Nezam Haider

Localization of Apoptotic Macrophages at the Site of Plaque Rupture in Sudden Coronary Death

Although apoptosis is a well-recognized phenomenon in chronic atherosclerotic disease, its role in sudden coronary death, in particular, acute plaque rupture is unknown. Culprit lesions from 40 cases of sudden coronary death were evaluated. Cases were divided into two mechanisms of death: ruptured plaques with acute thrombosis (n = 25) and stable plaques with and without healed myocardial infarction (n = 15). Apoptotic cells were identified by staining of fragmented DNA and confirmed in select cases by gold conjugate labeling combined with ultrastructural analysis. Additional studies were performed to examine the expression and activation of two inducers of apoptosis, caspases-1 and -3. Ruptured plaques showed extensive macrophage infiltration of the fibrous cap, in particular at rupture sites contrary to stable lesions, which contained fewer inflammatory cells. Among the culprit lesions, the overall incidence of apoptosis in fibrous caps was significantly greater in ruptured plaques (P < 0.001) and was predominantly localized to the CD68-positive macrophages. Furthermore, apoptosis at plaque rupture sites was more frequent than in areas of intact fibrous cap (P = 0. 028). Plaque rupture sites demonstrated a strong immunoreactivity to caspase-1 within the apoptotic macrophages; staining for caspase-3 was weak. Immunoblot analysis of ruptured plaques demonstrated caspase-1 up-regulation and the presence of its active p20 subunit whereas stable lesions showed only the precursor; nonatherosclerotic control segments were negative for both precursor and active enzyme. These findings demonstrate extensive apoptosis of macrophages limited to the site of plaque rupture. The proteolytic cleavage of caspase-1 in ruptured plaques suggests activation of this apoptotic precursor. Whether macrophage apoptosis is essential to acute plaque rupture or is a response to the rupture itself remains to be determined.

Role of Caspases in Ox-LDL–Induced Apoptotic Cascade in Human Coronary Artery Endothelial Cells

Abstract— Oxidized low-density lipoprotein (ox-LDL) induces apoptosis in endothelial cells. However, steps leading to ox-LDL–induced apoptosis remain unclear. We examined the role of ox-LDL and its newly described receptor LOX-1 in the expression of intracellular pro- and antiapoptotic proteins and caspase pathways in human coronary artery endothelial cells (HCAECs). Cells were cultured and treated with different concentrations (10 to 80 &mgr;g/mL) of ox-LDL for different times (2 to 24 hours). Ox-LDL induced apoptosis in HCAECs in a concentration- and time-dependent manner. Ox-LDL also activated caspase-9 and caspase-3, but not caspase-8. After ox-LDL treatment, there was a significant release of activators of caspase-9, including cytochrome c and Smac from mitochondria to cytoplasmic compartment, and their release was not affected by treatment of cells with inhibitors of either caspase-8 or caspase-9. Ox-LDL also decreased expression of antiapoptotic proteins Bcl-2 and c-IAP (inhibitory apoptotic protein)-1, which are involved in the release of cytochrome c and Smac and activation of caspase-9, in a concentration- and time-dependent manner. On the other hand, ox-LDL did not change the expression of Fas-associated death domain-like interleukin-1&bgr;–converting enzyme-inhibitory protein (FLIP) and proapoptotic protein Fas, which are required for the activation of caspase-8. Further, ox-LDL did not cause the truncation of Bid, which implies the activation of caspase-8. In other experiments, pretreatment of HCAECs with the caspase-9 inhibitor z-LEHD-fmk, but not the caspase-8 inhibitor z-IETD-fmk, blocked ox-LDL–induced activation of caspase-3 and apoptosis. As expected, pretreatment with the caspase-3 inhibitor DEVD-CHO inhibited ox-LDL–induced activation of caspase-3 and resultant apoptosis. The proapoptotic effects of ox-LDL were mediated by its receptor LOX-1, because pretreatment of HCAECs with antisense-LOX-1, but not sense-LOX-1, blocked these effects of ox-LDL. These findings suggest that ox-LDL through its receptor LOX-1 decreases the expression of antiapoptotic proteins Bcl-2 and c-IAP-1. This is followed by activation of apoptotic signaling pathway, involving release of cytochrome c and Smac and activation of caspase-9 and then caspase-3.

