Fast clearing RGD‐based near‐infrared fluorescent probes for in vivo tumor diagnosis

A fast clearing hydrophilic near‐infrared (NIR) dye ICG‐Der‐02 was used to constitute tumor targeting contrast agents. Cell adhesion molecule integrin α_vβ₃ served as the target receptor because of its unique expression on almost all sprouting tumor vasculatures. The purpose of this study was to synthesize and compare the properties of integrin α_vβ₃‐targeted, fast clearing NIR probes both in vitro and in vivo for tumor diagnosis. ICG‐Der‐02 was covalently conjugated to three kinds of RGD peptide including linear, monoeric cyclic and dimeric RGD to form three RGD‐based NIR probes. The integrin receptor specificities of these probes were evaluated in vitro by confocal microscopy. The dynamic bio‐distribution and elimination ratse were in vivo real‐time monitored by a near‐infrared imaging system in normal mice. Further, the in vivo tumor targeting abilities of the RGD‐based NIR probes were compared in α_vβ₃‐positive MDA‐MB‐231, U87MG and α_vβ₃‐negtive MCF‐7 xenograft mice models. Three RGD‐based NIR probes were successfully synthesized with good optical properties. In vitro cellular experiments indicated that the probes have a clear binding affinity to α_υβ₃‐positive tumor cells, with a cyclic dimeric RGD probe owing the highest integrin affinity. Dynamic bio‐distributions of these probes showed a rapid clearing rate through the renal pathway. In vivo tumor targeting ability of the RGD‐based porbes was demonstrated on MDA‐MB‐231 and U87MG tumor models. As expected, the c(RGDyK)₂‐ICG‐Der‐02 probe displayed the highest tumor‐to‐normal tissue contrast. The in vitro and in vivo block experiments confirmed the receptor binding specificity of the probes. The hydrophilic dye‐labeled NIR probes exhibited a fast clearing rate and deep tissue penetration capability. Further, the α_υβ₃ receptor affinity of the three RGD‐based NIR probes followed the order of dimer cyclic > monomer cyclic > linear. The results demonstrate potent fast clearing probes for in vivo early tumor diagnosis. Copyright © 2012 John Wiley & Sons, Ltd.     

Optical imaging detection of microscopic mammary cancer in ErbB‐2 transgenic mice through the DA364 probe binding αvβ3 integrins

Despite spontaneous tumor growth in genetically engineered mice being one of the most recognized tools for the in vivo evaluation of novel diagnostic and therapeutic anticancer compounds, monitoring early stage lesions in live animals is a goal that has yet to be achieved. A large number of targets for the molecular imaging of various diseases have been identified and many imaging technologies, including optical techniques are emerging. One of the most commonly exploited targets in tumor imaging is α_vβ₃ integrin, which plays an important role in the expansion, invasiveness and metastatic capability of a number of cancers, including breast cancer. The aim of this study was to set up an optical imaging method for the early detection of autochthonous mammary cancer in female BALB/c mice transgenic for the rat‐ErbB‐2 oncogene (BALB‐neuT). We show that DA364, a near‐infrared fluorescence arginine–glycine–aspartic acid cyclic probe, was taken up by neoplastic mammary glands and that its uptake increased with cancer progression. By contrast, the nonaccumulation of DA364 in the healthy mammary glands of virgin and lactating wild‐type mice suggests that the probe specifically targets breast cancers. Comparisons of optical imaging with whole‐mount and histological findings showed that DA364 allows the noninvasive visualization of atypical hyperplasia and microscopic foci of in situ carcinoma 2 months before mammary lesions become detectable by palpation. Moreover, DA364 was successfully used to monitor the outcome of anticancer vaccination. Therefore, it can be considered a promising early detection tool in near‐infrared noninvasive optical imaging for the early diagnosis of breast cancer. Copyright © 2013 John Wiley & Sons, Ltd.     

