Ultrasound Evaluation of Anti-Vascular Endothelial Growth Factor–Induced Changes in Vascular Response Following Tendon Injury

While vascular ingrowth is necessary for tendon healing, hypervascularization following tendon injury is not always believed to be beneficial, and there is now evidence showing beneficial results of anti-angiogenic treatments in the context of tendon healing. However, the dose-dependency of anti-angiogenic–altered vascular response, as well as methods for evaluating these changes in vivo, has not been fully investigated. Therefore, the objective of this study was to evaluate if in vivo ultrasound imaging can detect dose-dependent, anti-angiogenic treatment–induced changes in vascularity in rat Achilles tendon after injury. Color Doppler ultrasound revealed an increase in vascularity in a low-dosage group, while photoacoustic imaging demonstrated a decrease in vascularity in mid- and high-dosage groups. Histologic staining supported the decrease in vascularity observed in the mid-dosage group. This study demonstrates dose-dependent vascular alterations from the delivery of an anti-angiogenic factor after tendon injury that can be detected through ultrasound imaging methods.     

A Lipopeptide-Based αvβ3 Integrin-Targeted Ultrasound Contrast Agent for Molecular Imaging of Tumor Angiogenesis

The design and fabrication of targeted ultrasound contrast agents are key factors in the success of ultrasound molecular imaging applications. Here, we introduce a transformable αvβ₃ integrin-targeted microbubble (MB) by incorporation of iRGD-lipopeptides into the MB membrane for non-invasive ultrasound imaging of tumor angiogenesis. First, the iRGD-lipopeptides were synthesized by conjugating iRGD peptides to distearoylphosphatidylethanolamine–polyethylene glycol 2000–maleimide. The resulting iRGD-lipopeptides were used for fabrication of the iRGD-carrying αvβ₃ integrin-targeted MBs (iRGD-MBs). The binding specificity of iRGD-MBs for endothelial cells was found to be significantly stronger than that of control MBs (p < 0.01) under in vitro static and dynamic conditions. The binding of iRGD-MBs on the endothelial cells was competed off by pre-incubation with the anti-αv or anti-β₃ antibody (p < 0.01). Ultrasound images taken of mice bearing 4T1 breast tumors after intravenous injections of iRGD-MBs or control MBs revealed strong contrast enhancement within the tumors from iRGD-MBs but not from the control MBs; the mean acoustic signal intensity was 10.71 ± 2.75 intensity units for iRGD-MBs versus 1.13 ± 0.18 intensity units for the control MBs (p < 0.01). The presence of αvβ₃ integrin was confirmed by immunofluorescence staining. These data indicate that iRGD-MBs can be used as an ultrasound imaging probe for the non-invasive molecular imaging of tumor angiogenesis, and may have further implications for ultrasound image-guided tumor targeting drug delivery.     

Characterization and Evaluation of 64Cu-Labeled A20FMDV2 Conjugates for Imaging the Integrin αvβ6

Purpose

The integrin α_vβ₆ is overexpressed in a variety of aggressive cancers and serves as a prognosis marker. This study describes the conjugation, radiolabeling, and in vitro and in vivo evaluation of four chelators to determine the best candidate for ⁶⁴Cu radiolabeling of A20FMDV2, an α_vβ₆ targeting peptide.

Procedures

Four chelators were conjugated onto PEG₂₈-A20FMDV2 (1): 11-carboxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-methanephosphonic acid (CB-TE1A1P), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and 4,4′-((3,6,10,13,16,19-hexazazbicyclo[6.6.6]ico-sane-1,8-diylbis(aza-nediyl))bis(methylene)dibenzoic acid (BaBaSar). All peptides were radiolabeled with ⁶⁴Cu in ammonium acetate buffer at pH 6 and formulated to pH 7.2 in PBS for use. The radiotracers were evaluated using in vitro cell binding and internalization assays and serum stability assays. In vivo studies conducted include blocking, biodistribution, and small animal PET imaging. Autoradiography and histology were also conducted.

