Faculty Bibliography

Optical coherence tomography (OCT) is a noninvasive, high-resolution imaging technology capable of delivering real-time, near-histologic images of tissues. Mustard gas is a vesicant-blistering agent that can cause severe and lethal damage to airway and lungs. The ability to detect and assess airway injury in the clinical setting of mustard exposure is currently limited. The purpose of this study is to assess the ability to detect and monitor progression of half-mustard [2-chloroethylethylsulfide (CEES)] airway injuries with OCT techniques. A ventilated rabbit mustard exposure airway injury model is developed. A flexible fiber optic OCT probe is introduced into the distal trachea to image airway epithelium and mucosa in vivo. Progression of airway injury is observed over eight hours with OCT using a prototype time-domain superluminescent diode OCT system. OCT tracheal images from CEES exposed animals are compared to control rabbits for airway mucosal thickening and other changes. OCT detects the early occurrence and progression of dramatic changes in the experimental group after exposure to CEES. Histology and immunofluorescence staining confirms this finding. OCT has the potential to be a high resolution imaging modality capable of detecting, assessing, and monitoring treatment for airway injury following mustard vesicant agent exposures.

Cardiomyocyte apoptosis has been implicated in the pathogenesis of heart failure (HF). This study was performed in patients with left ventricular (LV) volume overload at different stages in the development of HF to correlate apoptotic gene expression with LV echocardiographic phenotype. LV biopsies were procured from 24 cardiac surgical patients selected from 4 distinct clinical groups (n = 6) in the progression from preserved LV function to HF. Group I consisted of control patients with normal LV function (e.g., with atrial myxoma), group II had aortic regurgitation with LV hypertrophy and preserved systolic function (ejection fraction >50%), group III had aortic regurgitation with LV dysfunction (ejection fraction 30% to 40%), and group IV had end-stage HF (ejection fraction <20%). Biopsies were used to measure mRNA expression of the genetic regulators of mitochondrial (Bad, Bax, Bcl-2, Bcl-xL, and p53) and death-receptor- (Fas and tumor necrosis factor receptor 1 [TNFR1]) mediated apoptotic pathways by reverse transcription-polymerase chain reaction. Caspase activity was determined using specific fluorogenic peptide substrates and immunohistochemistry. Evidence for apoptosis was obtained using terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling and in situ oligo ligation assays. Expression of proapoptotic factors (Bax, p53, TNFR1), antiapoptotic mitochondrial factor (Bcl-xL), and caspases 3, 8, and 9 increased progressively during the transition from preserved LV function to HF (p <0.05, analysis of variance). No significant difference was found for Bad, Bcl-2, or Fas. No evidence of DNA fragmentation was identified. In conclusion, activation of the cardiomyocyte apoptotic cascade occurs during the development of volume overload-induced HF. Mitochondrial (Bax, p53, caspase 9) and death-receptor mediated (TNFR1, caspase 8) pathways are upregulated but without completion of DNA fragmentation.

UNLABELLED:Matrix metalloproteinases (MMPs) are expressed in atherosclerotic plaques and play an important role in plaque instability. METHODS:Using (99m)Tc-labeled broad-spectrum MMP inhibitor (MPI), we performed noninvasive imaging of MMP expression with micro-SPECT/micro-CT in mice deficient in apolipoprotein E (ApoE(-/-), n = 14), mice deficient in low-density-lipoprotein receptor (LDLR(-/-), n = 14), and C57/BL6 mice as controls (n = 7). Seven ApoE(-/-) and 7 LDLR(-/-) received a high-cholesterol diet. After in vivo imaging, aortas were explanted, ex vivo images acquired, and the percent injected dose of MPI per gram (%ID/g) determined, followed by histologic characterization of atherosclerotic lesions. RESULTS:MPI uptake was noninvasively visualized in atherosclerotic lesions by micro-SPECT, with confirmation by micro-CT of anatomic location and aortic calcification. %ID/g in each part of the aorta was highest in ApoE(-/-) that were fed a high-cholesterol diet, followed by LDLR(-/-) that were fed a high-cholesterol diet, ApoE(-/-) that were fed normal chow, and LDLR(-/-) that were fed normal chow. The control mice had minimal MPI uptake. A significant correlation was noted between %ID/g and % area positive for macrophages (r = 0.81, P = 0.009), MMP-2 (r = 0.65, P = 0.013), and MMP-9 (r = 0.62, P = 0.008). CONCLUSION/CONCLUSIONS:This study demonstrates the usefulness of molecular imaging for noninvasive assessment of the extent of MMP expression in various transgenic mouse models of atherosclerosis receiving a normal or hyperlipidemic diet. It is conceivable that such a strategy may be translationally developed for identification of unstable atherosclerotic plaques.

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.