RGD‐ligand mimetic antagonists of integrin αIIbβ3 paradoxically enhance GPVI‐induced human platelet activation

Summary. Background: The integrin α_IIbβ₃ is the major mediator of platelet aggregation and has, therefore, become an important target of antithrombotic therapy. Antagonists of α_IIbβ₃, for example abciximab, tirofiban and eptifibatide, are used in the treatment of acute coronary syndromes. However, in addition to effective blockade of the integrin, binding of can induce conformational changes in the integrin and can also induce integrin clustering. This class effect of RGD‐ligand mimetics might, therefore, underlie paradoxical platelet activation and thrombosis previously reported. Objectives: To examine the components of signaling pathways and functional responses in platelets that may underlie this phenomenon of paradoxical platelet activation. Methods: We assessed the effect of lotrafiban, and other α_IIbβ₃ antagonists including the clinically used drug tirofiban, on tyrosine phosphorylation of key signaling proteins in platelets by immunoblotting and also platelet functional outputs such as cytosolic calcium responses, phosphatidylserine exposure (pro‐coagulant activity) and dense granule release. Results: In all cases, no effect of α_IIbβ₃ antagonists were observed on their own, but these integrin antagonists did lead to a marked potentiation of glycoprotein VI (GPVI)‐associated FcR γ‐chain phosphorylation, activation of Src family kinases and Syk kinase. This correlated with increased dense granule secretion, cytosolic calcium response and exposure of phosphatidylserine on the platelet surface. P2Y₁₂ antagonism abolished the potentiated phosphatidylserine exposure and dense granule secretion but not the cytosolic calcium response. Conclusions: These data provide a mechanism for enhancement of platelet activity by α_IIbβ₃ inhibitors, but also reveal a potentially important signaling pathway operating from the integrin to GPVI signaling.     

A novel Tc‐99 m and fluorescence labeled peptide as a multimodal imaging agent for targeting angiogenesis in a murine tumor model

The serine–aspartic acid–valine (SDV) peptide binds specifically to integrin α_Vβ₃. In the present study, we successfully developed a TAMRA–GHEG–ECG–SDV peptide labeled with both Tc‐99 m and TAMRA to target the integrin α_Vβ₃ of tumor cells; furthermore, we evaluated the diagnostic performance of Tc‐99 m TAMRA–GHEG–ECG–SDV as a dual‐modality imaging agent for tumor of the murine model. TAMRA–GHEG–ECG–SDV was synthesized using Fmoc solid‐phase peptide synthesis. Radiolabeling of TAMRA–GHEG–ECG–SDV with Tc‐99 m was done using ligand exchange methods. Labeling stability and cytotoxicity studies were performed. Gamma camera imaging, biodistribution and ex vivo imaging studies were performed in murine models with HT‐1080 and HT‐29 tumors. A tumor tissue slide was prepared and analyzed using confocal microscopy. After radiolabeling procedures with Tc‐99 m, the Tc‐99 m TAMRA–GHEG–ECG–SDV complexes were prepared in high yield (>99%). In the gamma camera imaging study, a substantial uptake of Tc‐99 m TAMRA–GHEG–ECG–SDV into HT‐1080 tumor (integrin α_Vβ₃ positive) and low uptake of Tc‐99 m TAMRA–GHEG–ECG–SDV into HT‐29 tumor (integrin α_Vβ₃ negative) were demonstrated. A competition study revealed that HT‐1080 tumor uptake was effectively blocked by the co‐injection of an excess concentration of SDV. Specific uptake of Tc‐99 m TAMRA–GHEG–ECG–SDV was confirmed by biodistribution, ex vivo imaging and confocal microscopy studies. Our in vivo and in vitro studies revealed substantial uptake of Tc‐99 m TAMRA–GHEG–ECG–SDV in the integrin α_Vβ₃‐positive tumor. Tc‐99 m TAMRA–GHEG–ECG–SDV could be a good candidate for a dual‐modality imaging agent targeting tumor angiogenesis. Copyright © 2016 John Wiley & Sons, Ltd.     

Improvements in endotoxemic syndromes using a disintegrin,rhodostomin, through integrin αvβ3‐dependent pathway