Results

All radiotracers were radiolabeled in good radiochemical purity (>95 %) under mild conditions (37–50 °C for 15 min) with high specific activity (0.58–0.60 Ci/μmol). All radiotracers demonstrated α_vβ₆-directed cell binding (>46 %) with similar internalization levels (>23 %). The radiotracers ⁶⁴Cu-CB-TE1A1P-1 and ⁶⁴Cu-BaBaSar-1 showed improved specificity for the α_vβ₆ positive tumor in vivo over ⁶⁴Cu-DOTA-1 and ⁶⁴Cu-NOTA-1 (+/? tumor uptake ratios—3.82?+/-?0.44, 3.82?±?0.41, 2.58?±?0.58, and 1.29?±?0.14, respectively). Of the four radiotracers, ⁶⁴Cu-NOTA-1 exhibited the highest liver uptake (10.83?±?0.1 % ID/g at 4 h).

Conclusions

We have successfully conjugated, radiolabeled, and assessed the four chelates CB-TE1A1P, DOTA, NOTA, and BaBaSar both in vitro and in vivo. However, the data suggests no clear “best candidate” for the ⁶⁴Cu-radiolabeling of A20FMDV2, but instead a trade-off between the different properties (e.g., stability, selectivity, pharmacokinetics, etc.) with no obvious effects of the individual chelators.     

Correction to: Evaluation of Two Optical Probes for Imaging the Integrin αvβ6− In Vitro and In Vivo in Tumor-Bearing Mice

Molecular Imaging and Biology - This article was updated to correct the axes in Figures&nbsp;4e and 5d.     

Selective Imaging of Vascular Endothelial Growth Factor Receptor-1 and Receptor-2 in Atherosclerotic Lesions in Diabetic and Non-diabetic ApoE<Superscript>−/−</Superscript> Mice

Purpose

Plaque vulnerability is associated with inflammation and angiogenesis, processes that rely on vascular endothelial growth factor (VEGF) signaling via two receptors, VEGFR-1 and VEGFR-2. We have recently reported that enhanced uptake of scVEGF-PEG-DOTA/Tc-99m (scV/Tc) single photon emission computed tomography (SPECT) tracer that targets both VEGFR-1 and VEGFR-2, identifies accelerated atherosclerosis in diabetic relative to non-diabetic ApoE^?/? mice. Since VEGFR-1 and VEGFR-2 may play different roles in atherosclerotic plaques, we reasoned that selective imaging of each receptor can provide more detailed information on plaque biology.

Procedures

Recently described VEGFR-1 and VEGFR-2 selective mutants of scVEGF, named scVR1 and scVR2, were site-specifically derivatized with Tc-99m chelator DOTA via 3.4 kDa PEG linker, and their selectivity to the cognate receptors was confirmed in vitro. scVR1 and scVR2 conjugates were radiolabeled with Tc-99m to specific activity of 110 ± 11 MBq/nmol, yielding tracers named scVR1/Tc and scVR2/Tc. 34–40 week old diabetic and age-matched non-diabetic ApoE^?/? mice were injected with tracers, 2–3 h later injected with x-ray computed tomography (CT) contrast agent and underwent hybrid SPECT/CT imaging. Tracer uptake, localized to proximal aorta and brachiocephalic vessels, was quantified as %ID from. Tracer uptake was also quantified as %ID/g from gamma counting of harvested plaques. Harvested atherosclerotic arterial tissue was used for immunofluorescent analyses of VEGFR-1 and VEGFR-2 and various lineage-specific markers.

Results

Focal, receptor-mediated uptake in proximal aorta and brachiocephalic vessels was detected for both scVR1/Tc and scVR2/Tc tracers. Uptake of scVR1/Tc and scVR2/Tc was efficiently inhibited only by “cold” proteins of the same receptor selectivity. Tracer uptake in this area, expressed as %ID, was higher in diabetic vs. non- diabetic mice for scVR1/Tc (p = 0.01) but not for scVR2/Tc. Immunofluorescent analysis revealed enhanced VEGFR-1 prevalence in and around plaque area in diabetic mice.

Conclusions

Selective VEGFR-1 and VEGFR-2 imaging of atherosclerotic lesions may be useful to explore plaque biology and identify vulnerability.

