Ultrasound/Optical Dual‐Modality Imaging for Evaluation of Vulnerable Atherosclerotic Plaques with Osteopontin Targeted Nanoparticles

Because of the high mortality of coronary atherosclerotic heart diseases, it is necessary to develop novel early detection methods for vulnerable atherosclerotic plaques. Phenotype transformation of vascular smooth muscle cells (VSMCs) plays a vital role in progressed atherosclerotic plaques. Osteopontin (OPN) is one of the biomarkers for phenotypic conversion of VSMCs. Significant higher OPN expression is found in foam cells along with the aggravating capacity of macrophage recruitment due to its arginine‐glycine‐aspartate sequence and interaction with CD44. Herein, a dual‐modality imaging probe, OPN targeted nanoparticles (Cy5.5‐anti‐OPN‐PEG‐PLA‐PFOB, denoted as COP‐NPs), is constructed to identify the molecular characteristics of high‐risk atherosclerosis by ultrasound and optical imaging. Characterization, biocompatibility, good binding sensibility, and specificity are evaluated in vitro. For in vivo study, apolipoprotein E deficien (ApoE^?/?) mice fed with high fat diet for 20–24 weeks are used as atherosclerotic model. Ultrasound and optical imaging reveal that the nanoparticles are accumulated in the vulnerable atherosclerotic plaques. OPN targeted nanoparticles are demonstrated to be a good contrast agent in molecular imaging of synthetic VSMCs and foam cells, which can be a promising tool to identify the vulnerable atherosclerotic plaques.     

傅立叶变换离子回旋共振质谱分析与鉴定骨桥蛋白的多肽片段

采用高效液相色谱(HPLC)SCX及C4色谱柱从人乳样品的乳清部分分离与纯化出骨桥蛋白(Osteopontin, OPN),在一定条件下将其用胰蛋白酶酶解后,利用高分辨傅立叶变换离子回旋共振质谱(FT-ICR-MS),结合微喷雾(Nano-spray)技术,并借助Mascot软件数据库,获得了OPN相关多肽片段GDSVVYGLR和QNLLAPQTLPSK的质谱结构信息,分析了其裂解机理.研究表明,利用FT-ICR-MS并结合HPLC等化学方法,可以高效、快速地分离与纯化人乳样品中的OPN,借助Mascot软件数据库进行质谱分析,能够简洁、准确地鉴定相关多肽片段的结构信息,为OPN多肽片段不同翻译后修饰位点的确定与其生物学活性关系的研究提供了理论依据.     

MC3T3-E1 cell adhesion to hydroxyapatite with adsorbed bone sialoprotein, bone osteopontin, and bovine serum albumin

Native bone tissue is composed of a complex matrix of collagen, non-collagenous proteins, and hydroxyapatite (HAP). Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the non-collagenous protein family termed the SIBLING (small integrin-binding ligand, N-linked glycoproteins) proteins, which are primarily found in mineralized tissues. Previously, OPN was shown to exhibit a preferential orientation for MC3T3-E1 cell adhesion when it was specifically bound to collagen, while the MC3T3-E1 cell adhesion was shown to be dependant on the conformational flexibility of BSP specifically bound to collagen. Additionally, OPN was shown to play a greater role than BSP for cell binding to collagen. In this work, the orientations and conformations of BSP and OPN specifically bound to HAP are probed under similar conditions. Radiolabeled adsorption isotherms were obtained for BSP and OPN on HAP formed from a simulated body fluid, and the results show that HAP has the capacity to bind significantly more BSP than OPN. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of adsorbed BSP and OPN specifically bound to HAP. It was found that there is a preference for cell binding to HAP with adsorbed BSP as compared to OPN, but not to a statistically significant level. However, the maximum cell binding was observed on HAP substrates with adsorbed heat denatured bovine serum albumin (BSA). The influence of BSA on cell binding was shown to be concentration dependant and it is believed that the adsorbed BSA modulates the proliferation state of the bound cells.     