Summary. Background and objectives: Septic shock is a major cause of morbidity and mortality in intensive care units, but there is still no effective therapy for the patients. We evaluated the effects of rhodostomin (Rn), an Arg‐Gly‐Asp‐containing snake venom disintegrin, on lipopolysaccharide (LPS)‐activated phagocytes in vitro and LPS‐induced endotoxemia in vivo. Methods and results: Rn inhibited adhesion, migration, cytokine production and mitogen‐activated protein kinase (MAPK) activation of macrophage induced by LPS. Flow cytometric analysis revealed that Rn specifically blocked anti‐αv mAb binding to RAW264.7. Besides inhibiting MAPK activation of THP‐1, Rn bound to LPS‐activated THP‐1 and specifically blocked anti‐αvβ3 mAb binding to THP‐1. Binding assays proved that integrin αvβ3 was the binding site for rhodostomin on phagocytes. Rn reversed the enhancement of fibronectin and vitronectin on LPS‐induced monocyte adhesion and cytokine release. Transfection of integrin αv siRNA also inhibited LPS‐induced activation of monocyte, and Rn exerted no further inhibitory effect. Furthermore, Rn significantly decreased the production of tumor necrosis factor‐α (TNF‐a), interleukin (IL)‐6, ‐1β and ‐10 and attenuated cardiovascular dysfunction, including blood pressure and heart pulse, and thrombocytopenia in LPS‐induced endotoxemic mice. Rn also protected against tissue inflammation as evidenced by histological examination. Conclusions: Rn may interact with αvβ3 integrin of monocytes/macrophages leading to interfere with the activation of phagocytes triggered by LPS. These results suggest that the protective function of Rn in LPS‐induced endotoxemia may be attributed to its anti‐inflammation activities in vivo.     

Vicinal thiols are required for activation of the αIIbβ3 platelet integrin

Summary. Background: Closely spaced thiols in proteins that interconvert between the dithiol form and disulfide bonds are called vicinal thiols. These thiols provide a mechanism to regulate protein function. We previously found that thiols in both αIIb and β3 of the αIIbβ3 fibrinogen receptor were required for platelet aggregation. Methods and Results: Using p‐chloromercuribenzene sulfonate (pCMBS) we provide evidence that surface thiols in αIIbβ3 are exposed during platelet activation. Phenylarsine oxide (PAO), a reagent that binds vicinal thiols, inhibits platelet aggregation and labeling of sulfhydryls in both αIIb and β3. For the aggregation and labeling studies, binding of PAO to vicinal thiols was confirmed by reversal of PAO binding with the dithiol reagent 2,3‐Dimercapto‐1‐propanesulfonic acid (DMPS). In contrast, the monothiol β‐mercaptoethanol did not reverse the effects of PAO. Additionally, PAO did not inhibit sulfhydryl labeling of the monothiol protein albumin, confirming the specificity of PAO for vicinal thiols in αIIbβ3. As vicinal thiols represent redox sensitive sites that can be regulated by reducing equivalents from the extracellular or cytoplasmic environment, they are likely to be important in regulating activation of αIIbβ3. Additionally, when the labeled integrin was passed though a lectin column containing wheat germ agglutinin and lentil lectin a substantial amount of non‐labeled αIIbβ3 eluted separately from the labeled receptor. This suggests that two populations of integrin exist on platelets that can be distinguished by thiol labeling. Conclusion: A vicinal thiol‐containing population of αIIbβ3 provides redox sensitive sites for regulation of αIIbβ3.     

In Vivo Near-Infrared Fluorescence Imaging of Integrin αvβ3 in an Orthotopic Glioblastoma Model

Purpose Expression of cell adhesion molecule integrin α_vβ₃ is significantly up-regulated during tumor growth, and sprouting of tumor vessels and correlates well with tumor aggressiveness. The purpose of this study was to visualize tumor integrin α_vβ₃ expression in vivo by using near-infrared fluorescence (NIRF) imaging of Cy5.5-linked cyclic arginine–glycine–aspartic acid (RGD) peptide in an orthotopic brain tumor model.Procedures U87MG glioma cells transfected with the firefly luciferase gene were stereotactically injected into nude mice in the right frontal lobe. Bioluminescence imaging (BLI) using d-luciferin substrate and small animal magnetic resonance imaging (MRI) using gadolinium contrast enhancement were conducted weekly after tumor cell inoculation to monitor intracranial tumor growth. Integrin α_vβ₃ expression was assessed by using a three-dimensional optical imaging system (IVIS 200) 0–24 hours after administration of 1.5 nmol monomeric Cy5.5-RGD via the tail vein. Animals were injected intravenously with both Texas Red–tomato lectin and Cy5.5-RGD prior to sacrifice to visualize peptide localization to tumor vasculature using histology.Results Fluorescence microscopy demonstrated specific Cy5.5-RGD binding to both U87MG tumor vessels and tumor cells with no normal tissue binding. NIRF imaging showed highest tumor uptake and tumor to normal brain tissue ratio two hours postinjection (2.64 ± 0.20). Tumor uptake of Cy5.5-RGD was effectively blocked by using unlabeled c(RGDyK), and injection of Cy5.5 dye alone showed nonspecific binding.Conclusions Optical imaging via BLI and NIRF offer a simple, effective, and rapid technique for noninvasive in vivo monitoring and semiquantitative analysis of intracranial tumor growth and integrin α_vβ₃ expression. This study suggests that NIRF via fluorescently labeled RGD peptides may provide enhanced surveillance of tumor angiogenesis and anti-integrin treatment efficacy in orthotopic brain tumor models.     