     

Evaluation of Two Optical Probes for Imaging the Integrin αvβ6− In Vitro and In Vivo in Tumor-Bearing Mice

Purpose

The purpose of this study was to develop and evaluate two α_vβ₆-targeted fluorescent imaging agents. The integrin subtype α_vβ₆ is significantly upregulated in a wide range of epithelial derived cancers, plays a key role in invasion and metastasis, and expression is often located at the invasive edge of tumors. α_vβ₆-targeted fluorescent imaging agents have the potential to guide surgical resection leading to improved patient outcomes. Both imaging agents were based on the bi-PEGylated peptide NH₂-PEG₂₈-A20FMDV2-K16R-PEG₂₈ (1), a peptide that has high affinity and selectivity for the integrin α_vβ₆: (a) 5-FAM-X-PEG₂₈-A20FMDV2-K16R-PEG₂₈ (2), and (b) IRDye800-PEG₂₈-A20FMDV2-K16R-PEG₂₈ (3).

Procedures

Peptides were synthesized using solid-phase peptide synthesis and standard Fmoc chemistry. Affinity for α_vβ₆ was evaluated by ELISA. In vitro binding, internalization, and localization of 2 was monitored using confocal microscopy in DX3puroβ₆ (α_vβ₆+) and DX3puro (α_vβ₆?) cells. The in vivo imaging and ex vivo biodistribution of 3 was evaluated in three preclinical mouse models, DX3puroβ₆/DX3puro and BxPC-3 (α_vβ₆+) tumor xenografts and a BxPC-3 orthotopic pancreatic tumor model.

Results

Peptides were obtained in >?99% purity. IC₅₀ values were 28 nM (2) and 39 nM (3). Rapid α_vβ₆-selective binding and internalization of 2 was observed. Fluorescent intensity (FLI) measurements extracted from the in vivo images and ex vivo biodistribution confirmed uptake and retention of 3 in the α_vβ₆ positive subcutaneous and orthotopic tumors, with negligible uptake in the α_vβ₆-negative tumor. Blocking studies with a known α_vβ₆-targeting peptide demonstrated α_vβ₆-specific binding of 3.

Conclusion

Two fluorescence imaging agents were developed. The α_vβ₆-specific uptake, internalization, and endosomal localization of the fluorescence agent 2 demonstrates potential for targeted therapy. The selective uptake and retention of 3 in the α_vβ₆-positive tumors enabled clear delineation of the tumors and surgical resection indicating 3 has the potential to be utilized during image-guided surgery.

     

Integrin αvβ3 as a PET Imaging Biomarker for Osteoclast Number in Mouse Models of Negative and Positive Osteoclast Regulation

Purpose

The goal of this study was to determine the specificity of ⁶⁴Cu-CB-TE2A-c(RGDyK) (⁶⁴Cu-RGD) for osteoclast-related diseases, such as Paget's disease or rheumatoid arthritis.

Procedures

C57BL/6 mice were treated systemically with osteoprotegerin (OPG) for 15?days or RANKL for 11?days to suppress and stimulate osteoclastogenesis, respectively. The mice were then imaged by positron emission tomography/computed tomography using ⁶⁴Cu-RGD, followed by determination of serum TRAP5b and bone histology. Standard uptake values were determined to quantify ⁶⁴Cu-RGD in bones and other tissues.

Results

Mice treated with OPG showed decreased bone uptake of ⁶⁴Cu-RGD at 1, 2, and 24?h post-injection of the tracer (p?<?0.01 for all time points) compared to vehicle controls, which correlated with a post-treatment decrease in serum TRAP5b. In contrast, mice treated with RANKL showed significantly increased bone uptake at 2?h post-injection of ⁶⁴Cu-RGD (p?<?0.05) compared to the vehicle control group, corresponding to increased serum TRAP5b and OC numbers as determined by bone histology.

Conclusions

These data demonstrate that ⁶⁴Cu-RGD localizes to areas in bone with increased osteoclast numbers and support the use of ⁶⁴Cu-RGD as an imaging biomarker for osteoclast number that could be used to monitor osteoclast-related pathologies and their treatments.     

Radiolabeled RGD Tracer Kinetics Annotates Differential αvβ3 Integrin Expression Linked to Cell Intrinsic and Vessel Expression

Purpose

The purpose of this paper is to study the association between RGD binding kinetics and α_vβ₃ integrin receptor density in the complex tumor milieu.