OBJECTIVES/OBJECTIVE:Using molecular imaging techniques, we examined interstitial alterations during postmyocardial infarction (MI) remodeling and assessed the efficacy of antiangiotensin and antimineralocorticoid intervention, alone and in combination. BACKGROUND:The antagonists of the renin-angiotensin-aldosterone axis restrict myocardial fibrosis and cardiac remodeling after MI and contribute to improved survival. Radionuclide imaging with technetium-99m-labeled Cy5.5 RGD imaging peptide (CRIP) targets myofibroblasts and indirectly allows monitoring of the extent of collagen deposition post-MI. METHODS:CRIP was intravenously administered for gamma imaging after 4 weeks of MI in 63 Swiss-Webster mice and in 6 unmanipulated mice. Of 63 animals, 50 were treated with captopril (C), losartan (L), spironolactone (S) alone, or in combination (CL, SC, SL, and SCL), 8 mice received no treatment. Echocardiography was performed for assessment of cardiac remodeling. Hearts were characterized histopathologically for the presence of myofibroblasts and thick and thin collagen fiber deposition. RESULTS:Acute MI size was similar in all groups. The quantitative CRIP percent injected dose per gram uptake was greatest in the infarct area of untreated control mice (2.30 +/- 0.14%) and decreased significantly in animals treated with 1 agent (C, L, or S; 1.71 +/- 0.35%; p = 0.0002). The addition of 2 (CL, SC, or SL 1.31 +/- 0.40%; p < 0.0001) or 3 agents (SCL; 1.16 +/- 0.26%; p < 0.0001) demonstrated further reduction in tracer uptake. The decrease in echocardiographic left ventricular function, strain and rotation parameters, as well as histologically verified deposition of thin collagen fibers, was significantly reduced in treatment groups and correlated with CRIP uptake. CONCLUSIONS:Radiolabeled CRIP allows for the evaluation of the efficacy of neurohumoral antagonists after MI and reconfirms superiority of combination therapy. If proven clinically, molecular imaging of the myocardial healing process may help plan an optimal treatment for patients susceptible to heart failure.

Several groups, including ours, have reported that annexin A2 (ANXA2) expression is reduced in most prostate cancer (CaP). More recently, however, we reported that ANXA2 is expressed in some high-grade tumors, but the biologic consequence of this is currently unknown. To elucidate the function of ANXA2 in CaP, we reduced its expression in DU145 cells using shRNA and tested the impact on characteristics of malignancy. Reduction of ANXA2 suppressed anchorage-dependent and -independent cell growth without affecting invasiveness. Interestingly, interleukin-6 (IL-6) secretion was reduced concomitantly with the reduction of ANXA2 but independently of S100A10. IL-6 expression was restored when wild type but not mutant ANXA2 was reexpressed in these cells. In a retrospective study of radical prostatectomy specimens from patients with nonmetastatic CaP, 100% of patients with ANXA2-positive tumors (n = 4) had a biochemical relapse while only 50% of patients with ANXA2 negative tumors (n = 20) relapsed, suggesting that ANXA2 expression in prostate tumors may be predictive of biochemical relapse. Significant cytoplasmic staining of ANXA2 was detected in 3 of 4 ANXA2-positive tumors, whereas ANXA2 is localized to the plasma membrane in benign prostatic glands. These finding, taken together, suggests a possible mechanism whereby ANXA2 expression positively contributes to an aggressive phenotype in a subset of CaP and suggest that ANXA2 has markedly different functions depending on its cellular context. Finally, this is the first description of a role for ANXA2 in IL-6 expression, and ANXA2 represents a new therapeutic target for reducing IL-6 in high-grade prostate cancer.

OBJECTIVES/OBJECTIVE:The purpose of this study was to evaluate interstitial alterations in myocardial remodeling using a radiolabeled Cy5.5-RGD imaging peptide (CRIP) that targets myofibroblasts. BACKGROUND:Collagen deposition and interstitial fibrosis contribute to cardiac remodeling and heart failure after myocardial infarction (MI). Evaluation of myofibroblastic proliferation should provide indirect evidence of the extent of fibrosis. METHODS:Of 46 Swiss-Webster mice, MI was induced in 41 by coronary artery occlusion, and 5 were unmanipulated. Of the 41 mice, 6, 6, and 5 received intravenous technetium-99m labeled CRIP for micro-single-photon emission computed tomography imaging 2, 4, and 12 weeks after MI, respectively; 8 received captopril or captopril with losartan up to 4 weeks after MI. Scrambled CRIP was used 4 weeks after MI in 6 mice; the remaining 10 of 46 mice received unradiolabeled CRIP for histologic characterization. RESULTS:Maximum CRIP uptake was observed in the infarct area; quantitative uptake (percent injected dose/g) was highest at 2 weeks (2.75 +/- 0.46%), followed by 4 (2.26 +/- 0.09%) and 12 (1.74 +/- 0.24%) weeks compared with that in unmanipulated mice (0.59 +/- 0.19%). Uptake was higher at 12 weeks in the remote areas. CRIP uptake was histologically traced to myofibroblasts. Captopril alone (1.78 +/- 0.31%) and with losartan (1.13 +/- 0.28%) significantly reduced tracer uptake; scrambled CRIP uptake in infarct area (0.74 +/- 0.17%) was similar to CRIP uptake in normal myocardium. CONCLUSIONS:Radiolabeled CRIP allows for noninvasive visualization of interstitial alterations during cardiac remodeling, and is responsive to antiangiotensin treatment. If proven clinically feasible, such a strategy would help identify post-MI patients likely to develop heart failure.

