Performance of a new fluorescence‐labeled MMP inhibitor to image tumor MMP activity in vivo in comparison to an MMP‐activatable probe

Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade structural components of the extracellular matrix and participate in pathologies such as cancer and inflammatory disorders. The development of novel contrast agents for optical imaging of MMP activity in vivo is of great interest. The commonly used near‐infrared fluorescence‐compatible agents are dye‐quenched probes that do not emit fluorescence until they interact with MMPs. In contrast, fluorescent synthetic low‐molecular‐weight MMP inhibitors have not been systematically employed. The aim of this study was to evaluate the performance of our recently developed Cy5.5‐labeled MMP inhibitor to image MMP activity in tumors in vivo compared with activatable fluorescent MMP‐sensing probes. The dynamic uptake of Cy5.5‐AF489 into four different tumor entities was analyzed in xenografted mice by intravenous injection and subsequent fluorescence reflectance imaging. Tumors were characterized in regard to their MMP‐2 and ?9 mRNA expressions (qRT‐PCR analysis), proteins (immunohistochemistry) and gelatinase/collagenase activities (in situ zymography). Cy5.5‐AF489 was compared with MMPSense^TM 680 and MMPSense^TM 750 FAST, two commercially available MMP‐activatable probes. Cy5.5‐AF489 showed a specific tumor uptake, which was blocked by pre‐injection of the unlabeled MMPI, and discriminated between tumors with high or low MMP‐2/‐9 expressions. Our optical probe facilitated faster visualization of MMP‐active tumors accompanied by excellent tumor‐to‐background ratios when compared with activatable probes. The MMP inhibitor Cy5.5‐AF489 permits fast in vivo imaging of MMP expression/activity in tumors. Given its small molecular weight and non‐peptidic structure, translational imaging from a preclinical application to a diagnostic tool for MMP‐related diseases seems feasible. Copyright © 2012 John Wiley & Sons, Ltd.     

Angiostatin K1‐3 induces E‐selectin via AP1 and Ets1: a mediator for anti‐angiogenic action of K1‐3

Summary. Background: Angiostatin, a circulating angiogenic inhibitor, is an internal fragment of plasminogen and consists of several isoforms, K1‐3 included. We previously showed that K1‐3 was the most potent angiostatin to induce E‐selectin mRNA expression. The purpose of this study was to identify the mechanism responsible for K1‐3‐induced E‐selectin expression and investigate the role of E‐selectin in the anti‐angiogenic action of K1‐3. Methods and results: Quantitative real time RT‐PCR and Western blotting analyses confirmed a time‐dependent increase of E‐selectin mRNA and protein induced by K1‐3. Subcellular fractionation and immunofluorescence microscopy showed the co‐localization of K1‐3‐induced E‐selectin with caveolin 1 (Cav1) in lipid rafts in which E‐selectin may behave as a signaling receptor. Promoter‐driven reporter assays and site‐directed mutagenesis showed that K1‐3 induced E‐selectin expression via promoter activation and AP1 and Ets‐1 binding sites in the proximal E‐selectin promoter were required for E‐selectin induction. The in vivo binding of both protein complexes to the proximal promoter was confirmed by chromatin immunoprecipitation (ChIP). Although K1‐3 induced the activation of ERK1/2 and JNK, only repression of JNK activation attenuated the induction of E‐selectin by K1‐3. A modulatory role of E‐selectin in the anti‐angiogenic action of K1‐3 was manifested by both overexpression and knockdown of E‐selectin followed by cell proliferation assay. Conclusions: We show that K1‐3 induced E‐selectin expression via AP1 and Ets‐1 binding to the proximal E‐selectin promoter (?356/+1), which was positively mediated by JNK activation. Our findings also demonstrate E‐selectin as a novel target for the anti‐angiogenic therapy.     