Procedures

We assessed α_vβ₃ in vitro and by ⁶⁸Ga-DOTA-[c(RGDfK)]₂ positron emission tomography (PET) in tumors with varying α_vβ₃.

Results

Intrinsic α_vβ₃ expression decreased in the order of M21?>>>?MDA-MB-231?>?M21L in cells. Tumor volume of distribution by PET, V _T, was significantly higher in M21 compared to isogenic M21L tumors (0.40?±?0.01 versus 0.25?±?0.02; p?<?0.01) despite similar microvessel density (MVD) likely due to higher α_vβ₃. V _T for MDA-MB-231 (0.40?±?0.04) was comparable to M21 despite lower α_vβ₃ but in keeping with the higher MVD, suggesting superior tracer distribution.

Conclusions

This study demonstrates that radioligand binding kinetics of PET data can be used to discriminate tumors with different α_vβ₃ integrin expression—a key component of the angiogenesis phenotype—in vivo.     

Vascular Delivery of rAAVrh74.MCK.GALGT2 to the Gastrocnemius Muscle of the Rhesus Macaque Stimulates the Expression of Dystrophin and Laminin α2 Surrogates

Overexpression of GALGT2 in skeletal muscle can stimulate the glycosylation of α dystroglycan and the upregulation of normally synaptic dystroglycan-binding proteins, some of which are dystrophin and laminin α2 surrogates known to be therapeutic for several forms of muscular dystrophy. This article describes the vascular delivery of GALGT2 gene therapy in a large animal model, the rhesus macaque. Recombinant adeno-associated virus, rhesus serotype 74 (rAAVrh74), was used to deliver GALGT2 via the femoral artery to the gastrocnemius muscle using an isolated focal limb perfusion method. GALGT2 expression averaged 44 ± 4% of myofibers after treatment in macaques with low preexisting anti-rAAVrh74 serum antibodies, and expression was reduced to 9 ± 4% of myofibers in macaques with high preexisting rAAVrh74 immunity (P < 0.001; n = 12 per group). This was the case regardless of the addition of immunosuppressants, including prednisolone, tacrolimus, and mycophenolate mofetil. GALGT2-treated macaque muscles showed increased glycosylation of α dystroglycan and increased expression of dystrophin and laminin α2 surrogate proteins, including utrophin, plectin1, agrin, and laminin α5. These experiments demonstrate successful transduction of rhesus macaque muscle with rAAVrh74.MCK.GALGT2 after vascular delivery and induction of molecular changes thought to be therapeutic in several forms of muscular dystrophy.     

Synergism Between Circulating Tumor Necrosis Factor Receptor 2 and HbA1c in Determining Renal Decline During 5–18 Years of Follow-up in Patients With Type 1 Diabetes and Proteinuria

OBJECTIVE

We studied the serum concentration of tumor necrosis factor receptor 2 (TNFR2) and the rate of renal decline, a measure of the intensity of the disease process leading to end-stage renal disease (ESRD).

RESEARCH DESIGN AND METHODS

A cohort of 349 type 1 diabetic patients with proteinuria was followed for 5–18 years. Serum TNFR2, glycated hemoglobin A_1c (HbA_1c), and other characteristics were measured at enrollment. We used a novel analytic approach, a joint longitudinal-survival model, fitted to serial estimates of glomerular filtration rate (eGFR) based on serum creatinine (median seven per patient) and time to onset of ESRD (112 patients) to estimate the rate of renal decline (eGFR loss).

RESULTS

At enrollment, all patients had chronic kidney disease stage 1–3. The mean (±SD) rate of eGFR loss during 5–18 years of follow-up was −5.2 (±4.9) mL/min/1.73 m²/year. Serum TNFR2 was the strongest determinant of renal decline and ESRD risk (C-index 0.79). The rate of eGFR loss became steeper with rising concentration of TNFR2, and elevated HbA_1c augmented the strength of this association (P = 0.030 for interaction). In patients with HbA_1c ≥10.1% (87 mmol/mol), the difference in the rate of eGFR loss between the first and fourth quartiles of TNFR2 was 5.4 mL/min/1.73 m²/year, whereas it was only 1.9 in those with HbA_1c <7.9% (63 mmol/mol).

