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Cerebral malaria (CM) is a life-threatening diffuse encephalopathy caused by Plasmodium falciparum, in which the destruction of the blood–brain barrier (BBB) is the main cause of death. However, increasing evidence has shown that antimalarial drugs, the current treatment for CM, do little to protect against CM-induced BBB damage. Therefore, a means to alleviate BBB dysfunction would be a promising adjuvant therapy for CM. The adhesion molecule CD146 has been reported to be expressed in both endothelial cells and proinflammatory immune cells and mediates neuroinflammation. Here, we demonstrate that CD146 expressed on BBB endothelial cells but not immune cells is a novel therapeutic target in a mouse model of experimental cerebral malaria (eCM). Endothelial CD146 is upregulated during eCM development and facilitates the sequestration of infected red blood cells (RBCs) and/or proinflammatory lymphocytes in CNS blood vessels, thereby promoting the disruption of BBB integrity. Mechanistic studies showed that the interaction of CD146 and Galectin-9 contributes to the aggregation of infected RBCs and lymphocytes. Deletion of endothelial CD146 or treatment with the anti-CD146 antibody AA98 prevents severe signs of eCM, such as limb paralysis, brain vascular leakage, and death. In addition, AA98 combined with the antiparasitic drug artemether improved the cognition and memory of mice with eCM. Taken together, our findings suggest that endothelial CD146 is a novel and promising target in combination with antiparasitic drugs for future CM therapies.

Anti-angiogenesis drug therapy is ineffective in treating uveal melanoma since it only targets angiogenesis leaving vasculogenic mimicry aside. There is no effective clinical strategy targeting vasculogenic mimicry, yet. We show here that CD146 is a novel target to inhibit uveal melanoma progression since it regulates both uveal melanoma angiogenesis and vasculogenic mimicry activity.CD146 inhibition was achieved with its specific siRNAs or antibody AA98. Tube formation and migration of primary human retinal microvascular endothelial cells and tube-like structure formation, migration, invasion of uveal melanoma cells were evaluated after CD146 inhibition. The underlying mechanisms were investigated by Western blot and immunofluorescence. Finally, uveal melanoma cells were injected subretinally into the eyes of nude mice and AA98 was administrated. Tumor size was revealed by HE staining, and angiogenesis and vasculogenic mimicry were evaluated with CD31-PAS staining.CD146 inhibition induced declines in tube formation and migration of primary human retinal microvascular endothelial cells and tube-like structure formation of uveal melanoma cells. CD146 mediated VEGFR/AKT/p38/NF-κB and FAK/VE-cadherin signal cascades were partially responsible for these biological effects. CD146 blockade by siRNA or AA98 also resulted in inhibition of migration and invasion as well as EMT process of uveal melanoma cells. The physiological relevance of such declines was confirmed by showing that AA98 treatment markedly suppressed the tumor growth, angiogenesis and vasculogenic mimicry induced by implantation of uveal melanoma cells into the eyes of nude mice.CD146 is a novel mediator of both angiogenesis and vasculogenic mimicry in uveal melanoma. Its antibody AA98 has the potency to be developed as a new antibody drug for treating uveal melanoma. Our results warrant further assessment of CD146 as a potential target to improve therapeutic management of uveal melanoma in a clinical setting.

Initiation of T cell receptor (TCR) signaling involves the activation of the tyrosine kinase LCK; however, it is currently unclear how LCK is recruited and activated. Here, we have identified the membrane protein CD146 as an essential member of the TCR network for LCK activation. CD146 deficiency in T cells substantially impaired thymocyte development and peripheral activation, both of which depend on TCR signaling. CD146 was found to directly interact with the SH3 domain of coreceptor-free LCK via its cytoplasmic domain. Interestingly, we found CD146 to be present in both monomeric and dimeric forms in T cells, with the dimerized form increasing after TCR ligation. Increased dimerized CD146 recruited LCK and promoted LCK autophosphorylation. In tumor models, CD146 deficiency dramatically impaired the antitumor response of T cells. Together, our data reveal an LCK activation mechanism for TCR initiation. We also underscore a rational intervention based on CD146 for tumor immunotherapy.

