondence: Martin A. Alpert, MD, Room CE338, University of Missouri Health Sciences Center, Five Hospital Drive, Columbia, MO 65212. E-mail: alpertm@health.missouri.edu

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