Faculty Bibliography

OBJECTIVES/OBJECTIVE:A pressure overload model was developed to simulate aortic stenosis and assess caspase activity during the transition to heart failure. BACKGROUND:Cardiomyocyte apoptosis is implicated in the pathogenesis of heart failure, and caspase activation is central to this pathophysiological process. METHODS:A total of 10 sheep were banded with variable aortic constriction devices, progressively inflated to increase left ventricular (LV) afterload. Serial LV endomyocardial biopsy samples were obtained to measure caspase activity and presence of apoptosis. RESULTS:Over the first 3 to 4 weeks, hypertrophy developed in the sheep (LV mass index 90.8 +/- 4.9 g/m2 vs. 44.0 +/- 3.0 g/m2, p < 0.01), followed by gradual dilatation of the left ventricle (diastolic LV internal diameter 4.23 +/- 0.08 cm vs. 3.39 +/- 0.07 cm, p < 0.01). Ventricular function remained stable until 7 to 8 weeks after banding, when there was significant deterioration (fractional shortening 18.3 +/- 2.4% vs. 46.9 +/- 2.6%, p < 0.01), associated with clinical heart failure. Serial LV endomyocardial biopsy samples were obtained at each echocardiographically defined stage (LV hypertrophy, LV dilation, and LV failure). Activity of caspases-3, -8, and -9 (measured by specific fluorogenic peptide substrates and immunohistochemistry) increased progressively, particularly with the onset of myocardial dysfunction (caspase-3 7.92 +/- 1.19 vs. 1.00 +/- 0.15, caspase-8 1.94 +/- 0.21 vs. 1.00 +/- 0.04, caspase-9 5.87 +/- 0.97 vs. 1.00 +/- 0.18 relative fluorescent units, p < 0.05). No evidence of deoxyribonucleic acid (DNA) fragmentation, however, was identified by immunohistochemical assays. CONCLUSIONS:Activation of cardiomyocyte caspase enzymes occurs during the transition to heart failure, without completion of apoptotic DNA fragmentation. Increased activity of caspase-8 and -9 suggests both mitochondrial and death-receptor mediated pathways are involved in this pathological process. Further knowledge of these pathways may stimulate development of apoptosis-based strategies for slowing progression of heart failure in aortic stenosis patients.

UNLABELLED:Transgenic mice such as apolipoprotein E-deficient (apoE(-/-)) and low-density-lipoprotein receptor-deficient (LDLR(-/-)) mice exhibit hypercholesterolemia and develop complex atherosclerotic lesions similar to those seen in humans. Radiolabeled annexin A5 has been successfully used to noninvasively image experimental and clinical atherosclerotic disease. We evaluated the feasibility of annexin A5 imaging in transgenic apoE(-/-) and LDLR(-/-) mice with or without a cholesterol diet. METHODS:Thirty-three mice (mean age, 62 +/- 0.9 wk old) were used. Of these 33 mice, apoE(-/-) mice with the cholesterol diet for 4 mo (n = 5) and without the cholesterol diet (n = 8) and LDLR(-/-) mice with the cholesterol diet for 6 mo (n = 7) and without the cholesterol diet (n = 7) were compared with 6 normal wild-type (C57BL/6) mice with the same genetic background. (99m)Tc-annexin A5 was injected in 31 animals for noninvasive imaging using micro-SPECT/CT. After in vivo micro-SPECT/CT, aortas were explanted to acquire ex vivo images and calculate the percentage injected dose per gram (%ID/g) annexin uptake, followed by histologic and immunohistochemical characterization. For the evaluation of precise target localization, biotinylated annexin A5 was injected in the remaining 2 normally fed apoE(-/-) mice. RESULTS:Aortic lesions were clearly visualized noninvasively by micro-SPECT and aorta calcification was detectable by micro-CT. The quantitative uptake of annexin A5 was highest in the cholesterol-fed apoE(-/-) (0.88 +/- 0.27 %ID/g) mice, followed by the normal chow-fed apoE(-/-) (0.60 +/- 0.16 %ID/g), the cholesterol-fed LDLR(-/-) (0.59 +/- 0.14 %ID/g), the chow-fed LDLR(-/-) (0.40 +/- 0.31 %ID/g), and the control (0.15 +/- 0.05 %ID/g) mice. The histologic extent of atherosclerosis paralleled radiotracer uptake, and immunohistochemical studies revealed a significant correlation between radiotracer uptake and both macrophage infiltration and the extent of apoptosis. Intravenously injected biotinylated annexin A5 localized in apoptotic and nonapoptotic macrophages. CONCLUSION/CONCLUSIONS:This study demonstrates the feasibility of noninvasive imaging of atherosclerosis with radiolabeled annexin A5 in transgenic mouse models of human atherosclerosis.