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease of cardiopulmonary units. No cure is currently available due to an incomplete understanding of vascular remodeling. Here we identify CD146-hypoxia-inducible transcription factor 1 alpha (HIF-1α) cross-regulation as a key determinant in vascular remodeling and PAH pathogenesis. CD146 is markedly upregulated in pulmonary artery smooth muscle cells (PASMCs/SMCs) and in proportion to disease severity. CD146 expression and HIF-1α transcriptional program reinforce each other to physiologically enable PASMCs to adopt a more synthetic phenotype. Disruption of CD146-HIF-1α cross-talk by genetic ablation of Cd146 in SMCs mitigates pulmonary vascular remodeling in chronic hypoxic mice. Strikingly, targeting of this axis with anti-CD146 antibodies alleviates established pulmonary hypertension (PH) and enhances cardiac function in two rodent models. This study provides mechanistic insights into hypoxic reprogramming that permits vascular remodeling, and thus provides proof of concept for anti-remodeling therapy for PAH through direct modulation of CD146-HIF-1α cross-regulation., Vascular remodelling contributes to the development of pulmonary hypertension (PH). Here Luo and colleagues find that increases in CD146 levels drive vascular remodelling in PH through a cross-talk with hypoxia inducible factor (HIF) signalling, and show that inhibition of CD146 can attenuate disease progression.

Engineered nanocarriers have been widely developed for tumor theranostics. However, the delivery of imaging probes or therapeutic drugs to the tumor pre-formation site for early and accurate detection and therapy remains a major challenge. Here, by using tailor-functionalized human H-ferritin (HFn), we developed a triple-modality nanoprobe IRdye800-M-HFn and achieved the early imaging of tumor cells before the formation of solid tumor tissues. Then, we developed an HFn-doxorubicin (Dox) drug delivery system by loading Dox into the HFn protein cage and achieved early-stage tumor therapy. The intravenous injection of HFn nanoprobes enabled the imaging of tumor cells as early as two days after tumor implantation, and the triple-modality imaging techniques, namely, near-infrared fluorescence molecular imaging (NIR-FMI), magnetic resonance imaging (MRI), and photoacoustic imaging (PAI), ensured the accuracy of detection. Further exploration indicated that HFn could specifically penetrate into pre-solid tumor sites by tumor-associated inflammation-mediated blood vessel leakage, followed by effective accumulation in tumor cells by the specific targeting property of HFn to transferrin receptor 1. Thus, the HFn-Dox drug delivery system delivered Dox into the tumor pre-formation site and effectively killed tumor cells at early stage. IRDye800-M-HFn nanoprobes and HFn-Dox provide promising strategies for early-stage tumor diagnosis and constructive implications for early-stage tumor treatment.

The mechanism of obesity-related metabolic dysfunction involves the development of systemic inflammation, largely mediated by macrophages. Switching of M1-like adipose tissue macrophages (ATMs) to M2-like ATMs, a population of macrophages associated with weight loss and insulin sensitivity, is considered a viable therapeutic strategy for obesity-related metabolic syndrome. However, mechanisms for reestablishing the polarization of ATMs remain elusive. This study demonstrates that CD146

Optical imaging technologies improve clinical diagnostic accuracy of early gastric cancer (EGC). However, there was a lack of imaging agents exhibiting molecular specificity for EGCs. Here, we employed the dye labeled human heavy-chain ferritin (HFn) as imaging nanoprobe, which recognizes tumor biomarker transferrin receptor 1 (TfR1), to enable specific EGC imaging using confocal laser endomicroscopy (CLE). TfR1 expression was initially examined in vitro in gastric tumor cells and in vivo through whole-body fluorescence and CLE imaging in tumor-bearing mice. Subsequently, dye labeled HFn was topically applied to resected human tissues for EGC detection. CLE analysis of TfR1-targeted fluorescence imaging allowed distinction of neoplastic from non-neoplastic tissues (P 0.0001), and TfR1 expression level was found to correlate with EGC differentiation degrees (P 0.0001). Notably, the CLE evaluation correlated well with the immunohistochemical findings (κ-coefficient: 0.8023). Our HFn-nanoprobe-based CLE increases the accuracy of EGC detection and enables visualization of tumor margins and endoscopic resection.

