Mathew A. von Wronski

BR55: a lipopeptide-based VEGFR2-targeted ultrasound contrast agent for molecular imaging of angiogenesis.

Objectives:BR55, an ultrasound contrast agent functionalized with a heterodimer peptide targeting the vascular endothelial growth factor receptor 2 (VEGFR2), was evaluated in vitro and in vivo, demonstrating its potential for specific tumor detection. Materials and Methods:The targeted contrast agent was prepared by incorporation of a biospecific lipopeptide into the microbubble membrane. Experiments were performed in vitro to demonstrate the binding capacities of BR55 microbubbles on immobilized receptor proteins and on various endothelial or transfected cells expressing VEGFR2. The performance of BR55 microbubbles was compared with that of streptavidin-conjugated microbubbles targeted to the same receptor by coupling them to a biotinylated antibody. The specificity of BR55 binding to human and mouse endothelial cells was determined in competition experiments with the free lipopeptide, vascular endothelial growth factor (VEGF), or a VEGFR2-specific antibody. Molecular ultrasound imaging of VEGFR2 was performed in an orthotopic breast tumor model in rats using a nondestructive, contrast-specific imaging mode. Results:BR55 was shown to bind specifically to the immobilized recombinant VEGFR2 under flow (dynamic conditions). BR55 accumulation on the target over time was similar to that of microbubbles bearing a specific antibody. BR55 avidly bound to cells expressing VEGFR2, and the pattern of microbubble distribution was correlated with the pattern of receptor expression determined by immunocytochemistry. The binding of targeted microbubbles on cells was competed off by an excess of free lipopeptide, the natural ligand (VEGF) and by a VEGFR2-specific antibody (P < 0.001). Although selected for the human receptor, the VEGFR2-binding lipopeptide was also shown to recognize the rodent receptor. Tumor perfusion was assessed during the vascular phase of BR55, and then the malignant lesion was highlighted by specific accumulation of the targeted microbubbles on tumoral endothelium. The presence of VEGFR2 was confirmed by immunofluorescence staining of tumor cryosections. Conclusions:VEGFR2-targeted ultrasound contrast agents such as BR55 will likely prove useful in human for the early detection of tumors as well as for the assessment of response to specific treatments.

Tuftsin Binds Neuropilin-1 through a Sequence Similar to That Encoded by Exon 8 of Vascular Endothelial Growth Factor

Tuftsin, Thr-Lys-Pro-Arg (TKPR), is an immunostimulatory peptide with reported nervous system effects as well. We unexpectedly found that tuftsin and a higher affinity antagonist, TKPPR, bind selectively to neuropilin-1 and block vascular endothelial growth factor (VEGF) binding to that receptor. Dimeric and tetrameric forms of TKPPR had greatly increased affinity for neuropilin-1 based on competition binding experiments. On endothelial cells tetrameric TKPPR inhibited the VEGF165-induced autophosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) even though it did not directly inhibit VEGF binding to VEGFR-2. Homology between exon 8 of VEGF and TKPPR suggests that the sequence coded for by exon 8 may stabilize VEGF binding to neuropilin-1 to facilitate signaling through VEGFR-2. Given the overlap between processes involving neuropilin-1 and tuftsin, we propose that at least some of the previously reported effects of tuftsin are mediated through neuropilin-1.

Ultrasound molecular imaging of transient acute myocardial ischemia with a clinically translatable P- and E-selectin targeted contrast agent: correlation with the expression of selectins.