CD40 ligand shedding is regulated by interaction between matrix metalloproteinase‐2 and platelet integrin αIIbβ3

Summary. Background: CD40 ligand (CD40L, CD154) in the circulatory system is mainly contained in platelets, and surface‐expressed CD40L on activated platelets is subsequently cleaved by proteolytic activity to generate soluble CD40L (sCD40L). However, the enzyme responsible for the shedding of CD40L in activated platelets has not been clearly identified yet. We have recently found that molecular interaction of matrix metalloproteinase‐2 (MMP‐2) with integrin α_IIbβ₃ is required for the enhancement of platelet activation. Objectives: To elucidate the biochemical mechanism of MMP‐2‐associated sCD40L release. Methods: Localization of MMP‐2 and CD40L in platelets was analyzed by flow cytometry and fluorescence microscopy. The release of sCD40L from activated platelets was measured by enzyme‐linked immunosorbent assay. MMP‐2 binding to α_IIbβ₃ was analyzed by immunoprecipitation and western blotting. Recombinant hemopexin‐like domain and MMP‐2‐specific inhibitor were used to characterize the nature of MMP‐2 binding and catalytic activity. Results: It was revealed that interaction of MMP‐2 with α_IIbβ₃ is required for effective production of sCD40L in activated human platelets. Platelet activation and release of sCD40L were significantly affected by inhibition of platelet‐derived MMP‐2 activity or by inhibition of binding between the enzyme and the integrin. It was also found in platelet‐rich plasma that MMP‐2 activity is responsible for generating sCD40L. Conclusions: The results presented here strongly suggest that MMP‐2 interacts with α_IIbβ₃ to regulate the shedding of CD40L exposed on the surfaces of activated human platelets.     

Near-Infrared Fluorescence Imaging of Tumor Integrin αvβ3 Expression with Cy7-Labeled RGD Multimers

Purpose Cell adhesion molecule integrin α_vβ₃ is an excellent target for tumor interventions because of its unique expression on the surface of several types of solid tumor cells and on almost all sprouting tumor vasculatures. Here, we describe the development of near-infrared (NIR) fluorochrome Cy7-labeled RGD peptides for tumor integrin targeting.Procedures Mono-, di-, and tetrameric RGD peptides were synthesized and conjugated with Cy7. The integrin specificity of these fluorescent probes was tested in vitro for receptor binding assay and fluorescence microscopy and in vivo for subcutaneous U87MG tumor targeting.Results The tetrameric RGD peptide probe with the highest integrin affinity showed the highest tumor activity accumulation and strongest tumor-to-normal tissue contrast. This uptake is integrin-specific as the signal accumulated in the tumor can be effectively blocked by unconjugated RGD peptide antagonist of integrin α_vβ₃.Conclusions Noninvasive NIR fluorescence imaging is able to detect and semiquantify tumor integrin expression based upon the highly potent tetrameric RGD peptide probe.     

Dual In Vivo Quantification of Integrin-targeted and Protease-activated Agents in Cancer Using Fluorescence Molecular Tomography (FMT)

Purpose

Integrins, especially α_vβ₃ and α_vβ₅, are upregulated in tumor cells and activated endothelial cells and as such, serve as cancer biomarkers. We developed a novel near-infrared-labeled optical agent for the in vivo detection and quantification of α_vβ₃/α_vβ₅.