Nanomaterials with intrinsic enzyme-like activities (nanozymes), have been widely used as artificial enzymes in biomedicine. However, how to control their in vivo performance in a target cell is still challenging. Here we report a strategy to coordinate nanozymes to target tumor cells and selectively perform their activity to destruct tumors. We develop a nanozyme using nitrogen-doped porous carbon nanospheres which possess four enzyme-like activities (oxidase, peroxidase, catalase and superoxide dismutase) responsible for reactive oxygen species regulation. We then introduce ferritin to guide nitrogen-doped porous carbon nanospheres into lysosomes and boost reactive oxygen species generation in a tumor-specific manner, resulting in significant tumor regression in human tumor xenograft mice models. Together, our study provides evidence that nitrogen-doped porous carbon nanospheres are powerful nanozymes capable of regulating intracellular reactive oxygen species, and ferritinylation is a promising strategy to render nanozymes to target tumor cells for in vivo tumor catalytic therapy., If decorated with the right surface modifications, nanoparticles can function as Trojan horses, transporting cell death-facilitating compounds to tumor cells. Here, the authors prepare a particle with four enzyme-like activities and show that ferritin can direct nanoparticles to tumor cells.

Cerebral malaria is a lethal complication of malaria infection characterized by central nervous system dysfunction and is often not effectively treated by antimalarial combination therapies. It has been shown that the sequestration of the parasite-infected red blood cells that interact with cerebral vessel endothelial cells and the damage of the blood-brain barrier (BBB) play critical roles in the pathogenesis. In this study, we developed a ferritin nanozyme (Fenozyme) composed of recombinant human ferritin (HFn) protein shells that specifically target BBB endothelial cells (BBB ECs) and the inner Fe

Biomimetic design of nanomaterials with enzyme-like characteristics has emerged as a promising method for the generation of novel therapeutics. However, synthesis of nanomaterials while maintaining a high degree of control over both geometry and valency poses a prominent challenge. Herein, the authors introduce a nanomaterial-based synthetic biology strategy for accurate and quantitative tailoring of high-ordered nanostructures that uses a "bottom-up" hierarchical incorporation of protein building blocks. The assembled nano-oligomers possessed tunable protein motifs and multivalent binding domains, which facilitated prolonged blood circulation time, accumulation within tumor cells through direct targeting of cell receptors, and deep tumor tissue penetration via a transcytosis mechanism. Using these protein/protein nano-oligomers as scaffolds, the authors created a new series of artificial nano-scaled metalloenzymes (nanozymes) by the in situ incorporation of metal nanoclusters within the cavity of the protein nanocages. Nanozymes were capable of mimicking peroxidase-like activity and generated cytotoxic free radicals. Compared to nanozyme alone, the systemic delivery of oligomeric nanozymes demonstrated significantly enhanced therapeutic and anti-tumor benefits. This study shows a new insight into nanotechnology by taking advantage of synthetic biotechnology.

