Zoia Levashova

Analysis of In Situ and Ex Vivo Vascular Endothelial Growth Factor Receptor Expression During Experimental Aortic Aneurysm Progression

Objective—Mural inflammation and neovascularization are characteristic pathological features of abdominal aortic aneurysm (AAA) disease. Vascular endothelial growth factor receptor (VEGFR) expression may also mediate AAA growth and rupture. We examined VEGFR expression as a function of AAA disease progression in the Apolipoprotein E–deficient (Apo E−/−) murine AAA model. Methods and Results—Apo E−/− mice maintained on a high-fat diet underwent continuous infusion with angiotensin II at 1000 ng/kg/min (Ang II) or vehicle (Control) via subcutaneous osmotic pump. Serial transabdominal ultrasound measurements of abdominal aortic diameter were recorded (n=16 mice, 3 to 4 time points per mouse) for up to 28 days. Near-infrared receptor fluorescent (NIRF) imaging was performed on Ang II mice (n=9) and Controls (n=5) with scVEGF/Cy, a single-chain VEGF homo-dimer labeled with Cy5.5 fluorescent tracer (7 to 18 &mgr;g/mouse IV). NIRF with inactivated single chain VEGF/Cy tracer (scVEGF/In, 18 &mgr;g/mouse IV) was performed on 2 additional Ang II mice to control for nonreceptor-mediated tracer binding and uptake. After image acquisition and sacrifice, aortae were harvested for analysis. An additional AAA mouse cohort received either an oral angiogenesis inhibitor or suitable negative or positive controls to clarify the significance of angiogenesis in experimental aneurysm progression. Aneurysms developed in the suprarenal aortic segment of all Ang II mice. Significantly greater fluorescent signal was obtained from aneurysmal aorta as compared to remote, uninvolved aortic segments in Ang II scVEGF/Cy mice or AAA in scVEGF/In mice or suprarenal aortic segments in Control mice. Signal intensity increased in a diameter-dependent fashion in aneurysmal segments. Immunostaining confirmed mural VEGFR-2 expression in medial smooth muscle cells. Treatment with an angiogenesis inhibitor attenuated AAA formation while decreasing mural macrophage infiltration and CD-31+ cell density. Conclusion—Mural VEGFR expression, as determined by scVEGF/Cy fluorescent imaging and VEGFR-2 immunostaining, increases in experimental AAAs in a diameter-dependent fashion. Angiogenesis inhibition limits AAA progression. Clinical VEGFR expression imaging strategies, if feasible, may improve real-time monitoring of AAA disease progression and response to suppressive strategies.

Direct site-specific labeling of the Cys-tag moiety in scVEGF with technetium 99m.

Angiogenesis is a fundamental feature of tumor development, and therefore, the tracers for molecular imaging of specific angiogenic biomarkers are expected to be useful for diagnostics, patient monitoring, and drug development. We have created a new class of imaging agents based on the most important mediator of angiogenesis, vascular endothelial growth factor (VEGF). Our latest version is a single-chain (sc) VEGF protein containing an N-terminal Cys-tag designed for site-specific modification with a variety of imaging and therapeutic moieties. We have recently found that the Cys-tag itself can form a stable chelate with (99m)Tc using tin-tricine as an exchange reagent. This self-chelation approach yields a highly stable and fully functional form of radiolabeled scVEGF that can be used as a SPECT tracer for tumor angiogenesis. Also of note is that directly labeled scVEGF has less than one-half the nonspecific renal uptake of (99m)Tc-HYNIC-scVEGF. The simple production of scVEGF for direct chelation of (99m)Tc makes it a promising molecular imaging agent for the oncology clinic.

Molecular Imaging of Changes in the Prevalence of Vascular Endothelial Growth Factor Receptor in Sunitinib-Treated Murine Mammary Tumors

Several drugs targeting vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) are approved for cancer treatment. However, these drugs induce relatively modest and frequently unpredictable tumor responses. In this work, we explored whether noninvasive imaging of VEGFR, a direct target of antiangiogenic drugs, can provide real-time information on responses to the treatment with sunitinib, a small-molecule VEGFR inhibitor approved by the Food and Drug Administration. Methods: We imaged VEGFR in an orthotopic mammary tumor model during the course of treatment with sunitinib using a recently developed SPECT tracer, a 99mTc-labeled single-chain VEGF (scVEGF), that binds to and is internalized by VEGFR. Tumors from imaged mice were harvested and cryosectioned, and alternating sections were analyzed by autoradiography and immunohistochemistry to determine the expression of endothelial cell markers VEGFR-2 and CD31. Results: In vitro assays with endothelial cells overexpressing VEGFR-2 established that sunitinib does not inhibit VEGFR-2–mediated uptake of scVEGF-based tracers. SPECT and autoradiography with 99mTc-scVEGF of tumor cryosections revealed a 2.2- to 2.6-fold decrease in tracer uptake after 4 daily doses of sunitinib. However, once treatment was discontinued, tracer uptake rapidly (3 d) increased, particularly at the tumor edges. Immunohistochemical analysis of VEGFR-2 and CD31 supported SPECT and autoradiographic imaging findings, revealing the corresponding depletion of VEGFR-2– and CD31-positive endothelial cells from tumor vasculature during therapy and the rapid reemergence of VEGFR-2– and CD31-positive vasculature at the tumor edges after discontinuation of treatment. Conclusion: Our findings suggest that imaging with 99mTc-scVEGF might be useful for monitoring VEGFR responses to antiangiogenic treatment regimens.