Fluorescent molecular imaging of metastatic lymph node using near‐infrared emitting low molecular weight heparin modified nanoliposome based on enzyme‐substrate interaction

Tumor metastatic lymph node mapping has been widely used to predict the metastatic spread of primary tumor and guide the lymph node dissection in clinical practice. In this research, a new near‐infrared (NIR)‐emitting low molecular weight heparin (LMWHEP)‐modified Cy7‐loaded nanoliposome (LMWHEP‐NLips/Cy7) was developed and had the particle size of about 80 nm and the fluorescence intensity of about 2300, which is optimal for metastatic lymph node uptake and imaging. The NIR‐emitting nanoliposomes were designed by LMWHEP coating on the surface of Cy7‐loaded nanoliposome (NLips/Cy7) according to electrostatic attraction. The LMWHEP‐NLips/Cy7 with negligible cytotoxicity for Hela and RAW264.7 cells and was found to be fluorescent stability compared with the Cy7‐free dye at room temperature. The BALB/c nude mice bearing tumor lymphatic metastasis was established at eighth week post‐injection by subcutaneously injecting Hela cells suspension. Heparanase (HPA) expression concentrations quantitatively measured by ELISA kit respectively were 237.42U/mL, 214.82U/mL and 128.45U/mL in the extracellular Hela cells, metastatic popliteal and iliac lymph node. LMWHEP‐NLips/Cy7 successfully increased fluorescence signal in the metastatic lymph node compared with normal lymph node and achieve in vivo and ex vivo high fluorescence signal within 10 min and retention time up to 4 h post‐injection. Maximum mean fluorescence intensity of the LMWHEP‐NLips/Cy7 group was significantly more than NLips/Cy7 group (increase 2‐fold in the metastatic popliteal lymph node and 4.8‐fold in the metastatic iliac lymph node, p < 0.05). The experimental results demonstrating LMWHEP‐NLips/Cy7 have the potential utility as specific, biosafe and stable near‐infrared imaging contrast agents for HPA‐expression tumor metastatic lymph node mapping. Copyright © 2016 John Wiley & Sons, Ltd.     

Dual-Modality Micro-Positron Emission Tomography/Computed Tomography and Near-Infrared Fluorescence Imaging of EphB4 in Orthotopic Glioblastoma Xenograft Models

Purpose

In glioblastoma, EphB4 receptors, a member of the largest family of receptor tyrosine kinases, are overexpressed in both tumor cells and angiogenic blood vessels. The purpose of this study was to examine whether the EphB4-binding peptide TNYL-RAW labeled with both ⁶⁴Cu and near-infrared fluorescence dye Cy5.5 could be used as a molecular imaging agent for dual-modality positron emission tomography/computed tomography [PET/CT] and optical imaging of human glioblastoma in orthotopic brain tumor models.

Materials and Methods

TNYL-RAW was conjugated to Cy5.5 and the radiometal chelator 1,4,7,10-tetraazadodecane-N,N′,N″,N?-tetraacetic acid. The conjugate was then labeled with ⁶⁴Cu for in vitro binding and in vivo dual μPET/CT and optical imaging studies in nude mice implanted with EphB4-expressing U251 and EphB4-negative U87 human glioblastoma cells. Tumors and brains were removed at the end of the imaging sessions for immunohistochemical staining and fluorescence microscopic examinations.

Results

μPET/CT and near-infrared optical imaging clearly showed specific uptake of the dual-labeled TNYL-RAW peptide in both U251 and U87 tumors in the brains of the nude mice after intravenous injection of the peptide. In U251 tumors, the Cy5.5-labeled peptide colocalized with both tumor blood vessels and tumor cells; in U87 tumors, the tracer colocalized only with tumor blood vessels, not with tumor cells.

Conclusions

Dual-labeled EphB4-specific peptide could be used as a noninvasive molecular imaging agent for PET/CT and optical imaging of glioblastoma owing to its ability to bind to both EphB4-expressing angiogenic blood vessels and EphB4-expressing tumor cells.     

