Analysis of angiogenesis induced by local IGF-1 expression after myocardial infarction using microSPECT-CT imaging.

Insulin-like growth factor-1 (IGF-1) has been found to exert favorable effects on angiogenesis in prior animal studies. This study explored the long-term effect of IGF-1 on angiogenesis using microSPECT-CT in infarcted rat hearts after delivering human IGF-1 gene by adeno-associated virus (AAV). Myocardial infarction (MI) was induced in Sprague-Dawley rats by ligation of the proximal anterior coronary artery and a total of 10(11) AAV-CMV-lacZ (control) or IGF-1 vectors were injected around the peri-infarct area. IGF-1 expression by AAV stably transduced heart muscle for up to 16 weeks post-MI and immunohistochemistry revealed a remarkable increase in capillary density. A (99m)Tc-labeled RGD peptide (NC100692, GE Healthcare) was used to assess temporal and regional alpha(v) integrin activation. Rats were injected with NC100692 followed by (201)Tl chloride and in vivo microSPECT-CT imaging was performed. After imaging, hearts were excised and cut for quantitative gamma-well counting (GWC). NC100692 retention was significantly increased in hypoperfused regions of both lacZ and IGF-1 rats at 4 and 16 weeks post-MI. Significantly higher activation of alpha(v) integrin was observed in IGF-1 rats at 4 weeks after treatment compared with control group, although the activation was lower in the IGF-1 group at 16 weeks. Local IGF-1 gene delivery by AAV can render a sustained transduction and improve cardiac function post-MI. IGF-1 expression contributes to enhanced alpha(v) integrin activation which is linked to angiogenesis. MicroSPECT-CT imaging with (99m)Tc-NC100692 and quantitative GWC successfully assessed differences in alpha(v) integrin activation between IGF-1-treated and control animals post-MI.

Serial noninvasive targeted imaging of peripheral angiogenesis: validation and application of a semiautomated quantitative approach.

UNLABELLED: Previous studies by our group have demonstrated the feasibility of noninvasive imaging of alpha(v) integrin to assess temporal and spatial changes in peripheral and myocardial angiogenesis. In this study, we validate the reproducibility, accuracy, and applicability of a new semiautomated noninvasive approach for serial quantitative evaluation of targeted micro-SPECT/CT images of peripheral angiogenesis in wild-type and endothelial nitric oxide sythase (eNOS)-deficient (eNOS-/-) mice subjected to hindlimb ischemia. METHODS: Mice (n = 15) underwent surgical ligation of the right femoral artery to induce unilateral hindlimb ischemia. One week after ligation, a (99m)Tc-labeled cyclic-Arg-Gly-Asp peptide targeted at alpha(v) integrin (NC100692, n = 10) or a (99m)Tc-labeled negative control (AH-111744, n = 5) was injected, and 60 min later in vivo micro-SPECT/CT images were acquired. Mice were euthanized, tissue from proximal and distal hindlimb was excised for gamma-well counting (GWC) of radiotracer activity, and ischemic-to-nonischemic (I/NI) ratio was calculated. Micro-SPECT/CT images were analyzed using a new semiautomated approach that applies complex volumes of interest (VOIs) derived from segmentation of the micro-CT images onto micro-SPECT images to calculate I/NI activity ratios for the proximal and distal hindlimb. Studies were reprocessed for determination of intra- and interobserver variability. To compare 3-dimensional (3D) VOI analysis with traditional manual 2-dimensional region-of-interest (ROI) analysis of maximum-intensity-projection images, micro-SPECT images were summed onto a single anterior-posterior projection. Rectangular ROIs were manually drawn and I/NI ratio calculated. Our new 3D analysis approach was applied to additional groups of mice (eNOS-/-, n = 5; wild-type, n = 3) imaged before and 1 and 4 wk after femoral artery resection. RESULTS: Our new semiautomated approach for the evaluation of images of alpha(v) integrin targeted with micro-SPECT/CT demonstrated both a high intra- and interobserver variability (R(2) = 0.997) and an accuracy (R(2) = 0.780) for estimation of relative radiotracer activity relative to GWC. Analysis of serial micro-SPECT/CT images demonstrated a significant increase in relative NC100692 retention in the ischemic hindlimb of both wild-type and eNOS-/- mice at 1 wk after surgery. There was a significant (approximately 25%) decrease in radiotracer uptake in eNOS-/- mice relative to wild-type animals, which was not observed at baseline or 4 wk after ligation. CONCLUSION: A new semiautomated analysis of images of alpha(v) integrin targeted with micro-SPECT/CT provides a noninvasive approach for serial quantitative evaluation of peripheral angiogenesis. The reproducibility and accuracy of this approach allows for quantitative analysis of serial targeted molecular images of lower extremities, has applicability to other targeted SPECT or PET radiotracers, and may have implications for clinical imaging in patients with peripheral arterial disease.

NC-100717: a versatile RGD peptide scaffold for angiogenesis imaging.

