Annexin-V imaging for noninvasive detection of cardiac allograft rejection.

Heart transplant rejection is characterized pathologically by myocyte necrosis and apoptosis associated with interstitial mononuclear cell infiltration. Any one of these components can be targeted for noninvasive detection of transplant rejection. During apoptotic cell death, phosphatidylserine, a phospholipid that is normally confined to the inner leaflet of cell membrane bilayer, gets exteriorized. Technetium-99m-labeled annexin-V, an endogenous protein that has high affinity for binding to phosphatidylserine, has been administered intravenously for noninvasive identification of apoptotic cell death. In the present study of 18 cardiac allograft recipients, 13 patients had negative and five had positive myocardial uptake of annexin. These latter five demonstrated at least moderate transplant rejection and caspase-3 staining, suggesting apoptosis in their biopsy specimens. This study reveals the clinical feasibility and safety of annexin-V imaging for noninvasive detection of transplant rejection by targeting cell membrane phospholipid alterations that are commonly associated with the process of apoptosis.

Zinc-mediated reduction of apoptosis in cardiac allografts.

BACKGROUND: Apoptosis is thought to occur during immune-mediated acute rejection of cardiac allografts. In vitro studies have shown that zinc inhibits the activity of the proapoptotic enzyme caspase-3. We hypothesized that ZnCl(2) would reduce acute cardiac rejection in vivo via the blockade of caspase-3-dependent apoptosis. (99m)Tc-labeled annexin V was used to measure apoptosis in cardiac allografts through nuclear imaging. Annexin V binds to phosphatidylserines, which are externalized to the outer membrane of apoptotic cells. METHODS AND RESULTS: Twenty-seven PVG rat hearts were transplanted heterotopically into the abdomen of untreated ACI rats as controls (group 1). Fifteen were scanned and euthanized on postoperative day 4, and 12 were assessed for graft survival. Group 2 and 3 rats (n=15 each) received 1 and 5 mg/kg ZnCl(2) BID IP, respectively. Nine of each of these groups were scanned and euthanized on postoperative day 4, and 6 were studied for allograft survival. Group 4 rats (n=3) received isografts. Region-of-interest analysis demonstrated a dose-dependent reduction in (99m)Tc annexin uptake in ZnCl(2)-treated allografts: 2.43+/-0.37% for group 1, 1. 97+/-0.41% for group 2, 1.21+/-0.47% for group 3, and 0.55+/-0.19% for group 4 (ANOVA, P:=0.001). Graft survival times of 6.4+/-1.7, 9. 3+/-3.0, and 11.5+/-3.4 days for groups 1, 2, and 3, respectively, were also observed (ANOVA, P:=0.001). Caspase-3 activity in the allografts showed a 3.7-fold reduction in group 3 animals compared with group 1 animals (P:=0.004). CONCLUSIONS: Apoptosis that occurs in acute cardiac allograft rejection is reduced with ZnCl(2) in a dose-dependent manner via caspase-3 inhibition.

99mTc annexin V imaging of neonatal hypoxic brain injury.

BACKGROUND AND PURPOSE: Delayed cell loss in neonates after cerebral hypoxic-ischemic injury (HII) is believed to be a major cause of cerebral palsy. In this study, we used radiolabeled annexin V, a marker of delayed cell loss (apoptosis), to image neonatal rabbits suffering from HII. METHODS: Twenty-two neonatal New Zealand White rabbits had ligation of the right common carotid artery with reduction of inspired oxygen concentration to induce HII. Experimental animals (n=17) were exposed to hypoxia until an ipsilateral hemispheric decrease in the average diffusion coefficient occurred. After reversal of hypoxia and normalization of average diffusion coefficient values, experimental animals were injected with (99m)Tc annexin V. Radionuclide images were recorded 2 hours later. RESULTS: Experimental animals showed no MR evidence of blood-brain barrier breakdown or perfusion abnormalities after hypoxia. Annexin images demonstrated multifocal brain uptake in both hemispheres of experimental but not control animals. Histology of the brains from experimental animals demonstrated scattered pyknotic cortical and hippocampal neurons with cytoplasmic vacuolization of glial cells without evidence of apoptotic nuclei by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. Double staining with markers of cell type and exogenous annexin V revealed that annexin V was localized in the cytoplasm of scattered neurons and astrocytes in experimental and, less commonly, control brains in the presence of an intact blood-brain barrier. CONCLUSIONS: Apoptosis may develop after HII even in brains that appear normal on diffusion-weighted and perfusion MR. These data suggest a role of radiolabeled annexin V screening of neonates at risk for the development of cerebral palsy.