CONCLUSIONS

Circulating TNFR2 is a major determinant of renal decline in patients with type 1 diabetes and proteinuria. Elevated HbA_1c magnifies its effect. Although the mechanisms of this synergism are unknown, our findings allow us to stratify patients according to risk of ESRD.     

Evaluation of 6β-hydroxycortisol, 6β-hydroxycortisone, and a combination of the two as endogenous probes for inhibition of CYP3A4 in vivo

An endogenous probe for CYP3A activity would be useful for early identification of in vivo cytochrome P450 (CYP) 3A4 inhibitors. The aim of this study was to determine whether formation clearance (CL(f)) of the sum of 6β-hydroxycortisol and 6β-hydroxycortisone is a useful probe of CYP3A4 inhibition in vivo. In human liver microsomes (HLMs), the formation of 6β-hydroxycortisol and 6β-hydroxycortisone was catalyzed by CYP3A4, and itraconazole inhibited these reactions with half maximal inhibitory concentration (IC(50))(,u) values of 3.1 nmol/l and 3.4 nmol/l, respectively. The in vivo IC(50,u) value of itraconazole for the combined CL(f) of 6β-hydroxycortisone and 6β-hydroxycortisol was 1.6 nmol/l. The greater inhibitory potency in vivo is probably due to circulating inhibitory itraconazole metabolites. The maximum in vivo inhibition was 59%, suggesting that f(m,CYP3A4) for cortisol and cortisone 6β-hydroxylation is ~60%. Given the significant decrease in CL(f) of 6β-hydroxycortisone and 6β-hydroxycortisol after 200-mg and 400-mg single doses of itraconazole, this endogenous probe can be used to detect moderate and potent CYP3A4 inhibition in vivo.     

Dual-targeting of αvβ3 and galectin-1 improves the specificity of paramagnetic/fluorescent liposomes to tumor endothelium in vivo

Molecular imaging of angiogenesis requires a highly specific and efficient contrast agent for targeting activated endothelium. We have previously demonstrated that paramagnetic and fluorescent liposomes functionalized with two angiogenesis-specific ligands, the galectin-1-specific anginex (Anx) and the α(v)β(3) integrin-specific RGD, produce synergistic targeting effect in vitro. In the current study, we applied Anx and RGD dual-conjugated liposomes (Anx/RGD-L) for angiogenesis-specific MRI in vivo, focusing on the specificity and efficacy of liposome association with tumor endothelium. The targeting properties, clearance kinetics and biodistribution of Anx/RGD-L were investigated in B16F10 melanoma-bearing mice, and compared to liposomes functionalized with either Anx (Anx-L) or RGD (RGD-L). The contrast enhancement produced by dual- and single-targeted nanoparticles in the tumor was measured using in vivo T(1)-weighted MRI, complemented by ex vivo immunohistochemical evaluation of tumor tissues. Blood clearance kinetics of Anx/RGD-L was three-fold more rapid than for RGD-L, but comparable to Anx-L. Both dual- and single-targeted liposomes produced similar changes in MRI contrast parameters in tumors with high inter-tumor variability (ΔR(1)=0.04±0.03s(-1), 24h post-contrast). Importantly, however, the specificity of Anx/RGD-L association with tumor endothelium of 53±6%, assessed by fluorescence microscopy, was significantly higher compared to 43±9% (P=0.043) and 28±8% (P=0.0001) of Anx-L and RGD-L, respectively. In contrast, long-circulating RGD-L were on average 16% more efficient in targeting tumor endothelium compared to Anx/RGD-L. Significant differences were also found in the biodistribution of investigated contrast agents. In conclusion, synergistic targeting of α(v)β(3) and galectin-1 improved the specificity of the association of the liposomal contrast agent to tumor endothelium in vivo, providing therefore a more reliable MRI readout of the angiogenic activity.     