Procedures

A small peptidomimetic α_vβ₃ antagonist was synthesized, coupled to a near-infrared fluorescent (NIRF) dye, and tested for binding specificity using integrin-overexpressing cells, inhibition of vitronectin-mediated cell attachment, binding to tumor and endothelial cells in vitro, and competition studies. Pharmacokinetics, biodistribution, specificity of tumor targeting, and the effect of an antiangiogenic treatment were assessed in vivo.

Results

The integrin NIRF agent showed strong selectivity towards α_vβ_3/α_vβ₅ in vitro and predominant tumor distribution in vivo, allowing noninvasive and real-time quantification of integrin signal in tumors. Antiangiogenic treatment significantly inhibited integrin signal in vivo but had no effect on a cathepsin-cleavable NIR agent. Simultaneous imaging revealed different patterns of distribution reflecting the underlying differences in integrin and cathepsin biology during tumor progression.

Conclusions

NIRF-labeled integrin antagonists allow noninvasive molecular fluorescent imaging and quantification of tumors in vivo, improving and providing more refined approaches for cancer detection and treatment monitoring.     

A role for adhesion and degranulation‐promoting adapter protein in collagen‐induced platelet activation mediated via integrin α2β1

Summary. Background: Collagen‐induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α₂β₁. Adhesion and degranulation‐promoting adapter protein (ADAP) regulates α_IIbβ₃ in platelets and α_Lβ₂ in T cells, and is phosphorylated in GPVI‐deficient platelets activated by collagen. Objectives: To determine whether ADAP plays a role in collagen‐induced platelet activation and in the regulation and function of α₂β₁. Methods: Using ADAP^?/? mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. Results and Conclusions: Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP^?/? platelets. However, aggregation and signaling induced by collagen‐related peptide (CRP), a GPVI‐selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α₂β₁‐selective ligand GFOGER and to a peptide (III‐04), which supports adhesion that is dependent on both GPVI and α₂β₁, was reduced in ADAP^?/? platelets. An impedance‐based label‐free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non‐fluorescent differential‐interference contrast microscopy, which revealed reduced filpodia formation in ADAP^?/? platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen‐binding integrin α₂β₁. In addition, we found that ADAP^?/? mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild‐type animals. This may reflect increased removal of platelets from the circulation.     

A mutation in the β3 cytoplasmic tail causes variant Glanzmann thrombasthenia by abrogating transition of αIIbβ3 to an active state

Summary. Background: The cytoplasmic tails of α_IIb and β₃ regulate essential α_IIbβ₃ functions. We previously described a variant Glanzmann thrombasthenia mutation in the β₃ cytoplasmic tail, IVS14: ?3C>G, which causes a frameshift with an extension of β₃ by 40 residues. Objectives: The aim of this study was to characterize the mechanism by which the mutation abrogates transition of α_IIbβ₃ from a resting state to an active state. Methods: We expressed the natural mutation, termed 742ins, and three artificial mutations in baby hamster kidney (BHK) cells along with wild‐type (WT) α_IIb as follows: β₃‐742stop, a truncated mutant to evaluate the effect of deleted residues; β₃‐749stop, a truncated mutant that preserves the NPLY conserved sequence; and β₃‐749ins, in which the aberrant tail begins after the conserved sequence. Flow cytometry was used to determine ligand binding to BHK cells. Results and conclusions: Surface expression of α_IIbβ₃ of all four mutants was at least 60% of WT expression, but there was almost no binding of soluble fibrinogen following activation with activating antibodies (anti‐ligand‐induced‐binding‐site 6 [antiLIBS6] or PT25‐2). Activation of the α_IIbβ₃ mutants was only achieved when both PT25‐2 and antiLIBS6 were used together or following treatment with dithiothreitol. These data suggest that the ectodomain of the four mutants is tightly locked in a resting conformation but can be forced to become active by strong stimuli. These data and those of others indicate that the middle part of the β₃ tail is important for maintaining α_IIbβ₃ in a resting conformation.     

An αIIb mutation in patients with Glanzmann thrombasthenia located in the N‐terminus of blade 1 of the β‐propeller (Asn2Asp) disrupts a calcium binding site in blade 6

Summary. Background: Studies of Glanzmann thrombasthenia (GT)‐causing mutations has generated invaluable information on the formation and function of integrin αIIbβ₃. Objective: To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations. Methods and Results: Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the β‐propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbβ₃ was found in patients’ platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT β₃. Although the αIIbβ₃ was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbβ₃ crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of αIIbβ₃. Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbβ₃ surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbβ₃, αVβ₃ harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVβ₃ to calcium chelation compared with αIIbβ₃. Conclusion: The new GT causing mutation highlights the importance of calcium binding domains in the β‐propeller for intracellular trafficking of αIIbβ₃. The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.     