Specific E‐selectin targeting with a superparamagnetic MRI contrast agent

Targeting of the endothelial inflammatory adhesion molecule E‐selectin by magnetic resonance imaging (MRI) was performed with a superparamagnetic contrast agent in the context of in vitro and in vivo models of inflammation. The specific contrast agent was obtained by grafting a synthetic mimetic of sialyl Lewis^x (sLe^x), a natural ligand of E‐selectin expressed on leukocytes, on the dextran coating of ultrasmall particles of iron oxide (USPIO). This new contrast agent, called USPIO‐g‐sLe^x, was tested, in vitro, on cultured human umbilical vein endothelial cells (HUVECs) stimulated to express inflammatory adhesion molecules, and in vivo, on a mouse model of hepatitis. In vitro, HUVECs were stimulated with the pro‐inflammatory cytokine tumor necrosis factor alpha (TNF‐α) and were then incubated with USPIO‐g‐sLe^x or ungrafted USPIO. In vivo, hepatitis was induced on NMRI mice by injection of concanavalin A (Con A). USPIO‐g‐sLe^x and ungrafted USPIO were injected intravenously. In vitro results showed an extensive retention of USPIO‐g‐sLe^x on TNF‐α stimulated HUVECs. Image intensity and R₂ measurements performed on T₂‐weighted MR images demonstrated a significantly higher binding of USPIO‐g‐sLe^x on stimulated HUVECs. In vivo, USPIO are known to pass through the fenestrae of the liver and to be captured by Kupffer cells, inducing a loss of signal intensity on T₂‐weighted MR images. Unexpectedly, when injected to Con A‐treated mice, USPIO‐g‐sLe^x induced a significantly lower attenuation of liver signal intensity than USPIO or USPIO‐g‐sLe^x injected to healthy mice, or USPIO injected to Con A‐treated mice, suggesting that the specific contrast media is retained extracellularly by an interaction with E‐selectin overexpressed on the vascular endothelium. Both in vitro and in vivo results therefore indicate that USPIO‐g‐sLe^x is recognizing endothelial E‐selectin. USPIO‐g‐sLe^x is thus well suited for the MRI diagnosis of inflammation and for the in vitro evaluation of endothelial cells activation. Copyright © 2006 John Wiley & Sons, Ltd.     

In vivo tumor imaging in mice with near-infrared labeled endostatin

Endostatin is a potent inhibitor of angiogenesis currently in phase I clinical trials. Imaging technologies that use near-infrared fluorescent probes are well suited to the laboratory setting. The goal of this study was to determine whether endostatin labeled with a near-infrared probe (Cy5.5) could be detected in an animal and whether it would selectively localize to a tumor. Endostatin was conjugated to Cy5.5 monofunctional dye and injected into mice bearing Lewis lung carcinoma tumors (350 mm2). Mice were imaged at various time points while under sedation using a lightproof box affixed to a fluorescent microscope mounted with a filter in the near-infrared bandwidth consistent with Cy5.5 fluorescence. After i.p. injection, endostatin-Cy5.5 was absorbed producing a near-infrared fluorescent image within the tumors at 18 h reaching a maximum at 42 h after injection. No signal was emitted from mice injected with unlabeled endostatin or Cy5.5 dye alone or those that received no injection. Further results show that a dose response exists with injection of endostatin-Cy5.5. Mimicking the clinical route of administration, an i.v. injection had a peak signal emission at 3 h but also persisted to 72 h. Finally, to determine the intratumoral binding site for endostatin, we performed immunofluorescence on tumor specimens and demonstrated that endostatin binds to tumor vasculature and colocalizes with platelet/endothelial cell adhesion molecule 1 expression. This study demonstrates that endostatin covalently bound to Cy5.5 will migrate from a distant i.p. injection site to a tumor. These data indicate that endostatin-Cy5.5 is appropriate for selectively imaging tumors in uninjured experimental animals.     

Magnetic resonance molecular imaging of post‐stroke neuroinflammation with a P‐selectin targeted iron oxide nanoparticle

We have developed a magnetic resonance molecular imaging method using a novel iron‐oxide contrast agent targeted towards P‐selectin – MNP‐PBP (magnetic nanoparticle‐P‐selectin binding peptide) – to image endothelial activation following cerebral ischemia/reperfusion. MNP‐PBP consists of ～1000 PBP ligands (primary sequence: GSIQPRPQIHNDGDFEEIPEEYLQ GGSSLVSVLDLEPLDAAWL) conjugated to a 50 nm diameter aminated dextran iron oxide particle. In vitro P‐ and E‐selectin binding was assessed by competition ELISA. Transient focal cerebral ischemia was induced in male C57/BL 6 mice followed by contrast injection (MNP‐PBP; MNP‐NH2; Feridex; MNP‐PBP‐FITC) at 24 h after reperfusion and T₂ magnetic resonance imaging at 9.4 T was performed. Infarction and microvasculature accumulation of contrast agent was assessed in coronal brain sections. MNP‐PBP attenuated antibody binding to P‐selectin by 34.8 ± 1.7%. P‐selectin was preferentially increased in the infarct hemisphere and MNP‐PBP‐FITC accumulation in the infarct hemisphere microvasculature was observed. Compared with the nontargeted iron oxide agents MNP‐NH2 and Feridex, MNP‐PBP showed a significantly greater T₂ effect within the infarction. MR imaging of P‐selectin expression with a targeted iron oxide nanoparticle contrast agent may reveal early endothelial activation in stroke and other neuroinflammatory states. Copyright © 2009 John Wiley & Sons, Ltd.     

