Faculty Bibliography

OBJECTIVES/OBJECTIVE:The role of iron toward doxorubicin (DOX) cardiotoxicity was studied using a rodent model of dietary carbonyl iron loading. BACKGROUND:Doxorubicin, a commonly used anticancer drug, is known to cause serious and potentially life-threatening cardiotoxicity. Doxorubicin cardiotoxicity is thought to be mediated through free-radical injury. METHODS:Male Sprague Dawley rats fed iron-rich chow (n = 8) and regular chow (n = 8) were treated with DOX or saline (4 animals in each arm). Cardiotoxicity was assessed using mortality, weight changes, Tc-99m annexin-V imaging, histopathology, and immunohistochemistry. RESULTS:Animals fed iron-rich chow showed significantly higher DOX cardiotoxicity as evidenced by greater weight loss (107 +/- 14 g vs. 55 +/- 10 g weight loss, p < 0.05), higher annexin uptake (0.14 +/- 0.01% vs. 0.08 +/- 0.01% injected dose/g of myocardium, p < 0.05), more severe myocyte injury on electron microscopy, and significantly higher cleaved caspase-3 staining compared with regular chow fed rats given DOX. Feeding iron-rich chow alone did not result in any cardiotoxicity. CONCLUSIONS:Dietary iron loading resulted in a substantially increased DOX cardiotoxicity in rats. Body iron stores as well as its bioavailability in tissue may be important independent predictors of susceptibility to DOX cardiotoxicity in man. Further clinical studies are warranted.

INTRODUCTION/BACKGROUND:Inflammation plays an important role in vulnerability of atherosclerotic plaques to rupture and hence acute coronary events. The monocyte-macrophage infiltration in plaques leads to upregulation of cytokines and metalloproteinase enzymes. Matrix metalloproteinases result in matrix dissolution and consequently expansive remodeling of the vessel. They also contribute to attenuation of fibrous cap and hence susceptibility to rupture. Assessment of metalloproteinase expression and activity should provide information about plaque instability.

Masses of the spermatic cord are rare and can be neoplastic or inflammatory lesions. We present a case of a sperm granuloma of the inguinal vas deferens presenting as a recurrent incarcerated inguinal hernia in a 42-year-old man.

CONTEXT/BACKGROUND:Annexin II is a calcium-dependent phospholipid-binding protein that plays a role in many cellular functions, including apoptosis, signal transduction, and cellular motility. The protein is strongly expressed in normal prostatic epithelial glands, but its expression in benign prostatic lesions has not been reported. Although commonly underexpressed in prostate cancer, the association of reduced expression with pathologic grade and stage is unknown. OBJECTIVE:To compare annexin II expression in benign prostatic lesions with expression in high-grade prostatic intraepithelial neoplasia and prostate cancer, as well as to correlate expression levels with pathologic grade and stage. DESIGN/METHODS:A semi-quantitative assessment of annexin II expression was performed in radical prostatectomy specimens from 74 patients and prostate needle core biopsy specimens from 13 patients. Foci with normal prostatic glands, atrophic glands, basal cell hyperplasia, high-grade prostatic intraepithelial neoplasia, and prostatic adenocarcinoma were evaluated. RESULTS:Annexin II expression was present in more than 50% of glands in most (>85%) samples of benign prostatic epithelium, atrophic glands, and basal cell hyperplasia. In high-grade prostatic intraepithelial neoplasia, annexin II staining was markedly reduced in epithelial cells but not in basal cells. Annexin II was absent or focally present in moderately differentiated adenocarcinoma but was retained in poorly differentiated adenocarcinomas. CONCLUSIONS:Reduced annexin II expression may be a useful diagnostic biomarker to help identify small foci of moderately differentiated adenocarcinoma on needle core biopsy specimens since it is consistently expressed in benign prostatic glands. Re-expression of annexin II in poorly differentiated adenocarcinoma may provide prognostic information.

BACKGROUND:Aortic valve stenosis is the most frequent indication for valve replacement surgery, and is commonly associated with pathologic calcification. Previous investigations by our group have shown a strong association of transforming growth factor-beta1 (TGF-beta1)-related mechanisms with calcific aortic stenosis in both cell culture and clinical pathology studies. METHODS:In the present investigations we sought to investigate the sequence of events involved in TGF-beta1-initiated aortic valve interstitial cell calcification in cell culture, and to study related gene expression pattern differences comparing calcific aortic stenosis surgical specimens with normal aortic valve leaflets. RESULTS:Sheep aortic valve interstitial cells (SAVIC) in culture progressively calcified over 14 days after the addition of TGF-beta1 to a significantly greater extent than non-TGF-beta1 controls. The TGF-beta1-induced SAVIC calcification was associated with maximal levels of alkaline phosphatase by 72 hours. Annexin V positive apoptosis was increased in TGF-beta1-treated SAVIC cultures at 14 days compared with controls. Matrix metalloproteinase 9 per gel zymography was detectable only in SAVIC cultures treated with TGF-beta1 from seven days on. Matrix metalloproteinase 2 was present in all SAVIC cultures per gel zymograms, either with or without TGF-beta1, but the active form of matrix metalloproteinase 2 significantly increased over 14 days in response to TGF-beta1. Quantitative gene expression studies (re: RNA levels) of human aortic valve cusps obtained at cardiac surgery demonstrated a number of related trends, including upregulation of the expression of TGF-beta1, alkaline phosphatase, and matrix metalloproteinase 9 in calcified human aortic valves. CONCLUSIONS:Transforming growth factor-beta1 causes SAVIC to calcify due to an early maximal increase in alkaline phosphatase activity with associated apoptotic events and increased matrix metalloproteinase 9. These TGF-beta1-related mechanistic events may be of clinical relevance based upon the gene expression pattern changes observed in calcific aortic stenosis valve cusps.

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.