ObjectiveThe diagnosis of acute coronary syndrome remains challenging especially in patients without clear symptoms or electrocardiographic and/or biomarker features. A hallmark of ischemia/reperfusion is activation of endothelial cells leading to altered expression of molecular markers, including selectins. In this context, we aimed to validate the value of ultrasound molecular imaging for detecting transient myocardial ischemia by using a clinically translatable dual P- and E-selectin–targeted ultrasound contrast agent (UCA) and microbubble (MBselectin). Material and MethodsTransient (20 minutes) myocardial ischemia of rat heart was produced by ligation of the left anterior descending coronary artery ligation followed by 2-, 5-, or 24-hour reperfusion. Imaging of the transient ischemic event was achieved by the use of MBselectin. Performance of this clinically translatable targeted UCA was compared with that of antibody-targeted streptavidin MBs. Finally, immunohistochemistry staining of rat myocardial ischemic tissue was performed to assess expression of selectins accessible to targeted UCA. ResultsIn rats subjected to myocardial ischemia (20 minutes) followed by reperfusion (2 hours), injection of MBselectin produced high late phase (ie, 10-minute postinjection) ultrasound molecular imaging enhancement in the myocardium, which colocalized with the ischemic area. Late phase enhancement persisted 5 and 24 hours after reperfusion. Similarly, the use of MBP and MBE, comprising antibodies specific for P- and E-selectin, respectively, showed high late-phase enhancement within the ischemic area compared with remote myocardial tissue. Two and 5 hours after ischemia has resolved, a persistent expression of these 2 selectins was detected. After 24 hours of reperfusion, only MBE produced late phase enhancement within the ischemic myocardium. Immunohistochemical findings revealed that both P- and E-selectin were expressed and accessible on the surface of the activated endothelium 2 and 5 hours after the acute ischemic event, whereas only E-selectin remained accessible after 24 hours. ConclusionsUltrasound molecular imaging of transient myocardial ischemia using dual selectin-targeted UCA is able to monitor the time course of expression of selectins after resolution of the ischemic event, paving the way for a large clinical diagnostic window.

Binding of the C-terminal amino acids of VEGF121 directly with neuropilin-1 should be considered

Shraga-Heled et al. reported experimental results in The FASEB Journal indicating that the potency of VEGF121 is enhanced by the coexpression of neuropilin-1 (NP-1) with VEGFR-2 “. . . even though VEGF121 does not bind to neuropilins” (1). Although the authors do not propose a specific mechanism for the phenomenon, they suggest that it may be dependant on spontaneously formed VEGFR-2/NP-1 complexes. We would like to point out an initial report that we coauthored (2) and two subsequent reports (3, 4) indicating that the Cterminal peptide coded for by exon 8 in most isoforms of VEGF-A, including VEGF121 , binds to neuropilin-1. Until the appearance of those reports, the conservation of exon 8 (coding for only 6 amino acids) in the various VEGF-A splice forms had been frequently noted but rarely commented on. Although the interaction between the six C-terminal amino acids of VEGF-A and NP-1 is relatively modest on its own (micromolar affinity), with VEGF tightly bound to VEGFR-2, even a weak interaction between the exon 8-encoded sequence and a nearby neuropilin-1 could have a significant impact on VEGF signaling. When the exon 8-encoded amino acids are replaced, as in the case of VEGF165b, an angiogenesis inhibitor is created that no longer binds to NP-1 (4), indicating that the enhancement of VEGFR-2 signaling by NP-1 involves a direct interaction with the exon 8-encoded peptide. We believe that the binding of the C-terminal amino acids of VEGF121 directly with neuropilin-1 should be considered when interpreting the results reported by Shraga-Heled et al. as it could possibly hasten productive lines of research in the area.

Ultrasound molecular imaging of heterogeneous tumors to guide therapeutic ultrasound

Propagation through heterogeneous tissues may hamper the ability to correctly focus ultrasound, especially when targeting a tumor for drug delivery applications. To solve this issue, we propose to use ultrasound molecular imaging (USMI) for treatment planning of drug delivery triggered by focused ultrasound. In a model of orthotopic rat prostate tumor having heterogeneous B-mode appearance, we show that tumor extend can be precisely delineated with USMI targeting VEGFR-2 receptors. High intensity ultrasound bursts (Peak negative pressure −15 MPa) are then delivered within this delineated 3D volume to trigger the release of liposomal-encapsulated chemotherapy, by local initiation of inertial cavitation. Real time imaging show that cavitation is indeed restricted to the targeted area. In the animal group receiving both chemotherapy and cavitational ultrasound, a potentiation of the therapeutic effect of the drug is clearly observed. This study demonstrates experimentally that USMI is an effective imaging method to characterize heterogeneous tissues and to guide therapeutic ultrasound.