PECAM‐1‐targeted micron‐sized particles of iron oxide as MRI contrast agent for detection of vascular remodeling after cerebral ischemia

An increasing amount of studies have provided evidence for vascular remodeling, for example, angiogenesis, after cerebral ischemia, which may play a significant role in post‐stroke brain plasticity and recovery. Molecular imaging can provide unique in vivo whole‐brain information on alterations in the expression of specific endothelial markers. A possible target for molecular magnetic resonance imaging (MRI) of post‐stroke (neo)vascularization is platelet endothelial cell adhesion molecule‐1 (PECAM‐1). Here we describe significantly increased PECAM‐1 mRNA levels in ipsilesional brain tissue at 6 h, 24 h and 3 days after transient middle cerebral artery occlusion in mice, and elevated PECAM‐1 staining throughout the lesion at 3, 7 and 21 days post‐stroke. The potential of micron‐sized particles of iron oxide (MPIO) conjugated with PECAM‐1‐targeted antibodies, that is, αPECAM‐1‐MPIO, to expose stroke‐induced PECAM‐1 upregulation with molecular MRI was assessed. In vitro studies demonstrated that PECAM‐1‐expressing brain endothelial cells could be effectively labeled with αPECAM‐1‐MPIO, giving rise to a fourfold increase in MRI relaxation rate R₂. Injection of near‐infrared fluorescent dye‐labeled αPECAM‐1 showed target specificity and dose efficiency of the antibody for detection of brain endothelial cells at 3 days post‐stroke. However, in vivo molecular MRI at 3 and 7 days after stroke revealed no αPECAM‐1‐MPIO‐based contrast enhancement, which was corroborated by the absence of αPECAM‐1‐MPIO in post mortem brain tissue. This indicates that this molecular MRI approach, which has been proven successful for in vivo detection of other types of cell adhesion molecules, is not invariably effective for MRI‐based assessment of stroke‐induced alterations in expression of cerebrovascular markers. Copyright © 2013 John Wiley & Sons, Ltd.     

Molecular imaging of tumors with nanobodies and antibodies: Timing and dosage are crucial factors for improved in vivo detection

The utility of nanobodies and conventional antibodies for in vivo imaging is well known, but optimum dosing and timing schedules for one versus the other have not been established. We aimed to improve specific tumor imaging in vivo with nanobodies and conventional antibodies using near‐infrared fluorescence (NIRF) imaging. We used ARTC2 expressed on lymphoma cells as a model target antigen. ARTC2‐specific nanobody s+16a and conventional antibody Nika102 were labeled with NIRF‐dye AF680. In vivo NIRF‐imaging of ARTC2‐positive and ARTC2‐negative xenografts was performed over 24 h post‐injection of 5, 10, 25, or 50 µg of each conjugate. Specific target‐binding and tissue‐penetration were verified by NIRF imaging ex vivo, flow cytometry and fluorescence microscopy. NIRF‐imaging of s+16a⁶⁸⁰ in vivo revealed a six times faster tumor accumulation than of Nika102⁶⁸⁰. Using 50 µg of s+16a⁶⁸⁰ increased the specific signals of ARTC2‐positive tumors without increasing background signals, allowing a tumor‐to‐background (T/B) ratio of 12.4 ± 4.2 within 6 h post‐injection. Fifty micrograms of Nika102⁶⁸⁰ increased specific signals of ARTC2‐positive tumors but also of ARTC2‐negative tumors and background, thereby limiting the T/B ratio to 6.1 ± 2.0. Ten micrograms of Nika102⁶⁸⁰ only slightly reduced specific tumor signals but dramatically reduced background signals. Ex vivo analyses confirmed a faster and deeper tumor penetration with s+16a⁶⁸⁰. Using nanobody s+16a allowed same‐day imaging with a high T/B ratio, whereas antibody Nika102 gave optimal imaging results only 24 h post injection. Nanobody s+16a required a high dose, whereas antibody Nika102 had the best T/B‐ratio at a low dose. Therefore, timing and dosage should be addressed when comparing nanobodies and conventional antibodies for molecular imaging purposes. Copyright © 2015 John Wiley & Sons, Ltd.     