Annexin A5 Uptake in Ischemic Myocardium: Demonstration of Reversible Phosphatidylserine Externalization and Feasibility of Radionuclide Imaging

Ischemic insult to the myocardium is associated with cardiomyocyte apoptosis. Because apoptotic cell death is characterized by phosphatidylserine externalization on cell membrane and annexin-A5 (AA5) avidly binds to phosphatidylserine, we hypothesized that radiolabeled AA5 should be able to identify the regions of myocardial ischemia. Methods: Models of brief myocardial ischemia by the occlusion of the coronary artery for 10 min (I-10) and reperfusion for 180 min (R-180) for the detection of phosphatidylserine exteriorization using 99mTc-labeled AA5 and γ-imaging were produced in rabbits. 99mTc-AA5 uptake after brief ischemia was compared with an I-40/R-180 infarct model. Histologic characterization of both myocardial necrosis and apoptosis was performed in ischemia and infarct models. Phosphatidylserine exteriorization was also studied in a mouse model, and the dynamics and kinetics of phosphatidylserine exposure were assessed using unlabeled recombinant AA5 and AA5 labeled with biotin, Oregon Green, or Alexa 568. Appropriate controls were established. Results: Phosphatidylserine exposure after ischemia in the rabbit heart could be detected by radionuclide imaging with 99mTc-AA5. Pathologic characterization of the explanted rabbit hearts did not show apoptosis or necrosis. Homogenization and ultracentrifugation of the ischemic myocardial tissue from rabbit hearts recovered two thirds of the radiolabeled AA5 from the cytoplasmic compartment. Murine experiments demonstrated that the cardiomyocytes expressed phosphatidylserine on their cell surface after an ischemic insult of 5 min. Phosphatidylserine exposure occurred continuously for at least 6 h after solitary ischemic insult. AA5 targeted the exposed phosphatidylserine on cardiomyocytes; AA5 was internalized into cytoplasmic vesicles within 10–30 min. Twenty-four hours after ischemia, cardiomyocytes with internalized AA5 had restored phosphatidylserine asymmetry of the sarcolemma, and no detectable phosphatidylserine remained on the cell surface. The preadministration of a pan-caspase inhibitor, zVAD-fmk, prevented phosphatidylserine exposure after ischemia. Conclusions: After a single episode of ischemia, cardiomyocytes express phosphatidylserine, which is amenable to targeting by AA5, for at least 6 h. Phosphatidylserine exposure is transient and internalized in cytoplasmic vesicles after AA5 binding, indicating the reversibility of the apoptotic process.

Apoptosis in atherosclerosis. Does it contribute to plaque instability

Several reports have demonstrated apoptosis in the advanced human atheroma. Most clinical events however, are precipitated by plaque rupture, to a lesser extent erosion, and the development of occlusive thrombi. Whether the extent of apoptosis can influence lesion stability is not precisely known, however, there is emerging data supporting this role. Obvious difficulties arise when studying apoptosis in atherosclerotic plaques because of the complex nature of the disease and lack of an experimental model of plaque instability. This article applies a systematic approach to discuss the issue of apoptosis in context of early disease to complex symptomatic lesions that may become fatal.

Radiolabeled Monocyte Chemotactic Protein 1 for the Detection of Inflammation in Experimental Atherosclerosis

Chemotactic peptides, such as Monocyte Chemotactic Protein 1 (MCP-1), play a key role in transendothelial migration of mononuclear cells during the development and progression of atherosclerotic disease. Because atherosclerotic plaques that are precursors of acute coronary events harbor abundant macrophage infiltration, we hypothesized that the detection of a high concentration of MCP-1 receptors on inflammatory cells should noninvasively identify vulnerable plaques. Methods: Atherosclerotic lesions were induced by balloon deendothelialization of the abdominal aorta, which was followed by a 0.5% cholesterol diet for 16 wk in 7 New Zealand White rabbits; 5 unmanipulated rabbits, fed normal chow for 16 wk, were used as controls. Radionuclide imaging was performed immediately after intravenous 99mTc-labeled MCP-1 administration and 3 h later. At the end of imaging session, aortas were explanted and submitted for estimation of quantitative MCP-1 uptake (in percentage injected dose per gram, %ID/g) and pathologic characterization. Results: Atherosclerotic lesions were clearly visible in all hyperlipidemic animal γ-imaging. No tracer uptake was seen in the control rabbits. The mean quantitative MCP-1 uptake in atherosclerotic lesions was 4-fold higher than that of the aortic specimens from the control rabbits (0.065 ± 0.005 vs. 0.016 ± 0.006; P < 0.0001). Histology confirmed a strong correlation between MCP-1 uptake and the number of macrophages in American Heart Association type II−IV lesions (r = 0.87, P < 0.0001). Conclusion: Noninvasive radionuclide imaging of inflammation is feasible by MCP-1 in experimentally induced atherosclerosis. It is proposed that detection of the extent of inflammation in advanced atherosclerotic plaques may allow identification of unstable plaques.