Apoptosis and allograft rejection in the absence of CD8+ T cells.

BACKGROUND: The requirement for cytotoxic T lymphocytes during allograft rejection is controversial. We previously demonstrated that CD8+ T cells are not necessary for allograft rejection or for the induction of apoptosis in rat small intestinal transplantation. In this study, we examined the mechanisms of apoptosis and rejection after liver transplantation in the absence of CD8+ T cells. METHODS: Either Lewis or dark agouti rat liver grafts were transplanted into Lewis recipients to create syngeneic and allogeneic combinations. CD8+ T cells were depleted in an additional allogeneic group by treatment with OX-8 mAb on day -1 and day 1 after liver transplant. RESULTS: Apoptosis and rejection were observed in both the CD8+ T cell-depleted allogeneic and allogeneic grafts by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and radiolabeled-annexin V in vivo imaging. Granzyme B and FasL were expressed in all allogeneic transplants, including those depleted of CD8+ T cells, indicating that a mononuclear cell other than a CD8+ T cell can be the source of these molecules during allograft rejection. Activation of the caspase cascade was detected in all rejecting allografts. Caspases 3, 8, and 9 were activated at similar significantly elevated levels in both allogeneic and CD8+ T cell-depleted liver grafts. CONCLUSION: These data indicate that in the absence of CD8+ T cells an alternative pathway, associated with granzyme B and FasL expression and activation of the caspase cascade, can mediate apoptosis and graft rejection.

Imaging cyclophosphamide-induced intramedullary apoptosis in rats using 99mTc-radiolabeled annexin V.

UNLABELLED: Intramedullary apoptosis of hematopoietic tissue is believed to play a major role in the pathophysiology of myelodysplastic syndrome. Annexin V, a specific marker of the early to intermediate phases of apoptosis, has been applied to the in vitro study of bone marrow aspirates. A noninvasive measure of intramedullary apoptosis in vivo that could serially monitor the clinical progression of myelodysplastic syndrome may be helpful. METHODS: We used 99mTc-radiolabeled annexin V and radionuclide gamma camera imaging to serially study the sites, extent, and severity of intramedullary apoptosis induced by cyclophosphamide treatment. RESULTS: Intravenously administered radiolabeled annexin V localized preferentially in the femur, pelvis, vertebrae, and spleen; increased uptake in these organs was easily visualized as early as 8 h after injection of 100 mg/kg cyclophosphamide in 8- to 10-wk-old animals. Higher doses of cyclophosphamide (150 mg/kg) in animals of the same age increased annexin V uptake in the bone marrow and splenic tissue and delayed recovery of these organs as seen histologically compared with lower doses. Older animals, 5-6 mo old, showed a slower response to cyclophosphamide treatment and delayed recovery of bone marrow and splenic tissues. CONCLUSION: Radiolabeled annexin V can be used to detect and directly quantify the degree of intramedullary and splenic apoptosis in a noninvasive fashion using current clinical radionuclide imaging equipment. Annexin V imaging may be useful clinically in the diagnosis and management of myelodysplastic syndrome.

Imaging of apoptosis (programmed cell death) with 99mTc annexin V.