Cysteine-containing fusion tag for site-specific conjugation of therapeutic and imaging agents to targeting proteins

Targeted delivery of therapeutic and imaging agents requires conjugation of a corresponding payload to a targeting peptide or protein. The ideal procedure should yield a uniform preparation of functionally active conjugates and be translatable for development of clinical products. We describe here our experience with site-specific protein modification via a novel cysteine-containing fusion tag (Cys-tag), which is a 15-amino-acid (aa) N-terminal fragment of human ribonuclease I with the R4C substitution. Several Cys-tagged proteins and peptides with different numbers of native cysteines were expressed and refolded into functionally active conformation, indicating that the tag does not interfere with the formation of internal disulfide bonds. We also describe standardized procedures for site-specific conjugation of very different payloads, such as functionalized lipids and liposomes, radionuclide chelators and radionuclides, fluorescent dyes, drug-derivatized dendrimers, scaffold proteins, biotin, and polyethyleneglycol to Cys-tagged peptides and proteins, as well as present examples of functional activity of targeted conjugates in vitro and in vivo. We expect that Cys-tag would provide new opportunities for development of targeted therapeutic and imaging agents for research and clinical use.

Imaging Vascular Endothelial Growth Factor (VEGF) Receptors in Turpentine-Induced Sterile Thigh Abscesses with Radiolabeled Single-Chain VEGF

Angiogenesis plays a central role in the pathogenesis of chronic inflammatory disorders. Vascular endothelial growth factor (VEGF) and its receptors are the most important regulators of angiogenesis. We wished to determine whether labeled forms of single-chain VEGF (scVEGF) could be used to image VEGF receptors in a well-characterized model of sterile soft-tissue inflammation induced by intramuscular injection of turpentine. Methods: Anesthetized adult male Swiss–Webster mice received a 20-μL intramuscular injection of turpentine into the right thigh. At 4, 7, or 10 d later, groups of 3–5 mice were injected via the tail vein with 50 μg of either scVEGF that had been site specifically labeled with Cy5.5 (scVEGF/Cy) or inactivated scVEGF/Cy (inVEGF/Cy) and then examined by fluorescence imaging. At 3, 4, 6, 7, 9, 10, or 12 d, additional groups of 3–5 mice were injected via the tail vein with 74–111 MBq of 99mTc-scVEGF (or 99mTc-inVEGF) and then examined by SPECT imaging. Results: On days 3 through 10, both forms of scVEGF (scVEGF/Cy and 99mTc-scVEGF) showed significantly higher uptake (P < 0.05) in the right (abscessed) thigh than in the contralateral thigh (and higher uptake than the inactivated tracer). Peak uptake occurred on day 7 (3.67 ± 1.79 [ratio of uptake in abscessed thigh to uptake in normal thigh, mean ± SD] and 0.72 ± 0.01 for scVEGF/Cy and inVEGF/Cy, respectively, and 3.49 ± 1.22 and 1.04 ± 0.41 for 99mTc-scVEGF and 99mTc-inVEGF, respectively) and slowly decreased thereafter. Autoradiography revealed peak tracer uptake in the thick irregular angiogenic rim of the abscess cavity on day 9 (5.83 × 10−7 ± 9.22 × 10−8 and 5.85 × 10−8 ± 5.95 × 10−8 percentage injected dose per pixel for 99mTc-scVEGF and 99mTc-inVEGF, respectively); in comparison, a thin circumscribed rim of uptake was seen with 99mTc-inVEGF. Immunostaining revealed that VEGFR-2 (VEGF receptor) colocalized with CD31 (endothelial cell marker) at all time points in the abscess rim, whereas F4/80 (macrophage) immunostaining reached a maximum at day 7 and decreased by day 10. Conclusion: The uptake of scVEGF in turpentine-induced abscesses was specific and directly related to VEGFR-2 expression in the neovasculature of the angiogenic rim. Peak tracer uptake coincided with maximum macrophage infiltration, suggesting that scVEGF imaging may be useful for the detection, localization, and monitoring of chronic inflammation in bone, joints, or soft tissues.

Characterization of a Recombinant Form of Annexin VI for Detection of Apoptosis

We developed a recombinant form of human annexin VI called annexin VI-601 (M(r) 76,224) with the N-terminal extension of Ala-Gly-Gly-Cys-Gly-His to allow ready attachment of fluorescent or radioactive labels. The protein was produced by expression in E. coli and was purified by calcium-dependent membrane binding, anion-exchange chromatography, and heparin-Sepharose affinity chromatography. The protein could be readily labeled with iodoacetamidofluorescein and with (99m)Tc. The protein bound with high affinity to PS-containing phospholipid vesicles and to erythrocytes with exposed phosphatidylserine. Fluorescent annexin VI-601 readily detected apoptosis of Jurkat cells by flow cytometry at much lower calcium concentrations than those required for equivalent detection by annexin V. In vivo administration of radiolabeled protein showed that blood clearance was much slower than annexin V. In conclusion, annexin VI may have advantages over annexin V in certain situations for both in vitro and in vivo detection of apoptosis and therapeutic targeting of PS due to its lower calcium requirement for membrane binding and its higher molecular weight.

Additional file 1: Figure S1. of Targeted scVEGF/177Lu radiopharmaceutical inhibits growth of metastases and can be effectively combined with chemotherapy

Representative image of mouse with 4T1luc bone metastatic lesions. CT and high resolution MicroCT of bone lesion areas show significant pitting and large areas of osteolysis. (PDF 118 kb)