Molecular imaging of human ACE-1 expression in transgenic rats.

OBJECTIVES The aim of this study was to develop a molecular imaging strategy that can monitor myocardial angiotensin-converting enzyme (ACE)-1 upregulation as a function of progressive heart failure. BACKGROUND High-affinity technetium-99m-labeled lisinopril (Tc-Lis) has been shown to specifically localize in tissues that express ACE in vivo, such as the lungs. Whether Tc-Lis can also detect upregulation of ACE in the heart, by external in vivo imaging, has not been established. METHODS Twenty-one ACE-1 over-expressing transgenic (Tg) and 18 wild-type control rats were imaged using in vivo micro single-positron emission computed tomography (SPECT)-computed tomography (CT) at 10, 30, 60, and 120 min after Tc-Lis injection. A subgroup of rats received nonradiolabeled (cold) lisinopril before the Tc-Lis injection to evaluate nonspecific binding. After imaging, the rat myocardium was explanted, ex vivo images were acquired, and percent injected dose per gram gamma-well was counted, followed by an assessment of enzyme-linked immunosorbent assay-verified ACE activity and messenger ribonucleic acid expression. RESULTS On micro SPECT-CT, myocardial ACE-1 uptake was best visualized in Tg rats at 120 min after Tc-Lis injection. The quantitative uptake of Tc-Lis in the myocardium was 5-fold higher in mutant Tg than in control rats at each time point after tracer injection. The percent injected dose per gram uptake was 0.74 ± 0.13 in Tg myocardium at 30 min and was reduced substantially to 0.034 ± 0.003% when pre-treated with cold lisinopril (p = 0.029). Enzyme activity assay showed a >30-fold higher level of ACE-1 activity in the myocardium of Tg rats than in controls. The ACE-1 messenger ribonucleic acid was quantified, and lisinopril was found to have no effect on ACE-1 gene expression. CONCLUSIONS The Tc-Lis binds specifically to ACE, and the activity can be localized in Tg rat hearts that over-express human ACE-1 with a signal intensity that is sufficiently high to allow external imaging. Such a molecular imaging strategy may help identify susceptibility to heart failure and may allow optimization of pharmacologic intervention.

Adenine nucleotide translocase 1 deficiency results in dilated cardiomyopathy with defects in myocardial mechanics, histopathological alterations, and activation of apoptosis.

OBJECTIVES the aim of this study was to test the hypothesis that chronic mitochondrial energy deficiency causes dilated cardiomyopathy, we characterized the hearts of age-matched young and old adenine nucleotide translocator (ANT)1 mutant and control mice. BACKGROUND ANTs export mitochondrial adenosine triphosphate into the cytosol and have a role in the regulation of the intrinsic apoptosis pathway. Mitochondrial energy deficiency has been hypothesized, on the basis of indirect evidence, to be a factor in the pathophysiology of dilated cardiomyopathies. Ant1 inactivation should limit adenosine triphosphate for contraction and calcium transport, thereby resulting in early cardiac dysfunction with later dilation and heart failure. METHODS we conducted a multiyear study of 73 mutant (Ant1-/-) and 57 control (Ant1+/+) mice, between the ages of 2 and 21 months. Hearts were characterized by cardiac anatomy, echocardiographic imaging with velocity vector analysis, histopathology, and apoptosis assays. RESULTS the Ant1-/- mice developed a distinctive concentric dilated cardiomyopathy, characterized by substantial myocardial hypertrophy and ventricular dilation, with cardiac function declining earlier in age as compared to control mice. Left ventricular circumferential, radial, and rotational mechanics were reduced even in the younger mutants with preserved systolic function. Histopathologic analysis demonstrated increased myocyte hypertrophy, fibrosis, and calcification in the mutant mice as compared with control mice. Furthermore, increased cytoplasmic cytochrome c levels and caspase 3 activation were observed in the mutant mice. CONCLUSIONS our results demonstrate that mitochondrial energy deficiency is sufficient to cause dilated cardiomyopathy, confirming that energy defects are a factor in this disease. Energy deficiency initially leads to early mechanical dysfunction before a decline in left ventricular systolic function. Chronic energy deficiency with age then leads to heart failure. Our results now allow us to use the Ant1-/- mouse model for testing new therapies for ANT1 mutant patients.