Triple heterozygosity in the integrin αIIb subunit in a patient with Glanzmann's thrombasthenia

Summary. We report triple heterozygosity in the integrin α_IIb subunit in a 5‐year‐old Canadian girl with Glanzmann's thrombasthenia. The patient has a severe bleeding history possibly aggravated by low VWF suggestive of associated type 1 von Willebrand's disease. Platelet aggregation was absent or severely reduced for all physiologic agonists. Flow cytometry showed an ～ 4% residual surface expression of α_IIbβ₃. Western blotting confirmed a low platelet expression of both subunits. PCR‐SSCP and direct sequencing showed no abnormalities in the β₃ gene, but revealed a G→A transition at a splice site [IVS 19 (+1)] of exon 19 in the α_IIb gene. Of maternal inheritance, the splice site mutation was associated with intermediate levels of α_IIbβ₃ in carriers. Unexpectedly, two G→A transitions were detected in exon 29 of the α_IIb gene and led to V⁹⁵¹→M and A⁹⁵⁸→T amino acid substitutions. Family studies using restriction enzymes showed that both exon 29 mutations were paternal in origin and cosegregated across three generations. Transient expression in which mutated α_IIb was cotransfected with wild‐type β₃ in COS‐7 cells showed that V⁹⁵¹→M gave a much reduced surface expression of α_IIbβ₃ and a block in the maturation of pro‐α_IIb. In contrast, the A⁹⁵⁸ substitution appeared to be a novel polymorphism. Our studies highlight an unusual mixture of defects giving rise to severe bleeding in a child and describe the first pathological missense mutation affecting a C‐terminal residue of the calf‐2 domain of α_IIb.     

A novel PET tracer for the imaging of αvβ3 and αvβ5 integrins in experimental breast cancer bone metastases

The aim of this study was the evaluation of ⁶⁸Ga‐DOTA‐E‐[c(RGDfK)]₂ as a novel PET tracer to image αvβ3 and αvβ5 integrins. For this purpose, DOTA‐E‐[c(RGDfK)]₂ was labeled with ⁶⁸Ga, which was obtained from a ⁶⁸Ge/⁶⁸Ga generator, purified by solid‐phase extraction and the radiochemical purity analyzed by radio‐RP‐HPLC. ⁶⁸Ga‐DOTA‐E‐[c(RGDfK)]₂ was obtained reproducibly in radiochemical yields of 60 ± 6% and with an excellent radiochemical purity of >99%. In nude rats bearing bone metastases after injection of MDA‐MB‐231 human breast cancer cells, biodistribution studies were performed to evaluate the accumulation of the radiotracer in selected organs, blood and bone metastases 0.5, 1, 2 and 3 h post injection. A rapid uptake into the bone metastases and rapid blood clearance was observed, resulting in tumor–blood ratios of up to 26.6 (3 h post injection) and tumor–muscle ratios of up to 7.9 (3 h post injection). A blocking experiment with coinjected αvβ3/αvβ5 antagonist showed the tumor uptake to be receptor‐specific. In an initial in vivo micro PET evaluation of the tracer using the same animal model, the bone metastasis was clearly visualized. These results suggest that ⁶⁸Ga‐DOTA‐E‐[c(RGDfK)]₂ is a promising PET tracer suitable for the imaging of αvβ3 and αvβ5 integrins in bone metastases. This novel PET tracer should be further evaluated concerning its usefulness for early detection of bone metastases and monitoring treatment response of these lesions. Copyright © 2011 John Wiley & Sons, Ltd.     

