\u3csup\u3e99m\u3c/sup\u3eTc-Labeled C2A Domain of Synaptotagmin I as a Target-Specific Molecular Probe for Noninvasive Imaging of Acute Myocardial Infarction

Abstract: The exposure of phosphatidylserine (PtdS) is a common molecular marker for both apoptosis and necrosis and enables the simultaneous detection of these distinct modes of cell death. Our aim was to develop a radiotracer based on the PtdS-binding activity of the C2A domain of synaptotagmin I and assess 99mTc-C2A-GST (GST is glutathione S-transferase) using a reperfused acute myocardial infarction (AMI) rat model. Methods: The binding of C2A-GST toward apoptosis and necrosis was validated in vitro. After labeling with 99mTc via 2-iminothiolane thiolation, radiochemical purity and radiostability were tested. Pharmacokinetics and biodistribution were studied in healthy rats. The uptake of 99mTc-C2A-GST within the area at risk was quantified by direct γ-counting, whereas nonspecific accumulation was estimated using inactivated 99mTc-C2A-GST. In vivo planar imaging of AMI in rats was performed on a γ-camera using a parallel-hole collimator. Radioactivity uptake was investigated by region-of-interest analysis, and postmortem tetrazolium staining versus autoradiography. Results: Fluorescently labeled and radiolabeled C2A-GST bound both apoptotic and necrotic cells. 99mTc-C2A-GST had a radiochemical purity of \u3e98% and remained stable. After intravenous injection, the uptake in the liver and kidneys was significant. For 99mTc-C2A-GST, radioactivity uptake in the area at risk reached between 2.40 and 2.63 %ID/g (%ID/g is percentage injected dose per gram) within 30 min and remained plateaued for at least 3 h. In comparison, with the inactivated tracer the radioactivity reached 1.06 ± 0.49 %ID/g at 30 min, followed by washout to 0.52 ± 0.23 %ID/g. In 7 of 7 rats, the infarct was clearly identifiable as focal uptake in planar images. At 3 h after injection, the infarct-to-lung ratios were 2.48 ± 0.27, 1.29 ± 0.09, and 1.46 ± 0.04 for acute-infarct rats with 99mTc-C2A-GST, sham-operated rats with 99mTc-C2A-GST, and acute-infarct rats with 99mTc-C2A-GST-NHS (NHS is N-hydroxy succinimide), respectively. The distribution of radioactivity was confirmed by autoradiography and histology. Conclusion: The C2A domain of synaptotagmin I labeled with fluorochromes or a radioisotope binds to both apoptotic and necrotic cells. Ex vivo and in vivo data indicate that, because of elevated vascular permeability, both specific binding and passive leakage contribute to the accumulation of the radiotracer in the area at risk. However, the latter component alone is insufficient to achieve detectable target-to-background ratios with in vivo planar imaging

P120-Catenin Isoforms 1 and 3 Regulate Proliferation and Cell Cycle of Lung Cancer Cells via β-Catenin and Kaiso Respectively

<div><h3>Background</h3><p>The different mechanisms involved in p120-catenin (p120ctn) isoforms' 1/3 regulation of cell cycle progression are still not elucidated to date.</p> <h3>Methods and Findings</h3><p>We found that both cyclin D1 and cyclin E could be effectively restored by restitution of p120ctn-1A or p120ctn-3A in p120ctn depleted lung cancer cells. When the expression of cyclin D1 was blocked by co-transfection with siRNA-cyclin D1 in p120ctn depleted cells restoring p120ctn-1A or 3A, the expression of cyclin E was slightly decreased, not increased, implying that p120ctn isoforms 1 and 3 cannot up-regulate cyclin E directly but may do so through up-regulation of cyclin D1. Interestingly, overexpression of p120ctn-1A increased β-catenin and cyclin D1 expression, while co-transfection with siRNA targeting β-catenin abolishes the effect of p120ctn-1A on up-regulation of cyclin D1, suggesting a role of β-catenin in mediating p120ctn-1A's regulatory function on cyclin D1 expression. On the other hand, overexpression of p120ctn isoform 3A reduced nuclear Kaiso localization, thus decreasing the binding of Kaiso to KBS on the cyclin D1 promoter and thereby enhancing the expression of cyclin D1 gene by relieving the repressor effect of Kaiso. Because overexpressing NLS-p120ctn-3A (p120ctn-3A nuclear target localization plasmids) or inhibiting nuclear export of p120ctn-3 by Leptomycin B (LMB) caused translocation of Kaiso to the nucleus, it is plausible that the nuclear export of Kaiso is p120ctn-3-dependent.</p> <h3>Conclusions</h3><p>Our results suggest that p120ctn isoforms 1 and 3 up-regulate cyclin D1, and thereby cyclin E, resulting in the promotion of cell proliferation and cell cycle progression in lung cancer cells probably via different protein mediators, namely, β-catenin for isoform 1 and Kaiso, a negative transcriptional factor of cyclin D1, for isoform 3.</p> </div