E-选择素与脑卒中

脑卒中等多种疾病可引起E-选择素表达增加。在人体脑血管中,E-选择素主要是介导血液中中性粒细胞粘附。在人体脑卒中后,46%的患者可溶性E-选择素(sE)浓度在24～36h内升高,但大多数患者的sE在后续几天内降低到初始浓度。在多种动物局部大脑缺血模型中,使用抗E-选择素IgG抗体治疗后,动物存活率增加。由于对人体进行抗ICAM-1等其他粘附分子治疗的失败,以及E-选择素在脑卒中中具有的重要作用,因而人们越来越重视对E-选择素作用的拮抗,且强调使用人源性抗体。     

uMUC1-Targeting Magnetic Resonance Imaging of Therapeutic Response in an Orthotropic Mouse Model of Colon Cancer

Purpose

Noninvasive assessment of chemotherapeutic response in colon cancer would tremendously aid in therapeutic intervention of cancer patients and improve outcomes. The aim of the study was to evaluate the feasibility of a noninvasive assessment of chemotherapeutic response by magnetic resonance imaging utilizing underglycosylated mucin 1 (uMUC1) tumor antigen as a biomarker of therapeutic response in a colon cancer mouse model.

Procedures

The study was performed by applying molecular imaging approach based on targeting uMUC1 with specific dual-modality imaging probe (MN-EPPT). The probe consisted of dextran-coated iron oxide nanoparticles conjugated to the near infrared fluorescent dye Cy5.5 and to a uMUC1-specific peptide (EPPT) and was used for magnetic resonance imaging (MRI) and fluorescence optical imaging. An orthotopic murine model of colon cancer expressing human uMUC1 peptide (MC38 MUC1) was created along with the control model devoid of the antigen (MC38 neo). Animals received chemotherapy with 5-fluorouracil (5-FU) followed by MN-EPPT-enhanced MR and optical imaging.

Results

In vivo imaging of animals with uMUC1 expressing tumors after 5-FU therapy showed that the average deltaT2 was reduced by 7.27 ms (p?=?0.045) compared with animals in control groups indicating lower accumulation of MN-EPPT caused by uMUC1 downregulation. In vivo optical imaging, biodistribution, and fluorescence microscopy confirmed the MRI findings. Interestingly, we found that the group of animals that did not respond to chemotherapy (“progressive disease” per RECIST) showed higher accumulation of MN-EPPT compared to the group of responders (“stable disease”) consistent with proliferating tumor cells and increased antigen availability.

Conclusions

We believe that in application to over 50 % of human cancers expressing uMUC1, our results could provide insight into overall assessment of therapeutic response based on its expression as defined by non-invasive MN-EPPT-enhanced MRI.

     

Targeted ultrasound contrast agent for molecular imaging of inflammation in high‐shear flow

Targeted ultrasound contrast materials (gas‐filled microbubbles carrying ligands to endothelial selectins or integrins) have been investigated as potential molecular imaging agents. Such microbubbles normally exhibit good targeting capability at the slower flow conditions. However, in the conditions of vigorous flow, binding may be limited. Here, we describe a microbubble capable of efficient binding to targets both in slow and fast flow (exceeding 4 dyne/cm² wall shear stress) using a clustered polymeric form of the fast‐binding selectin ligand sialyl Lewis^X. Microbubbles were prepared from decafluorobutane gas and stabilized with a monolayer of phosphatidylcholine, PEG stearate and biotin‐PEG‐lipid. Biotinylated PSLe^x (sialyl Lewis^X polyacrylamide) or biotinylated anti‐P‐selectin antibody (RB40.34) was attached to microbubbles via a streptavidin bridge. In a parallel plate flow chamber targeted adhesion model, PSLe^x bubbles demonstrated specific adhesion, retention and slow rolling on P‐selectin‐coated plates. Efficiency of firm targeted adhesion to a P‐selectin surface (140 molecules/µm²) was comparable for antibody‐carrying bubbles and PSLe^x‐targeted bubbles at 0.68 dyne/cm² shear stress. At fast flow (4.45 dyne/cm²), PSLe^x‐targeted bubbles maintained their ability to bind, while antibody‐mediated targeting dropped more than 20‐fold. At lower surface density of P‐selectin (7 molecules/µm²), targeting via PSLe^x was more efficient than via antibody under all the flow conditions tested. Negative control casein‐coated plates did not retain bubbles in the range of flow conditions studied. To confirm echogenicity, targeted PSLe^x‐bubbles were visualized on P‐selectin‐coated polystyrene plates by ultrasound imaging with a clinical scanner operated in pulse inversion mode; control plates lacking targeted bubbles did not show significant acoustic backscatter. In vivo, in a murine model of inflammation in the femoral vein setting, targeting efficacy of intravenously administered PSLe^x‐microbubbles was comparable with targeting mediated by anti‐P‐selectin antibody, and significantly exceeded the accumulation of non‐targeted control bubbles. In the inflamed femoral artery setting, PSLe^x‐mediated microbubble targeting was superior to antibody‐mediated targeting. Copyright © 2006 John Wiley & Sons, Ltd.     