Collagen‐mimetic peptides mediate flow‐dependent thrombus formation by high‐ or low‐affinity binding of integrin α2β1 and glycoprotein VI

Summary. Background: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen‐derived triple‐helical peptides have identified the GXX’GER motif as an adhesive ligand for platelet integrin α2β1, and (GPO)_n as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). Objective: The potency was investigated of triple‐helical peptides, consisting of GXX’GER sequences within (GPO)_n or (GPP)_n motifs, to support flow‐dependent thrombus formation. Results: At a high‐shear rate, immobilized peptides containing both the high‐affinity α2β1‐binding motif GFOGER and the (GPO)_n motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co‐immobilized VWF was needed for thrombus formation. The (GPO)_n but not the (GPP)_n sequence induced GPVI‐dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low‐affinity (GASGER, GAOGER) α2β1‐binding motifs formed procoagulant thrombi only if both (GPO)_n and VWF were present. At a low‐shear rate, immobilized peptides with high‐ or low‐affinity α2β1‐binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)_n. Conclusions: Triple‐helical peptides with specific receptor‐binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high‐shear rate, either GPIb or high‐affinity (but not low‐affinity) GXX’GER mediates GPVI‐dependent formation of procoagulant thrombi. By extension, high‐affinity binding for α2β1 can control the overall platelet‐adhesive activity of native collagens.     

Imaging integrin alpha‐v‐beta‐3 expression in tumors with an 18F‐labeled dimeric RGD peptide

Integrin α_vβ₃ receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth. Many radiolabeled probes utilize the tight and specific association between the arginine–glycine–aspartatic acid (RGD) peptide and integrin α_vβ₃, but one main obstacle for any clinical application of these probes is the laborious multistep radiosynthesis of ¹⁸F. In this study, the dimeric RGD peptide, E‐[c(RGDfK)]₂, was conjugated with NODAGA and radiolabeled with ¹⁸F in a simple one‐pot process with a radiolabeling yield of 20%, the whole process lasting only 45 min. NODAGA‐E‐[c(RGDfK)]₂ labeled with ¹⁸F at a specific activity of 1.8 MBq nmol^?1 and a radiochemical purity of 100% could be achieved. The logP value of ¹⁸F‐labeled NODAGA‐E‐[c(RGDfK)]₂ was ?4.26 ± 0.02. In biodistribution studies, ¹⁸F‐NODAGA‐E‐[c(RGDfK)]₂ cleared rapidly from the blood with 0.03 ± 0.01 percentage injected dose per gram (%ID g^?1) in the blood at 2 h p.i., mainly via the kidneys, and showed good in vivo stability. Tumor uptake of ¹⁸F‐NODAGA‐E‐[c(RGDfK)]₂ (3.44 ± 0.20 %ID g^?1, 2 h p.i.) was significantly lower than that of reference compounds ⁶⁸Ga‐labeled NODAGA‐E‐[c(RGDfK)]₂ (6.26 ± 0.76 %ID g^?1; p <0.001) and ¹¹¹In‐labeled NODAGA‐E‐[c(RGDfK)]₂ (4.99 ± 0.64 %ID g^?1; p < 0.01). Co‐injection of an excess of unlabeled NODAGA‐E‐[c(RGDfK)]₂ along with ¹⁸F‐NODAGA‐E‐[c(RGDfK)]₂ resulted in significantly reduced radioactivity concentrations in the tumor (0.85 ± 0.13 %ID g^?1). The α_vβ₃ integrin‐expressing SK‐RC‐52 tumor could be successfully visualized by microPET with ¹⁸F‐labeled NODAGA‐E‐[c(RGDfK)]₂. In conclusion, NODAGA‐E‐[c(RGDfK)]₂ could be labeled rapidly with ¹⁸F using a direct aqueous, one‐pot method and it accumulated specifically in α_vβ₃ integrin‐expressing SK‐RC‐52 tumors, allowing for visualization by microPET. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitative 1H MRI, 19F MRI,and 19F MRS of cell‐internalized perfluorocarbon paramagnetic nanoparticles