An Evaluation of 2-deoxy-2-[18F]Fluoro-D-Glucose and 3′-deoxy-3′-[18F]-Fluorothymidine Uptake in Human Tumor Xenograft Models

Purpose

The aim of this study is to assess the variability of 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]-FDG) and 3??-deoxy-3??-[¹⁸F]-fluorothymidine ([¹⁸F]-FLT) uptake in pre-clinical tumor models and examine the relationship between imaging data and related histological biomarkers.

Procedures

[¹⁸F]-FDG and [¹⁸F]-FLT studies were carried out in nine human tumor xenograft models in mice. A selection of the models underwent histological analysis for endpoints relevant to radiotracer uptake. Comparisons were made between in vitro uptake, in vivo imaging, and ex vivo histopathology data using quantitative and semi-quantitative analysis.

Results

In vitro data revealed that [1-¹⁴C]-2-deoxy-d-glucose ([¹⁴C]-2DG) uptake in the tumor cell lines was variable. In vivo, [¹⁸F]-FDG and [¹⁸F]-FLT uptake was highly variable across tumor types and uptake of one tracer was not predictive for the other. [¹⁴C]-2DG uptake in vitro did not predict for [¹⁸F]-FDG uptake in vivo. [¹⁸F]-FDG SUV was inversely proportional to Ki67 and necrosis levels and positively correlated with HKI. [¹⁸F]-FLT uptake positively correlated with Ki67 and TK1.

Conclusion

When evaluating imaging biomarkers in response to therapy, the choice of tumor model should take into account in vivo baseline radiotracer uptake, which can vary significantly between models.     

Development and Characterization of Medical Phantoms for Ultrasound Imaging Based on Customizable and Mouldable Polyvinyl Alcohol Cryogel–Based Materials and 3-D Printing: Application to High-Frequency Cranial Ultrasonography in Infants

This work presents an affordable and easily customizable methodology for phantom manufacturing, which can be used to mimic different anatomic organs and structures. This methodology is based on the use of polyvinyl alcohol–based cryogels as a physical substitute for biologic soft tissues and of 3-D printed polymers for hard tissues, moulding and supporting elements. Thin and durable soft-tissue mimicking layers and multilayer arrangements can be obtained using these materials. Special attention was paid to the acoustic properties (sound speed, attenuation coefficient and mechanical impedance) of the materials developed to simulate soft tissues. These properties were characterized as a function of the additives concentration (propylene-glycol and alumina particles). The polyvinyl alcohol formulation proposed in this work is stable over several freeze-thaw cycles, allowing the manufacturing of multilayer materials with controlled properties. The manufacturing methodology presented was applied to the development of a phantom for high-frequency cranial ultrasonography in infants. This phantom was able to reproduce the main characteristics of the ultrasound images obtained in neonates through the anterior fontanel, down to 8-mm depth.     

Proposed Bedside Maneuver to Facilitate Accurate Anatomic Orientation for Correct Positioning of ECG Precordial Leads V1 and V2: A Pilot Study

Background

Misplacement of right precordial electrocardiogram (ECG) electrodes superiorly is a prevalent procedural error that may lead to false findings of T-wave inversion or QS complexes in V2—possibly triggering wasteful utilization of health care resources. Standard technique for proper placement of V1–V2 entails initial palpation for the sternal angle, pointing to the second intercostal space (ICS), followed by lead fixation at the fourth ICS.

Study Objective

Because adherence to this approach may be limited by lack of a visual landmark for the second ICS, we assessed an alternative technique.

Methods

The evaluated technique involved placement of the patient’s hand up against the base of his/her neck (H→N maneuver) to help demarcate visually a specific point “X” on the chest.

Results

Of 112 patients studied, “X” landed on the first rib in 2.7%, first ICS in 7.1%, second rib in 56.3%, second ICS in 33.0%, and third rib in 0.9%. Thus, in 89.3% (95% confidence interval 83.6–95.0%) of cases (93.3% of men, 84.6% of women; p = 0.13), the second ICS could be identified by H→N via the following simple rule: Utilize “X” if it overlies an ICS; or the immediately subjacent ICS if “X” overlies a rib.

Conclusion

The H→N maneuver provides a primarily visual approach to identifying the second ICS and, thereby, the fourth ICS for affixing V1–V2. If the present initial experience is confirmed, H→N might merit consideration as an educational tool to promote anatomically correct placement of these precordial leads, a prerequisite to diminishing the incidence of ECG procedure-related “septal ischemia/infarction.”     