A fluorescent chromophore TOTO‐3 as a ‘smart probe’ for the assessment of ultrasound‐mediated local drug delivery in vivo

Many potent anti‐cancer drugs have an intracellular mode of action, but are limited in crossing the cell membrane, resulting in a reduced clinical efficacy. Ultrasound (US) is known to facilitate the penetration of drugs into tumors cells. However (molecular) imaging techniques that monitor in vivo the underlying processes of US‐triggered drug delivery are lacking. The objective of this study was to demonstrate the feasibility of using a fluorescent nuclear acid stain (TOTO‐3) as a model drug to monitor in real‐time US‐mediated delivery by in vivo fluorescence imaging. Following co‐injection of TOTO‐3 and microbubbles US was applied to the tumor. The time course of the drug delivery process was monitored by fluorescence imaging. Immunohistological analysis and in vitro experiments were performed to investigate the results in more detail. A significant signal intensity enhancement of the US‐treated tumor was observed that indicates intracellular delivery of the dye. In the control tumor TOTO‐3 signal was strongly associated with macrophages, which was not the case for the sonicated tumor. The capability of macrophages to uptake TOTO‐3 was confirmed in vitro. This study demonstrates that an optical contrast agent with similar characteristics to an anti‐cancer drug may be used for continuous in vivo monitoring of the drug delivery process. Copyright © 2010 John Wiley & Sons, Ltd.     

Antigenic characterization of endothelial cell-derived microparticles and their detection ex vivo

Summary. Background: Endothelial activation and dysfunction are associated with several diseases. However, hardly any specific markers are available. Microparticles (MP) from endothelial cells (EC; EMP) were reported in patient groups and healthy individuals. The antibodies used to detect EMP, however, were mainly directed against antigens without EC specificity. Objectives: We evaluated the antigens on EC and EMP to establish proper markers for EMP detection. Methods: EMP were isolated from supernatants of resting and interleukin (IL)‐1α activated human umbilical vein EC (HUVEC; n = 3; 0–72 h), stained with annexin V and monoclonal antibodies, and analyzed by flow cytometry. Human platelet‐MP (PMP), the main MP population in plasma, were prepared in vitro. EMP and PMP were studied in plasma from systemic lupus erythematosus (SLE) patients (n = 11) and healthy individuals (n = 10). Results: Platelet–endothelial cell adhesion molecule‐1 (PECAM‐1), α_ν and β₃ were constitutively exposed on HUVEC, but (almost) absent on EMP (<15% positive for α_ν and β₃), or only exposed on a subpopulation (PECAM‐1; 30–60%). Activated HUVEC (>80%) and (subpopulations of) EMP exposed E‐selectin and tissue factor. PMP strongly exposed PECAM‐1, β₃, and glycoprotein (GP)Ib (CD42b), but not α_ν or E‐selectin. GPIb and P‐selectin (CD62P) were absent on EMP. Plasma samples contained 0.5% MP staining for E‐selectin and/or α_ν. Plasma from one SLE patient contained E‐selectin exposing MP (21%), but little α_ν‐positive MP. Conclusions: EC release EMP in vitro. The antigenic phenotype of EMP released from resting and IL‐1α‐stimulated EC differs among each other as well as from resting and stimulated EC, respectively. E‐selectin exposed on IL‐1α‐stimulated EC is a valid marker for EMP detection ex vivo to establish endothelial cell activation.     