UNLABELLED:Although apoptosis within atherosclerotic plaques is associated with plaque vulnerability and rupture, the role of inhibition of the apoptotic process is not clear. We evaluated the impact of dietary modification and statin therapy (measures known to favorably influence outcomes in coronary disease) on the incidence of apoptosis in experimental atherosclerotic lesions. METHODS:A total of 30 animals were studied; 1 group of 6 animals served as the controls (group 1), and the remaining 24 animals were subjected to balloon de-endothelialization of the abdominal aorta and a high-cholesterol diet. These atherosclerotic animals were randomized as follows: high-cholesterol diet for 4 mo (n=6; untreated atherosclerotic group [group 2]), high-cholesterol diet for 3 mo and normal chow diet for 1 mo (n=6; diet withdrawal group [group 3]), and high-cholesterol diet for 4 mo and simvastatin orally every day of the last month (n=6; statin therapy group [group 4]). 99mTc-Annexin A5 was used for noninvasive detection of apoptosis in groups 1-4. The remaining 6 rabbits on a high-cholesterol diet for 4 mo were studied with radiolabeled mutant annexin A5 (n=6; nonspecific control group [group 5]). Quantitative annexin A5 uptake in the abdominal aorta was determined and compared with the histologic and immunohistochemical characteristics of the atherosclerotic lesions. RESULTS:Maximum annexin A5 uptake (mean+/-SD, 0.051+/-0.009 percentage injected dose per gram [%ID/g] of tissue) was observed in the untreated atherosclerotic animals. The uptake was substantially reduced in the diet withdrawal (0.03+/-0.006%ID/g; P<0.0001) and statin therapy (0.03+/-0.006%ID/g; P<0.0001) groups. The plaques in the untreated high-cholesterol group demonstrated advanced atherosclerotic lesions. On the other hand, the diet withdrawal and statin therapy groups showed histologic characteristics of stabilization, including the resolution of macrophage infiltration and an increase in smooth muscle cell content. There was a marked reduction in the apoptosis of macrophages. No significant uptake of annexin A5 or mutant annexin A5 was seen in rabbits on the normal chow diet or atherosclerotic rabbits, respectively. CONCLUSION/CONCLUSIONS:Dietary modification and statin therapy in atherosclerosis lead to a reduction in apoptosis and contribute to plaque stabilization. It can be hypothesized that a reduction in apoptosis is a favorable process in atherosclerotic disease.

Primary vasculitis is the inflammation and necrosis of vessel walls not associated with infections, drugs, and autoimmune and lymphoproliferative disorders. It is important to make the correct diagnosis of different types of vasculitis, as their prognosis may be significantly different. Classification of vasculitis based on the size of the vessel is helpful, but there is often an overlap. Whereas the criteria proposed by the American College of Rheumatology are primarily clinical, the definitions set forth by the Chapel Hill Consensus Conference are based only on histologic observations. Correct diagnosis requires appropriate incorporation of the clinical history, laboratory parameters, and the histologic data. Incorporation of antineutrophil cytoplasmic antibodies in defining the pathogenesis of vasculitis has been particularly useful in diagnosing those small vessel vasculitides that are life threatening and need immediate intervention.