Forkhead box L2 is a target of miR‐133b and plays an important role in the pathogenesis of non‐small cell lung cancer

Abstract Background Forkhead box L2 (FOXL2) has been recognized as a transcription factor in the progression of many malignancies, but its role in non‐small cell lung cancer (NSCLC) remains unclear. This research clarified on the role of FOXL2 and the specific molecular mechanism in NSCLC. Methods RNA and protein levels were detected by quantitative real‐time polymerase chain reaction (qRT–PCR) and western blotting assays. Cell proliferation was examined by cell counting kit‐8 (CCK‐8) and clonogenic assays. Transwell and wound healing assays were used to detect cell invasion and migration. Cell cycle alterations were assessed by flow cytometry. The relationship between FOXL2 and miR‐133b was verified by dual‐luciferase reporter assays. In vivo metastasis was monitored in the tail vein‐injected mice. Results FOXL2 was upregulated in NSCLC cells and tissues. Downregulation of FOXL2 restrained cell proliferation, migration, and invasion and arrested the cell cycle of NSCLC cells. Moreover, FOXL2 promoted the epithelial–mesenchymal transition (EMT) process of NSCLC cells by inducing the transforming growth factor‐β (TGF‐β)/Smad signaling pathway. miR‐133b directly targeted the 3′‐UTR of FOXL2 and negatively regulated FOXL2 expression. Knockdown of FOXL2 blocked metastasis in vivo. Conclusions miR‐133b downregulates FOXL2 by targeting the 3′‐UTR of FOXL2, thereby inhibiting cell proliferation, EMT and metastasis induced by the TGF‐β/Smad signaling pathway in NSCLC. FOXL2 may be a potential molecular target for treating NSCLC

Triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1) reveals platelet activation in preeclampsia

Triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1) is a membrane protein receptor found in α-granules of megakaryocytes and platelets. Upon platelet activation TLT-1 is rapidly relocated to the surface of platelets. In plasma, a soluble form of TLT-1 (sTLT-1) is present. Plasma levels of sTLT-1 are significantly elevated in thrombotic diseases. In the present study, we investigated to whether TLT-1 reflects platelet activation in pregnant women with preeclampsia. We studied 30 preeclamptic patients who were matched with 30 normotensive pregnant women and 30 non-pregnant controls. Basal TLT-1, P-selectin, and CD63 expressions on platelets were analyzed with the use of flow-cytometry (FCM). Platelet reactivity was induced by thrombin receptor activation peptide and determined by FCM. Plasma concentrations of sTLT-1 and soluble P-selectin (sP-selectin) were measured by an enzyme-linked immunosorbent assay. Results show that basal platelet expression of TLT-1, P-selectin and CD63 were increased in women with preeclampsia (PE) compared with normotensive pregnant women (NP). Platelets from PE women and NP women were more responsive compared to from nonpregnant women controls (NC), and which was demonstrated by increased expression of TLT-1, P-selectin, and CD63 upon stimulation in vitro. Plasma concentration of sTLT-1 was greater in PE women compared to NP women and NC women. Plasma sP-selectin level was higher in pregnant women than in nonpregnant women, but there were no significant differences between PE and NP women. In summary, our results revealed that platelet activation is prominent in preeclampsia, TLT-1 reflects platelet activation and may be a useful indicator for preeclampsia

The chimeric monoclonal antibody MHCSZ-123 against human von Willebrand factor A3 domain inhibits high-shear arterial thrombosis in a Rhesus monkey model