Compensatory Adaptations of Structural Dynamics in an Intrinsically Disordered Protein Complex

Intrinsically disordered proteins (IDPs) play crucial roles in protein interaction networks and in this context frequently constitute important hubs and interfaces. Here we show by a combination of NMR and EPR spectroscopy that the binding of the cytokine osteopontin (OPN) to its natural ligand, heparin, is accompanied by thermodynamically compensating structural adaptations. The core segment of OPN expands upon binding. This “unfolding‐upon‐binding” is governed primarily through electrostatic interactions between heparin and charged patches along the protein backbone and compensates for entropic penalties due to heparin–OPN binding. It is shown how structural unfolding compensates for entropic losses through ligand binding in IDPs and elucidates the interplay between structure and thermodynamics of rapid substrate‐binding and ‐release events in IDP interaction networks.     

Expression analyses of human cleft palate tissue suggest a role for osteopontin and immune related factors in palatal development

Cleft lip and/or palate (CL/P) is a common congenital malformation with a complex etiology which is not fully elucidated yet. Epidemiological studies point to different etiologies in the cleft lip and palate subgroups, isolated cleft lip (CL), isolated cleft palate (CP) and combined cleft lip and palate (CLP). In order to understand the biological basis in these cleft lip and palate subgroups better we studied the expression profiles in human tissue from patients with CL/P. In each of the CL/P subgroups, samples were obtained from three patients and gene expression analysis was performed. Moreover, selected differentially expressed genes were analyzed by quantitative RT-PCR, and by immunohistochemical staining of craniofacial tissue from human embryos. Osteopontin (SPP1) and other immune related genes were significantly higher expressed in palate tissue from patients with CLP compared to CP and immunostaining in palatal shelves against SPP1, chemokine receptor 4 (CXCR4) and serglycin (PRG1) in human embryonic craniofacial tissue were positive, supporting a role for these genes in palatal development. However, gene expression profiles are subject to variations during growth and therefore we recommend that future gene expression in CL/P studies should use tissue from the correct embryonic time and place if possible, to overcome the biases in the presented study.     

无胶筛分毛细管电泳法检测微量蛋白质骨桥蛋白

采用非涂层毛细管,以150 mmol/L硼酸盐缓冲液为电泳缓冲液,30 g/L聚乙二醇(PEG)20000为筛分介质,经对分离条件进行优化,成功地建立了用无胶筛分毛细管电泳检测微量蛋白质的方法。用所建立的方法测定骨桥蛋白,其批内、批间迁移时间的相对标准偏差均小于5%,回收率大于95%,被检测样品中骨桥蛋白的含量与其峰面积呈良好的线性关系（相关系数为0.996）,最低检测限为0.079 g/L。考察了无血清饥饿培养对血管平滑肌细胞分泌骨桥蛋白的影响,结果表明无血清饥饿培养24 h骨桥蛋白的合成与分泌达到高峰,此后随时间延长含量随之降低,此结果与采用Western blot方法检测的结果一致。该方法具有进样量小（nL级)、检测速度快、可自动化等优点,是一种简便、快捷的检测微量蛋白质的好方法。     

An extended stable isotope‐labeled signature peptide internal standard for tracking immunocapture of human plasma osteopontin for LC‐MS/MS quantification

A stable isotope‐labeled signature peptide, whose sequence corresponds to the human osteopontin (hOPN) specific antibody epitope, was evaluated as an internal standard to compensate for immunocapture variability during quantification of hOPN by immunoaffinity‐coupled LC‐MS/MS. Immunocapture variability was induced by varying the antibody amount per well from 150 to 4500 ng and analysis was carried out with internal standards added before and after the immunocapture step. The immunocapture variability ranged from ?80.9 to 77.0% when the IS was added after immunocapture and from ?37.5 to 20.3% when the internal standard was added before immunocapture. The lower variability demonstrates the ability of stable labeled isotope internal standard peptide to compensate for variation during immunocapture. Copyright © 2015 John Wiley & Sons, Ltd.