In vivo molecular imaging with targeted MRI contrast agents will require sensitive methods to quantify local concentrations of contrast agent, enabling not only imaging‐based recognition of pathological biomarkers but also detection of changes in expression levels as a consequence of disease development, therapeutic interventions or recurrence of disease. In recent years, targeted paramagnetic perfluorocarbon emulsions have been frequently applied in this context, permitting high–resolution ¹H MRI combined with quantitative ¹⁹F MR imaging or spectroscopy, under the assumption that the fluorine signal is not altered by the local tissue and cellular environment. In this in vitro study we have investigated the ¹⁹F MR–based quantification potential of a paramagnetic perfluorocarbon emulsion conjugated with RGD–peptide to target the cell–internalizing α_νβ₃–integrin expressed on endothelial cells, using a combination of ¹H MRI, ¹⁹F MRI and ¹⁹F MRS. The cells took up the targeted emulsion to a greater extent than nontargeted emulsion. The targeted emulsion was internalized into large 1–7 µm diameter vesicles in the perinuclear region, whereas nontargeted emulsion ended up in 1–4 µm diameter vesicles, which were more evenly distributed in the cytoplasm. Association of the targeted emulsion with the cells resulted in different proton longitudinal relaxivity values, r₁, for targeted and control nanoparticles, prohibiting unambiguous quantification of local contrast agent concentration. Upon cellular association, the fluorine R₁ was constant with concentration, while the fluorine R₂ increased nonlinearly with concentration. Even though the fluorine relaxation rate was not constant, the ¹⁹F MRI and ¹⁹F MRS signals for both targeted nanoparticles and controls were linear and quantifiable as function of nanoparticle concentration. Copyright © 2010 John Wiley & Sons, Ltd.     

Platelet hyperreactivity and a prothrombotic phenotype in mice with a gain‐of‐function mutation in phospholipase Cγ2

Summary. Background: Agonist‐induced platelet activation involves different signaling pathways leading to the activation of phospholipase C (PLC) β or PLCγ2. Activated PLC produces inositol 1,4,5‐trisphosphate and diacylglycerol, which trigger Ca²⁺ mobilization and the activation of protein kinase C, respectively. PLCβ is activated downstream of Gq‐coupled receptors for soluble agonists with only short interaction times in flowing blood. In contrast, PLCγ2 becomes activated downstream of receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI or activated integrins. Objective and methods: We speculated that PLCγ2 activity might be optimized for sustained but submaximal signaling to control relatively slow platelet responses. To test this hypothesis, we analyzed platelets from mice heterozygous for a gain‐of‐function mutation in the Plcg2 gene (Plcg2^Ali5/+). Results: Plcg2^Ali5/+ platelets showed enhanced Ca²⁺ mobilization, integrin activation, granule secretion and phosphatidylserine exposure upon GPVI or C‐type lectin‐like receptor‐2 stimulation. Furthermore, integrin α_IIbβ₃ outside‐in signaling was markedly enhanced in the mutant platelets, as shown by accelerated spreading on different matrices and faster clot retraction. These defects translated into virtually unlimited thrombus formation on collagen under flow in vitro and a prothrombotic phenotype in vivo. Conclusions: These results demonstrate that the enzymatic activity of PLCγ2 is tightly regulated to ensure efficient but limited platelet activation at sites of vascular injury.     

Modification of MR molecular imaging probes with cysteine‐terminated peptides and their potential for in vivo tumour detection

One of the challenges facing superparamagnetic iron oxide (SPIO) nanoparticles is to improve their biological compatibility. While highly uniform SPIOs can be manufactured, the surfaces are hydrophobic as a result of the surfactants used in their fabrication. In this study, we developed a general strategy to fabricate an MR molecular imaging probe in one step by replacing hydrophobic surfactants with small peptides terminated with cysteine. The hydrophobic SPIO surface was transformed into a hydrophilic one by exchanging surface oleic acids with the peptides RGD–Cys or RGD–PEG–Cys. After the RGD–Cys and RGD–PEG–Cys peptide exchange, both RGD–Cys–SPIO and RGD–PEG–Cys–SPIO specifically targeted α_vβ₃‐expressing cells (A549) in vitro, with RGD–Cys–SPIO achieving this more efficiently. Furthermore, MR imaging of A549 tumors receiving RGD–Cys–SPIO or RGD–PEG–Cys–SPIO demonstrated that both the targeted particles could reach and label the α_vβ₃‐expressing tumor, much more efficiently than the non‐targeted particles (Cys–SPIO). Histology showed that the probes not only target the tumor neovasculature but also extravasate from vessels and address the tumor cells. Our study shows that directly replacing oleic acid with cysteine or cysteine‐terminated small peptides is a general strategy to transforming the hydrophobic surface of SPIO into a hydrophilic one, as well as providing targeting ligands. Such SPIOs are of interest as MR molecular imaging probes to detect for cancer in vivo. Copyright © 2010 John Wiley & Sons, Ltd.