UNLABELLED: Apoptosis (programmed cell death) is a critical element in normal physiology and in many disease processes. Phosphatidylserine (PS), one component of cell membrane phospholipids, is normally confined to the inner leaflet of the plasma membrane. Early in the course of apoptosis, this phospholipid is rapidly exposed on the cell's outer surface. Annexin V, an endogenous human protein, has a high affinity for membrane-bound PS. This protein has been labeled with fluorescein and has been used to detect apoptosis in vitro. We describe the use of radiolabeled annexin V to detect apoptosis in vivo. The results are compared to histologic and flow cytometric methods to identify cells and tissues undergoing apoptosis. METHODS: Annexin V was coupled to hydrazinonicotinamide (HYNIC) and radiolabeled with 99mTc. Bioreactivity of 99mTc-HYNIC annexin V was compared with fluorescein isothiocyanate (FITC)-labeled annexin V in cultures of Jurkat T-cell lymphoblasts and in ex vivo thymic cell suspensions undergoing apoptosis in response to different stimuli. In addition, the uptake of FITC annexin V and 99mTc-HYNIC annexin V was studied in heat-treated necrotic Jurkat T-cell cultures. In vivo localization of annexin V was studied in Balb/c mice injected with 99mTc-HYNIC annexin V before and after induction of Fas-mediated hepatocyte apoptosis with intravenously administered antiFas antibody. RESULTS: Membrane-bound radiolabeled annexin V activity linearly correlated to total fluorescence as observed by FITC annexin V flow cytometry in Jurkat T-cell cultures induced to undergo apoptosis in response to growth factor deprivation (N = 10, r2 = 0.987), antiFas antibody (N = 8, r2 = 0.836) and doxorubicin (N = 10, r2 = 0.804); and in ex vivo experiments on thymic cell suspensions with dexamethasone-induced apoptosis from Balb/c mice (N = 6, r2 = 0.989). Necrotic Jurkat T-cell cultures also demonstrated marked increases in radiopharmaceutical (4000-5000-fold) above control values. AntiFas antibody-treated Balb/c mice (N = 6) demonstrated a three-fold rise in hepatic uptake of annexin V (P < 0.0005) above control (N = 10), identified both by imaging and scintillation well counting. The increase in hepatic uptake in antiFas antibody-treated mice correlated to histologic evidence of fulminant hepatic apoptosis. CONCLUSION: These data suggest that 99mTc-HYNIC annexin V can be used to image apoptotic and necrotic cell death in vivo.

Radionuclide imaging of acute lung transplant rejection with annexin V.

STUDY OBJECTIVES: Early detection and treatment of lung transplant rejection is critical for preservation of pulmonary graft function. Damage to pulmonary allografts is mediated by apoptotic cell death induced by the alloreactive T lymphocytes that infiltrate lung grafts. Previous studies demonstrate that acute cardiac allograft rejection can be visualized using radiolabeled annexin V. This study was done to determine whether this technique could visualize acute rejection in a rodent model of unilateral orthotopic lung transplantation. DESIGN: Eighteen Sprague-Dawley ACI rats underwent removal of their left lung followed by orthotopic transplant of either an allogeneic (PVG, immunologically mismatched; N = 10) or a syngeneic (ACI, immunologically matched) pulmonary graft (N = 8). Animals were imaged 1 h after IV injection of 1 mCi (37.0 MBq) of (99m)Tc-annexin V 1 to 7 days after transplantation. RESULTS: Lungs receiving the allograft demonstrated moderate to marked mononuclear infiltration of the perivascular, interstitial, and peribronchial tissues. No mononuclear infiltrates were noted in the native right lungs nor in the syngeneic transplants. Region of interest image analysis revealed significant (p < 0.0005) increases of transplant to normal lung activity ratios 3 to 7 days after allograft surgery. The increased annexin V uptake in these lungs was confirmed at biodistribution assay (allograft 151% greater than isograft activity, p < 0.005). CONCLUSIONS: Acute experimental lung transplant rejection can be noninvasively identified using (99m)Tc-annexin V. Radiolabeled annexin V may be a clinically useful noninvasive screening tool for acute rejection.

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.

The influence of volumetric tumor doubling time, DNA ploidy, and histologic grade on the survival of patients with intracranial astrocytomas.