A heparin mimetic isolated from a marine shrimp suppresses neovascularization

Summary. Background: Choroidal neovascularization (CNV) is the main cause of severe visual loss in age‐related macular degeneration (AMD). Heparin/heparan sulfate are known to play important roles in neovascularization due to their abilities to bind and modulate angiogenic growth factors and cytokines. Previously, we have isolated from marine shrimp a heparin‐like compound with striking anti‐inflammatory action and negligible anticoagulant and hemorrhagic activities. Objectives: To investigate the role of this novel heparin‐like compound in angiogenic processes. Methods and Results: The anti‐angiogenic effect of this heparinoid in laser‐induced CNV and in vitro models is reported. The compound binds to growth factors (FGF‐2, EGF and VEGF), blocks endothelial cell proliferation and shows no cytotoxic effect. The decrease in proliferation is not related to cell death either by apoptosis or secondary necrosis. The results also showed that the heparinoid modified the 2‐D network organization in capillary‐like structures of endothelial cells in Matrigel and reduced the CNV area. The effect on CNV area correlates with decreases in the levels of VEGF and TGF‐β1 in the choroidal tissue. The low content of 2‐O‐sulfate groups in this heparinoid may explain its potent anti‐angiogenic effect. Conclusions: The properties of the shrimp heparinoid, such as potent anti‐angiogenic and anti‐inflammatory activities but insignificant anticoagulant or hemorrhagic actions, point to this compound as a compelling drug candidate for treating neovascular AMD and other angioproliferative diseases. A mechanism for the anti‐angiogenic effect of the heparinoid is proposed.     

Pharmacokinetics of contrast agents targeted to the tumor vasculature in molecular magnetic resonance imaging

Molecular magnetic resonance imaging (MRI) is increasingly used to investigate tumor angiogenic activity non‐invasively. However, the pharmacokinetic behavior and tumor penetration of the often large contrast agent particles is thus far unknown. Here, pharmacokinetic analysis of cyclic asparagine–glycine–arginine (cNGR) labeled paramagnetic quantum dots (pQDs) was developed to quantify the contrast agent's homing efficacy to activated endothelial cells of angiogenic tumor vessels using dynamic contrast‐enhanced (DCE) MRI. cNGR homes to CD13, an overexpressed aminopeptidase on angiogenic tumor endothelial cells. First, a two‐compartment pharmacokinetic model, comprising the blood space and endothelial cell surface, was compared with a three‐compartment model additionally including the extravascular–extracellular component. The resulting extravasation parameter was irrelevantly small and was therefore neglected. Next, the association constant K_a, the dissociation constant k_d and the fractional plasma volume v_P were determined from the time‐series data using the two‐compartment model. Magnitude and spatial distribution of the parameters were compared for cNGR‐labeled and unlabeled pQDs. The tumor area with significant K_a values was approximately twice as large for cNGR‐pQDs compared with unlabeled pQDs (p < 0.05), indicating more contrast agent binding for cNGR‐pQDs. Using cNGR‐pQDs, a two‐fold larger area with significant K_a was also found for the angiogenic tumor rim compared with tumor core (p < 0.05). It was furthermore found that both contrast agents perfused the tumor at all depths, thereby providing unequivocal evidence that rim/core differences can indeed be ascribed to stronger angiogenic activity in the rim. Summarizing, molecular DCE‐MRI with pharmacokinetic modeling provides unique information on contrast agent delivery and angiogenic activity in tumors. Copyright © 2010 John Wiley & Sons, Ltd.     

Decorin mimic promotes endothelial cell health in endothelial monolayers and endothelial–smooth muscle co‐cultures

Non‐specific cytotoxins, including paclitaxel and sirolimus analogues, currently utilized as anti‐restenotic therapeutics, affect not only smooth muscle cells (SMCs) but also neighbouring vascular endothelial cells (ECs). These drugs inhibit the formation of an intact endothelium following vessel injury, thus emphasizing the critical need for new candidate therapeutics. Utilizing our in vitro models, including EC monolayers and both hyperplastic and quiescent EC–SMC co‐cultures, we investigated the ability of DS–SILY₂₀, a decorin mimic, to promote EC health. DS–SILY₂₀ increased EC proliferation and migration by 1.5‐ and 2‐fold, respectively, which corresponded to increased phosphorylation of ERK‐1/2. Interestingly, IL‐6 secretion and the production of both E‐selectin and P‐selectin were reduced in the presence of 10 μm DS–SILY₂₀, even in the presence of the potent pro‐inflammatory cytokine platelet‐derived growth factor (PDGF). In hyperplastic and quiescent EC–SMC co‐cultures, DS–SILY₂₀ treatment reduced the secretion of IFNγ, IL‐1β, IL‐6 and TNFα, corresponding to a 23% decrease in p38 phosphorylation. E‐selectin and P‐selectin expression was further reduced following DS–SILY₂₀ treatment in both co‐culture models. These results indicate that DS–SILY₂₀ promotes EC health and that this decorin mimic could serve as a potential therapeutic to promote vessel healing following percutaneous coronary intervention (PCI). Copyright © 2015 John Wiley & Sons, Ltd.     