UNLABELLED:We used a model of porcine coronary atherosclerosis characterized by smooth muscle cell apoptosis to test the hypothesis that apoptosis of cells in the vascular wall of coronary arteries can be detected on SPECT images using a technetium-labeled radiotracer that targets apoptosis. METHODS:Eleven juvenile male swine received a high-fat diet combined with injury to 22 coronary vessels. After 51 +/- 9 d (mean +/- SD), the animals underwent coronary angiography, were injected with 403.3 +/- 48.1 MBq of 99mTc-annexin V, underwent SPECT, and were sacrificed. The coronary arteries underwent autoradiography and well counting, and immunostaining was performed for alpha-actin, caspase, and macrophages. RESULTS:Atherosclerotic lesions were predominantly of American Heart Association class II. Thirteen of the 22 injured vessels showed focal uptake of 99mTc-annexin V in vivo (scan positive), and 9 injured vessels and all control vessels showed no focal uptake (scan negative). The count ratios of the injured vessels to the control vessels were 2.38 +/- 0.61 for scan-positive vessels and 1.27 +/- 0.23 for scan-negative vessels (P < 0.001). The percentages of injected dose for the scan-positive and scan-negative vessels were 1.73 +/- 0.83 x 10(-3) and 0.68 +/- 0.20 x 10(-3), respectively (P < 0.001). Immunohistopathologic examination found that the cells undergoing apoptosis were smooth muscle cells. The apoptotic index (caspase-positive cells to total cells) was 63% +/- 7% for scan-positive vessels and 16% +/- 10% for scan-negative vessels (P < 0.001). Both the count ratio of injured vessels to control vessels and the percentage injected dose correlated significantly with death rate by regression analysis. CONCLUSION/CONCLUSIONS:Annexin is a noninvasive method to identify plaque apoptosis in the coronary vessels.

Endomyocardial biopsy (EMB) remains the "gold standard" for diagnosing rejection after cardiac transplantation. In addition, it has value in monitoring patients during treatment with doxorubicin. It also is important in the setting of acute-onset heart failure for the diagnosis of myocarditis, particularly giant cell myocarditis because earlier transplantation usually is undertaken in patients with giant cell morphologic features. EMB has a role in the unexplained cardiomyoapthy for excluding specific disease processes that might lead to similar morphofunctional changes but might be reversible or a contraindication for transplantation. This review focuses on the growing number of diseases that can be diagnosed by EMB in adult and pediatric age groups.

BACKGROUND:Myofibrillarlytic (MFL) cells are commonly observed in subendocardial myocardium in myocardial infarction. Because ischemic damage to myocytes is also known to induce apoptosis, we evaluated the prevalence of apoptosis in MFL cells in nine ischemic cardiomyopathic hearts explanted during transplantation. METHODS:Myocytes with partial or complete clearing of cytoplasm, observed commonly in the subendocardium, were recognized as MFL cells. Prevalence of apoptosis was defined by TUNEL and ISOL staining and further characterized by immunohistochemical staining for caspase-3, Bcl2, BCL-X(L), Bax, proliferating cell nuclear antigen (PCNA), and Ki67. RESULTS:Of 4131 MFL cells examined, 1305 (32%) possessed nuclei in a given histologic section; 1140 (88%) of the nucleated myocardial cells were TUNEL positive. Of 842 cells with normal appearance, 257 (31%) cells demonstrated nuclei in the given histologic section. TUNEL staining was observed in 5 (1.9%) in these control areas. All MFL cells stained positive for caspase 3. The antiapoptotic proteins, Bcl2 and BCL-X(L), demonstrated intense upregulation within and surrounding MFL cells, whereas pro-apoptotic protein Bax expression was only seen at control level. The MFL cells had Ki67 negative and PCNA positive nuclei. CONCLUSIONS:The present study demonstrates that the majority of MFL cells are apoptotic and are associated with upregulation of caspase 3. Simultaneous upregulation of Bcl2 represents a survival effort in these myocytes. This is consistent with the review of the literature that MFL cells are viable, persist in myocardium for long time and may be functionally reversible. Evidence for concurrent apoptosis and survival instinct represent a conceptual paradox and suggests that myocytes undergoing apoptosis should be amenable to reconstitution of function.