PURPOSE: To improve the prediction of individual survival in patients with intracranial astrocytomas through the analysis of volumetric tumor doubling time (VDt) and DNA ploidy. METHODS: A pilot study was retrospectively conducted on a group of 25 patients with intracranial astrocytomas in whom recurrent and/or progressive disease was observed on serial contrast-enhanced CT or MR examinations. VDt was computed using two or more data points from a semilogarithmic plot of tumor volume versus time. Size-adjusted survival was calculated using a method based on VDt and initial tumor volume to decrease the lead time bias attributable to differing tumor sizes at presentation. RESULTS: Slower VDt was associated with significantly longer survival and size-adjusted survival as determined by a univariate Cox proportional hazard analysis. Aneuploidy was a significant indicator of poor survival. Aneuploid and multiclonal astrocytomas had poor size-adjusted survivals compared with diploid astrocytomas. Grade IV astrocytomas had significantly poorer survival and size-adjusted survival compared with lower grades (I to III), which individually were not significantly correlated. However, grade IV histology was not a significant independent predictor of size-adjusted survival in a multivariate Cox model, whereas VDt and DNA ploidy remained significant. VDt also had a significant direct linear correlation to survival and size-adjusted survival. CONCLUSIONS: VDt and DNA ploidy were more sensitive than histologic grading as indicators of individual survival. Initial tumor size needs to be considered when staging and assessing survival in patients with intracranial astrocytomas.

Sonography, CT, and MR imaging: a prospective comparison of neonates with suspected intracranial ischemia and hemorrhage.

BACKGROUND AND PURPOSE: Sonography, CT, and MR imaging are commonly used to screen for neonatal intracranial ischemia and hemorrhage, yet few studies have attempted to determine which imaging technique is best suited for this purpose. The goals of this study were to compare sonography with CT and MR imaging prospectively for the detection of intracranial ischemia or hemorrhage and to determine the prognostic value(s) of neuroimaging in neonates suspected of having hypoxic-ischemic injury (HII). METHODS: Forty-seven neonates underwent CT (n = 26) or MR imaging (n = 24) or both (n = 3) within the first month of life for suspected HII. Sonography was performed according to research protocol within an average of 14.4 +/- 9.6 hours of CT or MR imaging. A kappa analysis of interobserver agreement was conducted using three independent observers. Infants underwent neurodevelopmental assessment at ages 2 months (n = 47) and 2 years (n = 26). RESULTS: CT and MR imaging had significantly higher interobserver agreement (P < .001) for cortical HII and germinal matrix hemorrhage (GMH) (Grades I and II) compared with sonography. MR imaging and CT revealed 25 instances of HII compared with 13 identified by sonography. MR imaging and CT also revealed 10 instances of intraparenchymal hemorrhage (>1 cm, including Grade IV GMH) compared with sonography, which depicted five. The negative predictive values of neuroimaging, irrespective of technique used, were 53.3% and 58.8% at the 2-month and 2-year follow-up examinations, respectively. CONCLUSION: CT and MR imaging have significantly better interobserver agreement for cortical HII and GMH/intraventricular hemorrhage and can reveal more instances of intraparenchymal hemorrhage compared with sonography. The absence of neuroimaging findings on sonograms, CT scans, or MR images does not rule out later neurologic dysfunction.

Noninvasive strategies to image cardiovascular apoptosis.

Apoptosis consists of a complex set of biochemical events initiated by an array of different stimuli and enzymatic pathways. There is a set of common morphologic and biochemical features of apoptosis that could be exploited as hot or cold targets to image cardiovascular apoptosis. First, the authors review the potential array of targets that can be used to identify apoptosis. Then, the authors examine the history and current status of radiolabeled annexin V, the agent currently used to image apoptosis.

Preliminary investigation of ultrasound scattering analysis to identify women at risk for later invasive cancer. I: Motivation and experimental technique for characterization of isolated breast tissue lobules.

A new experimental technique is described that allows the characterization of the angle-dependent ultrasonic scattering properties of isolated breast tissue lobules. A review of breast tissue micro-architecture is presented as background material. Measured estimates of the scatter angle-dependent differential scatter cross-sections (DSC) from 31 excised lobules (14 cancer in situ, 17 noncancer) were examined, and dominant trends described by statistical factors. Three factors were extracted, using principal component factor analysis, which collectively accounted for over 70% of the scatter angle-dependent variation exhibited by the measured data.

Preliminary investigation of ultrasound scattering analysis to identify women at risk for later invasive cancer. II: Extraction of dominant scattering angle-dependent trends from excised breast tissue.

