Katsuichi Ohtsuki

Technetium-99m HYNIC-annexin V: a potential radiopharmaceutical for the in-vivo detection of apoptosis

Abstract. Either inadequate or excessive apoptosis (programmed cell death) is associated with many diseases. A method to image apoptosis in vivo, rather than requiring histologic evaluation of tissue, could assist with therapeutic decision making in these disorders. Programmed cell death is associated with a well-choreographed series of events resulting in the cessation of normal cell function, and the ultimate disappearance of the cell. One component of apoptosis is signaling adjacent cells that this cell is committing suicide by externalizing phosphatidylserine to the outer leaflet of the cell membrane. Annexin V, a 32-kDa endogenous human protein, has a high affinity for membrane-bound phosphatidylserine. We have coupled annexin V with the bifunctional hydrazinonicotinamide reagent (HYNIC) to prepare technetium-99m HYNIC-annexin V and demonstrated localization of radioactivity in tissues undergoing apoptosis in vivo. In this report we describe the results of a series of experiments in mice and rats to characterize the biologic behavior of 99mTc-HYNIC- annexin V. Biodistribution studies were performed in groups of rats at 10–180 min after intravenous injection of 99mTc-HYNIC-annexin V. In order to estimate the degree of apoptosis required for localization of 99mTc-annexin V in vivo, mice were treated with dexamethasone at doses ranging from 1 to 20 mg/kg, 5 h prior to 99mTc-HYNIC-annexin V administration, to induce thymic apoptosis. Thymus was excised 1 h after radiolabeled HYNIC-annexin V injection; thymocytes were isolated, incubated with Hoechst 33342 followed by propidium iodide, and analyzed on a fluorescence-activated cell sorter. Each sorted cell population was counted in a scintillation counter. To test 99mTc-HYNIC-annexin V as a tracer for external radionuclide imaging of apoptotic cell death, radionuclide imaging of Fas-defective mice (lpr/lpr mice) and wild-type mice treated with the antibody to Fas (anti-Fas) was carried out 1 h post injection. Rat biodistribution studies demonstrated a blood clearance half-time of less than 10 min for 99mTc-HYNIC-annexin V. The kidneys had the highest concentration of radioactivity at all time points. Studies in the mouse thymus demonstrated a 40-fold increase in 99mTc-HYNIC-annexin V concentration in apoptotic thymocytes compared with the viable cell population. A correlation of r=0.78 was found between radioactivity and flow cytometric and histologic evidence of apoptosis. Imaging studies in the lpr/lpr and wild-type mice showed a substantial increase of activity in the liver of wild-type mice treated with anti-Fas, while there was no significant change, irrespective of anti-Fas administration, in lpr/lpr mice. Excellent images of hepatic apoptosis were obtained in wild-type mice 30 min after injection of 99mTc-HYNIC-annexin V. The imaging results were consistent with histologic analysis in these animals. In conlusion, these studies confirm the value of 99mTc-HYNIC-annexin V uptake as a marker for the detection and quantification of apoptotic cells in vivo.

Detection of Monocyte Chemoattractant Protein-1 Receptor Expression in Experimental Atherosclerotic Lesions An Autoradiographic Study

Background—Monocytes, a common component of atheroma, are attracted to the lesion site in response to chemotactic signals, particularly expression of monocyte chemoattractant peptide 1 (MCP-1). This study assessed the feasibility of using radiolabeled MCP-1 to identify monocytes and macrophages that have localized at sites of experimental arterial lesions. Methods and Results—The biodistribution of radiolabeled MCP-1 was determined in normal mice, and localization in experimental atheroma was determined in cholesterol-fed rabbits 4 weeks after arterial injury of the iliac artery (9 rabbits) and the abdominal aorta (1 rabbit). Vessels were harvested and autoradiographed after intravenous administration of 125I-labeled MCP-1 and Evans blue dye. The arteries were evaluated histologically by hematoxylin and eosin staining and immune staining with a monoclonal antibody specific for rabbit macrophages (RAM-11). 125I-MCP-1 has a blood clearance half-time of ≈10 minutes and circulates in association with cells. The liver, lungs, and kidneys had the highest concentration of 125I-MCP-1 at 5 and 30 minutes after tracer administration. Autoradiograms revealed accumulation of 125I-MCP-1 in the damaged artery wall, with an average ratio of lesion to normal vessel of 6:1 (maximum 45:1). The accumulation of 125I-MCP-1 in the reendothelialized (plaque formation) areas was greater than in the deendothelialized (Evans blue-positive) areas (6.55±2.26 versus 4.34±1.43 counts/pixel, P <0.05). The uptake of 125I-MCP-1 correlated with the number of macrophages per unit area (r =0.85, P <0.0001). Conclusions—Radiolabeled MCP-1 may be a useful tracer for imaging monocyte/macrophage-rich experimental atherosclerotic lesions.