Targeting the molecular pathways associated with angiogenesis offers great potential in detecting disease pathology using in vivo imaging technologies. Initiation of angiogenesis requires activation and migration of endothelial cells in order for neovascularization to proceed. Endothelial cells associate with the extracellular matrix through specific interactions with a variety of cell adhesion receptors known as integrins. Peptides containing the tripeptide sequence RGD are known to bind with high affinity to the alphavbeta3 and alphavbeta5 integrins associated with angiogenesis. We present herein the synthesis and in vitro binding affinity of the RGD-containing peptide NC-100717 and a range of molecular probes derived from this intermediate.

Noninvasive imaging of angiogenesis with a 99mTc-labeled peptide targeted at alphavbeta3 integrin after murine hindlimb ischemia.

BACKGROUND: Noninvasive imaging strategies play a critical role in assessment of the efficacy of angiogenesis therapies. The alpha(v)beta3 integrin is activated in angiogenic vessels and represents a potential target for noninvasive imaging of angiogenesis. METHODS AND RESULTS: We evaluated a 99mTc-labeled peptide (NC100692) targeted at alpha(v)beta3 integrin for imaging in an established murine model of angiogenesis induced by hindlimb ischemia. Control mice (n=9) or mice with surgical right femoral artery occlusion (n=29) were injected with NC100692 (1.5+/-0.2 mCi IV) at different times after femoral occlusion (1, 3, 7, and 14 days) for in vivo pinhole planar gamma camera imaging. Tissue from hindlimb proximal and distal to occlusion was excised for gamma well counting and for immunostaining. On in vivo pinhole images, increased focal NC100692 activity was seen distal to the occlusion at days 3 and 7. This increase in relative NC100692 activity was confirmed by gamma well counting. Lectin staining confirmed increased angiogenesis in the ischemic hindlimb at these time points. A fluorescent analogue of NC100692 was used to confirm specificity and localization of the targeted tracer in cultured endothelial cells. In addition, endothelial cell specificity was confirmed on tissue sections with the use of dual immunofluorescent staining of endothelium and the fluorescent analogue targeted at the alpha(v)beta3 integrin. CONCLUSIONS: A 99mTc-labeled peptide (NC100692) targeted at alpha(v)beta3 integrin selectively localized to endothelial cells in regions of increased angiogenesis and could be used for noninvasive serial "hot spot" imaging of angiogenesis. This targeted radiotracer imaging approach is a major advance in tracking therapeutic myocardial angiogenesis and has an important clinical potential.

In vivo imaging of human colon cancer xenografts in immunodeficient mice using a guanylyl cyclase C--specific ligand.

UNLABELLED: Guanylyl cyclase C (GC-C) is a transmembrane receptor expressed by human intestinal cells and primary and metastatic colorectal adenocarcinomas but not by extraintestinal tissues or tumors. The Escherichia coli heat-stable enterotoxin analog, STa (5--18), is a 14--amino acid peptide that selectively binds to the extracellular domain of GC-C with subnanomolar affinity. This study examined the utility of a radiolabeled conjugate of STa (5--18) to selectively target and image extraintestinal human colon cancer xenografts in vivo in nude mice. METHODS: The STa conjugate, ethoxyethyl-mercaptoacetamidoadipoylglycylglycine-STa (5--18) (NC100586), was synthesized and labeled with (99m)Tc to produce (99m)Tc-NC100586. This compound was intravenously administered to nude mice bearing human colon cancer xenografts, and specific targeting was evaluated by biodistribution and gamma camera imaging. RESULTS: In CD-1 nude mice, biodistribution and scintigraphic imaging analyses showed selective uptake of (99m)Tc-NC100586 into human colon cancer xenografts that express GC-C but not into normal tissues that do not express GC-C. Similarly, (99m)Tc-NC100586 injected intravenously into CD-1 nude mice with human colon cancer hepatic metastases selectively accumulated in those metastases, and about 5-mm foci of tumor cells were visualized after ex vivo imaging of excised livers. Accumulation of (99m)Tc-NC100586 in human colon cancer xenografts reflected binding to GC-C because (99m)Tc-NC100588, an inactive analog that does not bind to GC-C, did not selectively accumulate in cancer xenografts compared with normal tissues. Also, coadministration of excess unlabeled STa (5--18) prevented accumulation of (99m)Tc-NC100586 in human colon cancer xenografts. Furthermore, (99m)Tc-NC100586 did not selectively accumulate in Lewis lung tumor xenografts, which do not express GC-C. CONCLUSION: This study showed that intravenously administered STa (5--18) selectively recognizes and binds to GC-C expressed by human colon cancer cells in vivo. Also shown was the ability to exploit this selective interaction to target imaging agents to extraintestinal human colon tumors in nude mice. These results suggest the utility of STa and GC-C for the development of novel targeted imaging and therapeutic agents with high specificity for metastatic colorectal tumors in humans.