Normalized estimates of the scattering angle-dependent differential scattering cross-section (DSC) at 1.0 MHz were measured from 278 samples of excised breast tissue taken from 66 women. A comparison of results for samples that contained tissue structures previously associated with an increased probability of developing breast cancer to those that did not contain high-risk structures showed that the average magnitude of DSC estimates was insufficient to identify samples with high-risk lesions. Principal component factor analysis (PCFA) was applied to extract scattering angle-dependent trends common to the entire data base. The normalized estimates of the measured DSCs (NDSC) from tissue samples are compared to estimates previously obtained from isolated breast tissue lobules as well as with results from the PCFA. Results are presented that indicate that the dominant angle-dependent trends in the NDSC results are independent of the age of the patient and are similar to trends extracted from isolated breast tissue lobules. The breast tissue structure common to all of these specimens is the terminal duct.

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 99Tc(m)-labelled annexin have comparable affinity for apoptotic cells. In vivo studies with 99Tc(m)-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.

Nuclear medicine applications in molecular imaging: 2007 update.

This review examines several classes of radiolabeled agents, including analogs localizing in somatostatin, benzodiazepine and dopamine receptors; analogs of progesterone and estrogen; and agents localizing in lesions with hypoxia. It concludes the status of agents advocated for detecting angiogenesis and inflammation. The current clinical status of these agents, and their potential roles in diagnosis and treatment are discussed.

Annexin V SPECT imaging of phosphatidylserine expression in patients with dementia.

The authors sought to use radiolabeled annexin V, a marker of phosphatidylserine expression, to image Alzheimer dementia (AD). Four of five patients with AD had multifocal cortical annexin V uptake, whereas all seven non-AD and six control patients had normal SPECT. The mean cortex/cerebellar activity in patients with AD (1.4 +/- 0.6) was higher than that of non-AD dementia patients (0.7 +/- 0.2; p = 0.02). Radiolabeled annexin V may be useful for imaging AD.

Will imaging of apoptosis play a role in clinical care? A tale of mice and men.

Programmed cell death (apoptosis) plays a role in the pathophysiology of many diseases and in the outcome of treatment. Apoptosis is the likely mechanism behind the cytoreductive effects of standard chemotherapeutic and radiation treatments, rejection of organ transplants, cellular damage in collagen vascular disorders, and delayed cell death due to hypoxic-ischemic injury in myocardial infarction and neonatal hypoxic ischemic injury. Observations about the role of apoptosis have fueled the development of novel agents and treatment strategies specifically aimed at inducing or inhibiting apoptosis. Despite these research developments there are no clinical entities where specific measures of apoptosis are used in either diagnosis or patient management. Part of the difficulty in bridging the gap between the basic science understanding of apoptosis and the clinical application of this information is the lack of a sensitive marker to monitor programmed cell death in association with disease progression or regression. Technetium-99m labeled annexin V localizes at sites of apoptosis in-vivo, due to its nanomolar affinity for membrane bound phosphatidylserine. Radiolabeled annexin V imaging permits identification of the site and extent of apoptosis in experimental animals. Annexin V has been successfully used in animal models to image organ transplant rejection, characterize successful therapy of tumors, pinpoint acute myocardial infarction, and identify hypoxic ischemic brain injury of the newborn and adult. Early studies in human subjects suggest that 99mTc annexin imaging will be also be useful to identify rejection in transplant recipients, localize acute myocardial infarction, and characterize the effectiveness of a single treatment in patients with tumors. This review describes the imaging approaches to detect and monitor apoptosis in-vivo that are presently in early clinical trials. The preliminary data are extrapolated to identify conditions where apoptosis imaging may be valuable in clinical decision making. These conditions include: transplant rejection; hypoxic/ischemic injury of heart and brain; and determining the efficacy of therapy in cancer, heart failure and osteoporosis.

Non-invasive diagnosis of acute heart- or lung-transplant rejection using radiolabeled annexin V.

BACKGROUND: Apoptosis is a ubiquitous set of cellular processes by which superfluous or unwanted cells are eliminated in the body without harming adjacent healthy tissues. When apoptosis is inappropriate (too little or too much), a variety of human diseases can occur, including acute heart or lung transplant rejection. OBJECTIVE: Our group has developed a new radiopharmaceutical, radiolabeled annexin V, which can image apoptosis. RESULTS AND CONCLUSION: Here we briefly review the biomolecular basis of apoptosis and its role in acute rejection. We also describe the possible use of radiolabeled annexin V to screen children noninvasively for acute rejection following organ transplantation.