THE USE OF TECHNETIUM TC 99M ANNEXIN V FOR IN VIVO IMAGING OF APOPTOSIS DURING CARDIAC ALLOGRAFT REJECTION

OBJECTIVE Apoptosis, or programmed cell death, has been suggested as a mechanism of immunologic injury during cardiac allograft rejection. We tested the hypothesis that technetium Tc 99m annexin V, a novel radiopharmaceutical used to detect apoptosis, can be used to detect cardiac allograft rejection by nuclear imaging. METHODS Untreated ACI rats served as recipients of allogeneic PVG rat (n = 66) or syngeneic ACI rat (n = 30) cardiac grafts. Untreated recipient animals underwent 99mTc-annexin V imaging daily for 7 days. Region of interest analysis was used to quantify the uptake of 99mTc-annexin V. Immediately after imaging grafts were procured for histopathologic analysis and terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-biotin nick-end labeling of apoptotic nuclei. One group was treated with 10 mg/kg/d cyclosporine (INN: ciclosporin) commencing on day 4 after transplantation (n = 6). RESULTS Untreated allografts showed histologic signs of rejection 4 days after transplantation. Apoptotic nuclei could be demonstrated in myocytes, endothelial cells, and graft-infiltrating cells of all rejecting allografts. Nuclear imaging revealed a significantly greater uptake of 99mTc-annexin V in rejecting allogeneic grafts than in syngeneic grafts on day 4 (P = .05), day 5 (P < .001), day 6 (P < .001), and day 7 (P = .013) after transplantation. A correlation between the histologic grade of acute rejection and uptake of 99mTc-annexin V was observed (r2 = 0.87). After treatment of rejection with cyclosporine, no apoptotic nuclei could be identified in allografts and uptake of 99mTc-annexin V decreased to baseline. CONCLUSIONS Apoptosis occurs during acute cardiac allograft rejection and disappears after treatment of rejection. 99mTc-annexin V can be used to detect and monitor cardiac allograft rejection.

Apoptosis: the importance of nuclear medicine.

Apoptosis is a genetically controlled, energy-dependent process which removes unwanted cells from the body. Because of its orderly progression, apoptosis is also known as programmed cell death or cell suicide. Once initiated, apoptosis is characterized by a series of biochemical and morphological changes involving the cytoplasm, nucleus and cell membrane. Cytoplasmic changes include cytoskeletal disruption, cytoplasmic shrinkage and condensation; prominent changes in the nucleus include peripheral chromatin clumping and inter-nucleosomal DNA cleavage (DNA ladder formation); and membrane changes include the expression of phosphatidylserine on the outer surface of the cell membrane and blebbing (resulting in the formation of cell membrane-bound vesicles or apoptotic bodies). These events allow the cell to digest and package itself into membrane-bound packets containing autodigested cytoplasm and DNA, which can then be easily absorbed by adjacent cells or phagocytes. An endogenous human protein, annexin V (molecular weight approximately 35,000), has an affinity of about 10−9 M for phosphatidylserine exposed on the surface of apoptotic cells. Annexin V can be labelled with radionuclides such as iodine or technetium, or positron emitting agents. Experimental studies in cells confirm that fluorescence and 99Tcm-labelled annexin have comparable affinity for apoptotic cells. In vivo studies with 99Tcm-labelled annexin confirm that radiolabelled annexin V can be used to image apoptotic cells/tissues in vivo. In this article, we review experimental data using annexin V imaging and discuss its possible future use to identify apoptosis in vivo.

Clinical evaluation of hypertrophic cardiomyopathy by myocardial scintigraphy using 123I-labelled 15-(p-iodophenyl)-3-R, S-methylpentadecanoic acid (123I-BMIPP).

To evaluate the clinical utility of 123I-labelled 15-(p-iodophenyl)-3-R, S-methylpentadecanoic acid (123I-BMIPP), myocardial scintigraphy using 123I-BMIPP was performed in 13 patients with hypertrophic cardiomyopathy (HCM), and the findings were compared with those of 201Tl. The uptake of each tracer was scored visually from 4 (increased) to 0 (severely decreased) in a total of 13 segments in the apical and basal short axial images, and the long axial images of myocardial single photon emission tomography (SPET). In the comparison of the early images with 123I-BMIPP (taken 15 min after injection) and the 201Tl perfusion images, 70% of the segments showed uncoupled uptake, with the uptake of 123I-BMIPP more often being less. In the hypertrophic regions, 123I-BMIPP demonstrated significantly less uptake than 201Tl, especially in the delayed images (taken 3 h after injection). In the nonhypertrophic regions, there was no significant difference in uptake between the 123I-BMIPP early images and the 201Tl images; the 123I-BMIPP delayed images, however, demonstrated significantly less uptake than the 201Tl images. These results suggest that the uncoupling between the 123I-BMIPP and 201Tl uptakes and the washout (the relative decrease in activity from early to delayed images of 123I-BMIPP) reflect abnormal myocardial fatty acid metabolism in HCM.

Beneficial effect of gabexate mesilate on microscopic polyangiitis with renal dysfunction and pulmonary hemorrhage: A case report.

Corticosteroids and cyclophosphamide are the mainstay of the treatment of microscopic polyangiitis involving pulmonary hemorrhage or rapidly progressive glomerulonephritis. However, patients with advanced age are unable to tolerate this combined therapy, because of a relatively high incidence of side effects including infection, hemorrhagic cystitis, and bone marrow suppression. The authors encountered an 80-year-old patient with pulmonary hemorrhage and renal dysfunction ascribed to microscopic polyangiitis and achieved successful treatment by employing gabexate mesilate in addition to corticosteroids. The present case suggests that gabexate mesilate may be a therapeutic option for microscopic polyangiitis with progressive renal failure and pulmonary hemorrhage.

Long-term follow-up of myocardial perfusion and metabolic images in a patient with hypertrophic cardiomyopathy.

A 54-year-old woman was hospitalized in 1989 because of palpitations. She had a family history of hypertrophic cardiomyopathy. Hypertrophic cardiomyopathy was diagnosed by two-dimensional echocardiography, cardiac catheterization, and myocardial biopsy. A coronary arteriogram disclosed normal coronary arteries. She had received diltiazem and meliletine for 9 years. Her functional class, by the New York Heart Association classification, had worsened from II to III since 1995. Two-dimensional echocardiography showed a thrombus in the left ventricle in 1996, and warfarin and captopril were prescribed. Follow-up images showed that 1-123 β-methyl-p-iodophenyl-pentadecanoic acid uptake preceded that of TI-201.