TRAF-interacting Protein (TRIP): A Novel Component of the Tumor Necrosis Factor Receptor (TNFR)- and CD30-TRAF Signaling Complexes That Inhibits TRAF2-mediated NF-κB Activation

Through their interaction with the TNF receptor–associated factor (TRAF) family, members of the tumor necrosis factor receptor (TNFR) superfamily elicit a wide range of biological effects including differentiation, proliferation, activation, or cell death. We have identified and characterized a novel component of the receptor–TRAF signaling complex, designated TRIP (TRAF-interacting protein), which contains a RING finger motif and an extended coiled-coil domain. TRIP associates with the TNFR2 or CD30 signaling complex through its interaction with TRAF proteins. When associated, TRIP inhibits the TRAF2-mediated NF-κB activation that is required for cell activation and also for protection against apoptosis. Thus, TRIP acts as a receptor–proximal regulator that may influence signals responsible for cell activation/proliferation and cell death induced by members of the TNFR superfamily.Members of the TNF receptor (TNFR)¹ superfamily play important roles in the induction of diverse signals leading to cell growth, activation, and apoptosis (1). Whether the signals induced by a given receptor leads to cell activation or death is, however, highly cell-type specific and tightly regulated during differentiation of cells. For example, the TNFRs can exert costimulatory signals for proliferation of naive lymphocytes but also induce death signals required for deletion of activated T lymphocytes (1). The cytoplasmic domains of these receptors lack intrinsic catalytic activity and also exhibit no significant homology to each other or to other known proteins. Exceptions to this include Fas(CD95) and TNFR1 that share a significant homology within an 80–amino acid region of their cytoplasmic tails (called the “death domain”; 2, 3). Therefore, it is suggested that the TNFR family members can initiate different signal transduction pathways by recruiting different types of intracellular signal transducers to the receptor complex (1).Indeed, several types of intracellular signal transducers have been identified that initiate distinct signal transduction pathways when recruited to the members of TNFR superfamily (4–19). Recent biochemical and molecular studies showed that a class of signal-transducing molecules are recruited to Fas(CD95) or TNFR1 via interaction of the death domains (2, 3, 6, 12, 17, 20). For example, Fas(CD95) and TNFR1 recruit FADD(MORT1)/RIP or TRADD/FADD (MORT1)/RIP through the interactions of their respective death domains (2, 3, 6, 12, 17, 20, 21). Clustering of these signal transducers leads to the recruitment of FLICE/ MACH, and subsequently, to cell death (13, 14).The TNFR family members can also recruit a second class of signal transducers called TRAFs (TNFR-associated factor), some of which are responsible for the activation of NF-κB or JNK (9, 20, 22). TRAF proteins were identified by their biochemical ability to interact with TNFR2, CD40, CD30, or LT-βR (4, 5, 10, 11, 15, 23–27). These receptors interact directly with TRAFs via a short stretch of amino acids within their cytoplasmic tails, but do not interact with the death domain containing proteins (4, 5, 15, 24–27). To date, five members of the TRAF family have been identified as signaling components of the TNFR family members. All TRAF members contain a conserved TRAF domain, ∼230 amino acids in length, that is used for either homo- or heterooligomerization among the TRAF family, for interactions with the cytoplasmic regions of the TNFR superfamily, or for interactions with downstream signal transducers (4, 5, 8, 10, 11, 19, 23–25, 28). In addition to the TRAF domain, most of the TRAF family members contain an NH₂-terminal RING finger and several zinc finger structures, which appear to be important for their effector functions (4, 5, 10, 11, 23–25).Several effector functions of TRAFs were revealed by recent experiments based on a transfection system. TRAF2, first identified by its interaction with TNFR2 (4), was subsequently shown to mediate NF-κB activation induced by two TNF receptors, CD40 and CD30 (9, 28–30). TRAF5 was also implicated in NF-κB activation mediated by LTβR (10), whereas TRAF3 (also known as CRAF1, CD40bp, or LAP1; references 5, 11, 24, and 25) was shown to be involved in the regulation of CD40-mediated CD23 upregulation in B cells (5). The role of other TRAF members in the TNFR family–mediated signal transduction is not clear. They may possess some effector functions as yet to be revealed, or work as adapter proteins to recruit different downstream signal transducers to the receptor complex. For example, TRAF1 is required for the recruitment of members of the cellular inhibitor of apoptosis protein (c-IAP) family to the TNFR2-signaling complex (7). In addition to the signal transduction by the TNFR family members, TRAFs may regulate other receptor-mediated signaling pathways. For example, TRAF6 is a component of IL-1 receptor (IL1R)–signaling complex, in which it mediates the activation of NF-κB by IL-1R (31). Since TRAFs form homo- or heterooligomers, it is suggested that the repertoire of TRAF members in a given cell type may differentially affect the intracellular signals triggered by these receptors. This may be accomplished by the selective interaction of TRAFs with a specific set of downstream signal transducers. Although many aspects of TRAF-mediated effector functions leading to cellular activation have been defined, it needs to be determined whether TRAF proteins will also mediate the apoptotic signals induced by the “death-domain-less” members of the TNFR superfamily (1, 27, 32–36).Here we report the isolation and characterization of a novel component of the TNFR superfamily/TRAFs signaling complex, named TRIP (TRAF-interacting protein). TRIP associates with the receptor/TRAF signaling complex, and inhibits the TRAF2-mediated NF-κB activation. Biochemical studies indicate that TRIP associates with the TNFR2 or CD30 receptor complex via its interaction with TRAF proteins, suggesting a model which can explain why the ligation of these receptors can promote different cell fates: proliferation or death.     