Dynamic fluorescent imaging with indocyanine green for monitoring the therapeutic effects of photoimmunotherapy

A new type of monoclonal antibody (mAb)‐based, highly specific phototherapy (photoimmunotherapy; PIT) that uses a near‐infrared (NIR) phthalocyanine dye, IRDye700DX (IR700) conjugated with an mAb, has recently been described. NIR light exposure leads to immediate, target‐selective necrotic cell death. However, tumor shrinkage takes several days to occur, making it difficult to detect earlier changes in the tumor. In this study, Panitumumab targeting the epidermal growth factor receptor (EGFR1) conjugated to IR700 was used to treat EGFR‐expressing A431 tumor cells and in vivo xenografts. PIT was performed at varying doses of NIR light (10, 30, 50 and 100 J cm^?2) in xenograft tumors in mice. Indocyanine green (ICG) dynamic imaging was evaluated for monitoring cytotoxic effects for the first hour after PIT. Our results demonstrated a statistical difference (p < 0.05) in ICG intensity between control and PIT treated tumors in the higher light exposure groups (50 J cm^?2: 2.94 ± 0.35 vs 5.22 ± 0.92, p = 0.02; and 100 J cm^?2: 3.56 ± 0.96 vs 5.71 ± 1.43, p = 0.008) as early as 20 min post ICG injection. However, no significant difference (p > 0.05) in ICG intensity between control and PIT treated tumors was evident in the lower light exposure group at any time points up to 60 min (10 J cm^?2: 1.92 ± 0.49 vs 1.71 ± 0.3, p = 0.44; and 30 J cm^?2: 1.57 ± 0.35 vs 2.75 ± 0.59, p = 0.07). Similarly, the retention index (background to corrected uptake ratio of ICG) varied with light exposure. In conclusion, ICG may serve as a potential indicator of acute cytotoxic effects of mAb‐IR700‐induced PIT even before morphological changes can be seen in targeted tumors. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

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.     

Axl-Targeted Cancer Imaging with Humanized Antibody h173

Purpose

The tyrosine kinase receptor Axl is overexpressed in various types of cancer and correlated with cancer malignancy. Selective Axl blockade reduces tumor growth and metastasis. The purpose of this study was to examine whether the humanized anti-Axl antibody humanized 173 (h173) labeled with near-infrared fluorescence (NIRF) dye Cy5.5 could be applied as a molecular imaging probe for NIRF imaging of Axl expression in tumor models.

Procedures

NIRF dye Cy5.5 was conjugated to h173 or human normal immunoglobulin G (hIgG) control through amino groups. The resulting probes were evaluated in both A549 (Axl positive) and NCI-H249 (Axl negative) lung cancer xenografts through in vivo NIRF imaging. Ex vivo imaging and probe distribution assay were also carried out to confirm the in vivo imaging results.

Results

After conjugation, binding activity of h173-Cy5.5 was determined to be 97.75 %?±?2.09 % of the unmodified h173. In vitro fluorescence-activated cell sorting (FACS) and fluorescence microscopy analysis validated the specific binding of h173 toward Axl-positive A549 cells. h173-Cy5.5 was then applied to image Axl expression in vivo. In A549 (Axl positive) cancer xenografts, the tumor uptake of h173-Cy5.5 was significantly higher than that of the hIgG-Cy5.5 control (P?<?0.05) at late time points (1, 2, 3, 4, and 7 days). On the contrary, in NCI-H249 (Axl negative) cancer xenografts, the tumor uptake of both hIgG-Cy5.5 and h173-Cy5.5 was low and showed no significant difference (P?>?0.05) at all time points examined. Ex vivo imaging and immunofluorescence staining analysis further validated the in vivo imaging results.

Conclusions

Collectively, all in vitro, in vivo, and ex vivo data suggested that h173-Cy5.5 could serve as a valid probe for Axl-targeted cancer imaging, which could therefore aid in tumor diagnosis, prognosis, and treatment monitoring.