OBJECTIVES/OBJECTIVE:This study sought to evaluate the feasibility of noninvasive detection of matrix metalloproteinase (MMP) activity in experimental atherosclerosis using technetium-99m-labeled broad matrix metalloproteinase inhibitor (MPI) and to determine the effect of dietary modification and statin treatment on MMP activity. BACKGROUND:The MMP activity in atherosclerotic lesions contributes to the vulnerability of atherosclerotic plaques to rupture. METHODS:Atherosclerosis was produced in 34 New Zealand White rabbits by balloon de-endotheliazation of the abdominal aorta and a high-cholesterol diet. In addition, 12 unmanipulated rabbits were used as controls and 3 for blood clearance characteristics. In vivo micro-single-photon emission computed tomography (SPECT) imaging was performed after radiolabeled MPI administration. Subsequently, aortas were explanted to quantitatively measure percent injected dose per gram (%ID/g) MPI uptake. Histological and immunohistochemical characterization was performed and the extent of MMP activity was determined by gel zymography or enzyme-linked immunosorbent assays. RESULTS:The MPI uptake in atherosclerotic lesions (n = 18) was clearly visualized by micro-SPECT imaging; MPI uptake was markedly reduced by administration of unlabeled MPI before the radiotracer (n = 4). The MPI uptake was also significantly reduced after diet withdrawal (n = 6) and fluvastatin treatment (n = 6); no uptake was observed in normal control rabbits (n = 12). The %ID/g MPI uptake (0.10 +/- 0.03%) in the atherosclerotic lesions was significantly higher than the uptake in control aorta (0.016 +/- 0.004%, p < 0.0001). Uptake in fluvastatin (0.056 +/- 0.011%, p < 0.0005) and diet withdrawal groups (0.043 +/- 0.011%, p < 0.0001) was lower than the untreated group. The MPI uptake correlated with immunohistochemically verified macrophage infiltration (r = 0.643, p < 0.0001), and MMP-2 (r = 0.542, p < 0.0001) or MMP-9 (r = 0.578, p < 0.0001) expression in plaques. CONCLUSIONS:The present data show the feasibility of noninvasive detection of MMP activity in atherosclerotic plaques, and confirm that dietary modification and statin therapy reduce MMP activity.

PURPOSE/OBJECTIVE:While most laparoscopic nephron sparing surgery is performed using cold scissors, energy based devices may also be used. A criticism of this approach has been the potential thermal destruction of the cellular architecture at the tumor margin, precluding the ability to accurately determine whether tumor cells are present. We clinically characterized the histological appearance of tumor margins excised with cold scissors, and bipolar and ultrasonic shears. MATERIALS AND METHODS/METHODS:We evaluated 40 renal mass excisions performed by a total of 3 urologists at our institution between February 2003 and March 2007. There were 10 bipolar (5 mm LigaSure), 20 ultrasonic (Harmonic Scalpel) and 10 cold excisions. All slides were randomly evaluated twice by a single pathologist blinded to surgeon and excision method. Histological interpretation of the margin was scored as clear vs indeterminate. Variables, including margin fragmentation, artifact, extravascular blood clot, parenchymal hemorrhage, capillary congestion and vessel sealing, were assessed and scored on a scale of 0 to 3, that is 0--none, 1-1% to 25%, 2-26% to 50% and 3--greater than 50%. RESULTS:The pathologist was able to confidently identify cells at the margin as being malignant or benign in all cases. Histologically the ultrasonic scalpel demonstrated increased fragmentation and extravascular blood clotting compared with those of the other cutting methods (p <0.025 and <0.026, respectively). The ultrasonic scalpel also showed increased artifact depth compared to that of cold cutting (p <0.001). There were no statistical differences between the groups regarding margin artifact, parenchymal hemorrhage or capillary congestion. No statistical significance was observed in any variables between bipolar and cold cutting. CONCLUSIONS:Despite some degree of cellular damage the ability to determine whether cells at the margin were benign or malignant was not affected by using an energy based bipolar or ultrasonic device.