Comparison of [<Superscript>99m</Superscript>Tc]3PRGD<Subscript>2</Subscript> Imaging and [<Superscript>18</Superscript>F]FDG PET/CT in Breast Cancer and Expression of Integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript> in Breast Cancer Vascular Endothelial Cells

Purpose

This study aimed to investigate the value of ^99mtechnetium-three polyethylene glycol spacers-arginine-glycine-aspartic acid ([^99mTc]3PRGD₂) imaging in diagnosis and staging of breast cancer compared with 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) imaging, and to explore the expression of integrin α_vβ₃ in tumor vascular endothelial cells.

Procedures

Forty-two women with suspected breast cancer underwent both [^99mTc]3PRGD₂ imaging and [¹⁸F]FDG imaging. Visual analysis was used to assess primary breast lesion, axillary lymph node, and distant metastasis. The tumor-blood (T/B) ratios from [^99mTc]3PRGD₂ imaging and the maximum standardized uptake value (SUVmax) from [¹⁸F]FDG imaging were analyzed for breast lesions. Integrin α_vβ₃ was analyzed through immunohistochemistry.

Results

Forty-five breast lesions were found (malignant, n?=?38; benign, n?=?7). The sensitivity, specificity, and accuracy of [^99mTc]3PRGD₂ and [¹⁸F]FDG imaging in visual analysis for the breast lesion were 97.4, 87.5, and 95.6 % and 97.4, 71.4, and 93.3 %, respectively (P?>?0.05). For semi-quantitative analysis, no significant difference of the area under the curves (AUC) was found in the imaging using the two radiopharmaceuticals (0.880 and 0.955; Z?=?0.88, P?>?0.05). The sensitivity, specificity, and accuracy for axillary lymph node metastasis with [^99mTc]3PRGD₂ and [¹⁸F]FDG were 78.05, 99.36, and 94.92 % and 85.37, 98.72, and 95.64 %, respectively (P?>?0.05). Nine patients with distant metastases were all detected with the two radiopharmaceuticals. The expression of integrin α_vβ₃ was correlated with [^99mTc]3PRGD₂ uptake (r?=?0.582, P?=?0.001), which were significantly higher in the HER2-positive and stage III–IV patients (P?<?0.05).

Conclusions

The prospective study demonstrated that [^99mTc]3PRGD₂ imaging seems to be valuable for diagnosis of breast cancer and its staging. It may be less sensitive for detecting small lymph node metastatic lesions when compared with [¹⁸F]FDG imaging. Integrin α_vβ₃ in tumor microvessels was associated with the breast cancer subtype and its staging.