Abstract Background SZ-123, a murine monoclonal antibody that targets the human von Willebrand factor (VWF) A3 domain and blocks the binding of collagen, is a powerful antithrombotic. In a Rhesus monkey model of thrombosis, SZ-123 had no side effects, such as bleeding or thrombocytopenia. Methods The mouse/human chimeric version of SZ-123, MHCSZ-123, was developed and maintained inhibitory capacities in vitro and ex vivo after injection into monkeys. CHO-S cells were selected for stable expression of MHCSZ-123. Cell clones with high levels of MHCSZ-123 expression were screened with G418 then adapted to serum-free suspension culture. The antithrombotic effect of MHCSZ-123 on acute platelet-mediated thrombosis was studied in monkeys where thrombus formation was induced by injury and stenosis of the femoral artery, which allowed for cyclic flow reductions (CFRs). CFRs were measured in the femoral artery of anesthetized Rhesus monkeys before and after intravenous administration of MHCSZ-123. Ex vivo VWF binding to collagen, platelet aggregation, platelet counts, and template bleeding time were used as measurements of antithrombotic activity. In addition, plasma VWF and VWF occupancy were measured by ELISA. Results Injection of 0.1, 0.3, and 0.6 mg/kg MHCSZ-123 significantly reduced CFRs by 29.4%, 57.9%, and 73.1%, respectively. When 0.3 and 0.6 mg/kg MHCSZ-123 were administered, 46.6%–65.8% inhibition of ristocetin-induced platelet aggregation was observed between 15 and 30 min after injection. We observed minimal effects on bleeding time, minimal blood loss, and no spontaneous bleeding or thrombocytopenia. Conclusions The VWF-A3 inhibitor MHCSZ-123 significantly reduced thrombosis in Rhesus monkeys and appeared to be safe and well tolerated

FITC-Conjugated Cyclic RGD Peptides as Fluorescent Probes for Staining Integrin α_vβ₃/α_vβ₅ in Tumor Tissues

This study sought to evaluate FITC-conjugated cyclic RGD peptides (FITC-RGD₂, FITC-3P-RGD₂, and FITC-Galacto-RGD₂) as fluorescent probes for in vitro assays of integrin α_vβ₃/α_vβ₅ expression in tumor tissues. FITC-RGD₂, FITC-3P-RGD₂, and FITC-Galacto-RGD₂ were prepared, and their integrin α_vβ₃/α_vβ₅ binding affinity was determined using the displacement assay against ¹²⁵I-echistatin bound to U87MG glioma cells. IC₅₀ values of FITC-Galacto-RGD₂, FITC-3P-RGD₂, and FITC-RGD₂ were calculated to be 28 ± 8, 32 ± 7, and 89 ± 17 nM, respectively. The integrin α_vβ₃/α_vβ₅ binding affinity followed a general trend: FITC-Galacto-RGD₂ ∼ FITC-3P-RGD₂ > FITC-RGD₂. The xenografted tumor-bearing models were established by subcutaneous injection of 5 × 10⁶ tumor cells into shoulder flank (U87MG, A549, HT29, and PC-3) or mammary fat pad (MDA-MB-435) of each athymic nude mouse. Three to six weeks after inoculation, the tumor size was 0.1–0.3 g. Tumors were harvested for integrin α_vβ₃/α_vβ₅ staining, as well as hematoxylin and eosin (H&E) staining. Six human carcinoma tissues (colon cancer, pancreatic cancer, lung adenocarcinoma, squamous cell lung cancer, gastric cancer, and esophageal cancer) were obtained from recently diagnosed cancer patients. Human carcinoma slides were deparaffinized in xylene, rehydrated with ethanol, and then used for integrin α_vβ₃/α_vβ₅ staining, as well as H&E staining. It was found that the tumor staining procedures with FITC-conjugated cyclic RGD peptides were much simpler than those with the fluorescence-labeled integrin α_vβ₃ antibodies. Since FITC-RGD₂, FITC-3P-RGD₂, and FITC-Galacto-RGD₂ were able to co-localize with the fluorescence-labeled integrin β₃ antibody, their tumor localization and tumor cell binding are integrin α_vβ₃-specific. Quantification of the fluorescent intensity in five xenografted tumors (U87MG, MDA-MB-435, A549, HT29, and PC-3) and six human carcinoma tissues revealed an excellent linear relationship between the relative integrin α_vβ₃/α_vβ₅ expression levels determined with FITC-Galacto-RGD₂ and those obtained with the fluorescence-labeled anti-human integrin β₃ antibody. There was also an excellent linear relationship between the tumor uptake (%ID/g) of ^99mTc-3P-RGD₂ (an integrin α_vβ₃/α_vβ₅-targeted radiotracer) and the relative integrin α_vβ₃/α_vβ₅ expression levels from the quantification of fluorescent intensity in the tumor tissues stained with FITC-Galacto-RGD₂. These results suggest that FITC-conjugated cyclic RGD peptides might be useful to correlate the in vitro findings with the in vivo imaging data from an integrin α_vβ₃/α_vβ₅-targeted radiotracer. The results from this study clearly showed that the FITC-conjugated cyclic RGD peptides (particularly FITC-3P-RGD₂ and FITC-Galacto-RGD₂) are useful fluorescent probes for assaying relative integrin α_vβ₃/α_vβ₅ expression levels in tumor tissues