Effect of an antimicrobial agent on atherosclerotic plaques: assessment of metalloproteinase activity by molecular imaging.

OBJECTIVES Technetium-99m-labeled matrix metalloproteinase inhibitor (MPI) was used for the noninvasive assessment of matrix metalloproteinase (MMP) activity in atherosclerotic plaques after minocycline (MC) intervention. BACKGROUND MMP activity in atherosclerosis contributes to plaque instability. Some antimicrobial agents may attenuate MMP activity. METHODS Atherosclerotic lesions were produced in 38 rabbits with a high cholesterol diet for 4 months; 5 groups of rabbits, in the fourth month, received fluvastatin (FS) (n = 6), low-dose MC (n = 7), high-dose MC (n = 7), a combination of low-dose MC and FS (n = 6), or no intervention (n = 12); 8 unmanipulated rabbits were used as disease controls. Micro-single-photon emission computed tomography imaging was performed in all animals after intravenous MPI administration, followed by pathologic characterization of the aorta. A cell culture study evaluated the effect of MC on MMP production by activated human monocytes. RESULTS MPI uptake was visualized best in untreated atherosclerotic animals (percent injected dose per gram MPI uptake, 0.11 +/- 0.04%). MPI uptake was reduced in the FS (0.06 +/- 0.01%; p < 0.0001), high-dose MC (0.05 +/- 0.01%; p < 0.0001), and MC-FS (0.05 +/- 0.005%; p < 0.0001) groups. Low-dose MC did not resolve MPI uptake significantly (0.08 +/- 0.02; p = 0.167). There was no incremental benefit of the combination of MC and FS. MPI uptake showed a significant correlation with plaque MMP-2, and MMP-9 activity. MMP-9 release from tumor necrosis factor-alpha-activated macrophages was abrogated by incubation with MC. CONCLUSIONS Molecular imaging of MMP activity in atherosclerotic plaque allows for the study of the efficacy of therapeutic interventions. MC administration resulted in substantial reduction in plaque MMP activity and histologically verified plaque stabilization. MC was found to be equally effective as FS.

Adrenergic Excess, hNET1 Down-Regulation, and Compromised mIBG Uptake in Heart Failure: Poverty in the Presence of Plenty

An increased adrenergic drive contributes to augmented myocyte contractility and altered peripheral vasoreactivity in heart failure (HF) and appears intuitively compensatory ([1][1]). The increased adrenergic contents in the synaptic cleft eventually result in desensitization of the post-synaptic

Concurrent upregulation of endogenous proapoptotic and antiapoptotic factors in failing human hearts

Background Despite widespread activation of proapoptotic stimuli and mediators, the degree of apoptosis in failing hearts is not very high. Endogenous antiapoptotic mechanisms are thought to be triggered by the heart-failure process. We investigated whether activation of endogenous apoptosis inhibitors plays a part when death receptor and mitochondrial apoptotic pathways have been triggered.Methods We evaluated various proapoptotic and antiapoptotic factors in myocardial tissue specimens obtained from normal and explanted end-stage ischemic and dilated cardiomyopathic hearts. Caspases (CASPs) 3, 8 and 9, total and activated Bcl-2 homology domain 3-interacting domain death agonist, the X-linked inhibitor of apoptosis (XIAP), and DNA fragmentation factor (DFF) proteins were analyzed by western blotting. Expression of messenger RNA was measured by reverse-transcription polymerase chain reaction for the XIAP, DIABLO, CFLAR and DFF genes. We also assessed CASP3, CASP8 and CASP9 and DFF activity. Cytochrome c1 localization in myocytes was analyzed by immunohistochemistry and immunoelectron microscopy.Results We collected myocardial tissue from eight cardiomyopathic hearts and five normal hearts. Cytochrome c1 was released from mitochondria into the cytosol in the cardiomyopathic hearts but CASP9 was not activated. CASP8 activity was increased compared with that in normal myocardium. Although CASP3 was cleaved, activity was not greatly increased because of an increase in XIAP and decrease in DIABLO expression. DFF proteins were conspicuously absent.Conclusions Concurrent upregulation of endogenous antiapoptotic mechanisms can interrupt the apoptotic cascade and prevents cell loss despite the presence of multiple proapoptotic factors. This period might offer a therapeutic window for restoration of myocardial function in heart failure.