Could lymphocyte caspase-3 activity predict atherosclerotic plaque vulnerability?

JMB 2010; 29 (2) DOI: 10.2478/v10011-010-0010-2 UDK 577.1 : 61 ISSN 1452-8258 JMB 29: 73 –77, 2010 Original paper Originalni nau~ni rad COULD LYMPHOCYTE CASPASE-3 ACTIVITY PREDICT ATHEROSCLEROTIC PLAQUE VULNERABILITY? MOGU]NOST PREDVI\ANJA VULNERABILNOSTI ATEROSKLEROTSKOG PLAKA POMO]U AKTIVNOSTI LIMFOCITNE KASPAZE-3 Tatjana Risti}1, Vladan ]osi}1, Predrag Vlahovi}1, Marina Deljanin-Ili}2, Vidosava B. \or|evi}3 1Centre 2Institute for Medical Biochemistry, Clinical Centre, Ni{, Serbia for Cardiovascular and Rheumatic Diseases, Ni{ka Banja, Serbia 3Institute of Biochemistry, Medical Faculty, Ni{, Serbia Summary: Apoptotic cell death may play a critical role in a variety of cardiovascular diseases, especially in those developing on the basis of atherosclerosis. The goal of this study was to compare the activity of caspase-3 in different forms of ischemic heart disease and to correlate caspase-3 activity with inflammatory and lipid markers as well as risk factors. This enzyme activity was determined in peripheral blood mononuclear cells (PBMC) of 30 patients with stable angina pectoris (SAP), 27 with unstable angina (USAP), 39 with acute ST-elevation myocardial infarction (STEMI) and 27 healthy volunteers by a colorimetric commercially available ELISA method. In the SAP group caspase-3 activity was 0.093±0.036 mmol/mg protein, in patients with STEMI it was 0.110±0.062 mmol/mg protein, and both values were insignificantly higher in comparison with controls (0.092±0.022 mmol/mg protein). In PBMC of USAP patients caspase-3 activity (0.122±0.062 mmol/mg protein) was significantly higher (p<0.05) compared to the control group. In SAP patients caspase-3 activity showed a significant positive correlation with triglicerydes (p<0.05). Caspase-3 activity may be a valid parameter for assessing the atherosclerotic plaque activity, and a new target for therapeutic intervention. Keywords: ischemic heart disease, caspase-3, inflammatory markers, lipid markers Kratak sadr`aj: Smrt }elija apoptozom ima zna~ajnu ulogu u razli~itim kardiovaskularnim oboljenjima, posebno u onim koja se razvijaju na temelju ateroskleroze. Cilj ove studije bilo je pore|enje aktivnosti kaspaze-3 u razli~itim oblicima ishemijske bolesti srca i koreliranje njene aktivnosti sa inflamatornim, lipidnim markerima i faktorima rizika. Aktivnost kaspaze-3 odre|ivana je u mononuklearnim }elijama periferne krvi (M]PK) kolorimetrijskim komercijalnim ELISA testom. Enzimska aktivnost odre|ivana je kod 30 pacijenata sa stabilnom anginom pektoris (SAP), 27 sa nestabilnom anginom pektoris (NSAP), 39 sa akutnim infarktom miokarda sa elevacijom ST segmenta (STEMI) i 27 zdravih dobrovoljaca. U M]PK pacijenata sa SAP aktivnost enzima bila je 0,093±0,036 mmol/mg proteina, a kod pacijenata sa STEMI 0,110±0,062 mmol/mg proteina, i obe vrednosti su bile statisti~ki nezna~ajno vi{e u pore|enju sa kontrolnom grupom (0,092±0,022 mmol/mg proteina). U M]PK pacijenata sa NSAP aktivnost kaspaze-3 (0,122±0,062 mmol/mg proteina) bila je statisti~ki zna~ajno vi{a (p<0,05) u pore|enju sa kontrolnom grupom. U grupi pacijenata sa SAP postoji statisti~ki zna~ajna pozitivna korelacija kaspaze-3 i triglicerida (p<0,05). Aktivnost kaspaze-3 mo`e biti validan parametar u pra}enju aktivnosti ateroskleroti~nog plaka i nova meta za terapijske intervencije. Klju~ne re~i: ishemijska bolest srca, kaspaza-3, inflamatorni markeri, lipidni markeri Introduction Address for correspondence: Tatjana Risti} Center for Medical Biochemistry, Clinical Center, Ni{ Bulevar dr Zorana \in|i}a 48, 18000 Ni{, Serbia Phone: +381 18 4232210, +381 18 217261 e-mail: ristictªbankerinter.net Ischemic heart disease occurs on the basis of atherosclerosis (1). Atherosclerosis is a chronic inflammatory disease characterized by endothelial dysfunction and lipid and monocyte-derived macrophages accumulation within the vessel wall (2). Both the vessel wall and blood derived cells may undergo necrotic or apoptotic cell death during atherogenesis. 74 Risti} et al.: Caspase-3 activity and atherosclerosis Apoptosis occurs in atherosclerotic coronary arteries, and the significance of apoptosis depends on the stage of the plaque, localization and the cell type involved. In initial lesions, only a few cells undergo apoptosis. In advanced lesions, many cells die by programmed cell death (PCD). Apoptosis can be initiated by one of two pathways: the death receptor (extrinsic) pathway or mitochondrial activation of cytochrome c (intrinsic) pathway (3). In both pathways caspase-3 is activated as an executor molecule of apoptosis leading to cleavage of DNA and cell death (4). Caspases are expressed as proenzymes (5) and are activated following proteolytic processing and association of the large and small subunits (6). The activation of these enzymes may occur autocatalytically or in a cascade (7). The distribution of caspase-3 is consistent with its role as a downstream caspase that targets proteins including lamin A and B in the nuclear lamina, poly (ADP-ribose) polymerase and nuclear endonucleases (8) and actin as a cytoskeletal protein. Caspase-3 can cleave both the antiapoptotic protein bcl-2, releasing a fragment that promotes apoptosis, and ICAD (inhibitor of caspaseactivated deoxyribonuclease), releasing CAD, the nuclease that induces DNA fragmentation. In addition, caspase-3 also cleaves gelsolin, an actin-associated protein that may have influence on DNase activity (9). Caspase-3 is a cysteine protease located in both the cytoplasm and mitochondrial intermembrane space. In resting cells, a subset of caspase-3 zymogens is S-nitrosylated at the active site cysteine thus inhibiting enzyme activity. The majority of mitochondrial, but not cytoplasmatic, caspase-3 zymogens contain this inhibitory modification (10). During Fasinduced apoptosis, caspases are denitrosylated, allowing the catalytic site to function. Therefore, apoptosis is regulated by intracellular nitric oxide (NO) production. Since endothelial dysfunction may be caused by an accelerated inactivation of NO by reactive oxygen species (11), this may lead to the increased caspase-3 activation and cell death. The role of apoptosis in atherosclerotic plaque has been studied in animal and human tissue specimens (12). Caspase-3 activity and apoptosis rates are low in the normal vasculature (13). Apoptosis rate in atherosclerotic lesions is higher than in normal vasculature and correlates with disease activity: higher levels of apoptosis were seen in atherectomy specimens from patients with unstable angina compared to those with stable angina and among those with symptomatic vs. asymptomatic carotid plaques (14). Fas/APO-1 is documented in foam cells whose source are macrophages or smooth muscle cells (SMC). Also, CD3positive T lymphocytes found around foam cells have been found to express Fas/APO-1 (15). The goal of this study was to compare the activity of caspase-3 in different forms of ischemic heart disease and to correlate (...truncated)