// Dan Liu 1, 5 , Lei Du 2 , Dong Chen 3 , Zhongde Ye 1 , Hongxia Duan 1 , Tao Tu 1 , Jing Feng 1 , Yili Yang 4 , Quan Chen 2 , Xiyun Yan 1 1 Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 2 State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China 3 Department of Pathology, Beijing Anzhen Hospital, Capital Medical University, Beijing 100029, China 4 Department of Colorectal Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China 5 College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China Correspondence to: Xiyun Yan, email: yanxy@ibp.ac.cn Keywords: stemness, CD146, tumorigenesis, Wnt/β-catenin, colorectal cancer Received: September 09, 2015 Accepted: April 18, 2016 Published: June 09, 2016 ABSTRACT Cancer stemness drives tumor progression and drug resistance, representing a challenge to cancer eradication. Compelling evidence indicates that cancer cells can reenter the stem cell state due to the reprogramming of self-renewal machinery. Here, we show that CD146 knockdown induces stem cell properties in colorectal cancer (CRC) cells through activating canonical Wnt signaling. shRNA-mediated CD146 knockdown in CRC cells facilitates tumor initiation in serial xenotransplantation experiments. Moreover, upon CD146 knockdown, CRC cells show elevated expression of specific cancer stem cell (CSC) markers, increased sphere and clone formation as well as drug resistance in vitro . Mechanistically, our findings provide evidence that CD146 expression negatively correlates with canonical Wnt/β-catenin activity in CRC cell lines and primary CRC specimens. Knockdown of CD146 results in inhibition of NF-κB/p65-initiated GSK-3β expression, subsequently promoting nuclear translocation and activation of β-catenin, and as a consequence restoring stem cell phenotypes in differentiated CRC cells. Together, our data strongly suggest that CD146 functions as a suppressor of tumorigenesis and cancer stemness in CRC through inactivating the canonical Wnt/β-catenin cascade. Our findings provide important insights into stem cell plasticity and the multifunctional role of CD146 in CRC progression.

Development of enzyme mimics for the scavenging of excessive mitochondrial superoxide (O2 •- ) can serve as an effective strategy in the treatment of many diseases. Here, protein reconstruction technology and nanotechnology is taken advantage of to biomimetically create an artificial hybrid nanozyme. These nanozymes consist of ferritin-heavy-chain-based protein as the enzyme scaffold and a metal nanoparticle core as the enzyme active center. This artificial cascade nanozyme possesses superoxide dismutase- and catalase-like activities and also targets mitochondria by overcoming multiple biological barriers. Using cardiac ischemia-reperfusion animal models, the protective advantages of the hybrid nanozymes are demonstrated in vivo during mitochondrial oxidative injury and in the recovery of heart functionality following infarction via systemic delivery and localized release from adhesive hydrogels (i.e., cardiac patch), respectively. This study illustrates a de novo design strategy in the development of enzyme mimics and provides a promising therapeutic option for alleviating oxidative damage in regenerative medicine.

The vexing difficulty in distinguishing glioma from normal tissues is a major obstacle to prognosis. In an attempt to solve this problem, we used a joint strategy that combined targeted-cancer stem cells nanoparticles with precise photoacoustic and fluorescence navigation. We showed that traditional magnetic resonance imaging (MRI) did not represent the true morphology of tumors. Targeted nanoparticles specifically accumulated in the tumor area. Glioma was precisely revealed at the cellular level. Tumors could be non-invasively detected through the intact skull by fluorescence molecular imaging (FMI) and photoacoustic tomography (PAT). Moreover, PAT can be used to excise deep gliomas. Histological correlation confirmed that FMI imaging accurately delineated scattered tumor cells. The combination of optical PAT and FMI navigation fulfilled the promise of precise visual imaging in glioma detection and resection. This detection method was deeper and more intuitive than the current intraoperative pathology.

Photoacoustic imaging (PAI) is an attractive imaging modality, which is promising for clinical cancer diagnosis due to its advantages on deep tissue penetration and fine spatial resolution. However, few tumor catalytic/responsive PAI strategies are developed. Here, we design an exosome-like nanozyme vesicle for in vivo H

Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer deaths, primarily due to its high incidence of recurrence and metastasis. Considerable efforts have therefore been undertaken to develop effective therapies; however, effective anti-HCC therapies rely on identification of suitable biomarkers, few of which are currently available for drug targeting. Methods: GRP78 was identified as the membrane receptor of HCC-targeted peptide SP94 by immunoprecipitation and mass spectrum analysis. To develop an effective anti-HCC drug nanocarrier, we first displayed GRP78-targeted peptide SP94 onto the exterior surface of Pyrococcus furiosus ferritin Fn (HccFn) by genetic engineering approach, and then loaded doxorubicin (Dox) into the cavities of HccFn via urea-based disassembly/reassembly method, thereby constructing a drug nanocarrier called HccFn-Dox. Results: We demonstrated that HccFn nanocage encapsulated ultra-high dose of Dox (up to 400 molecules Dox/protein nanocage). In vivo animal experiments showed that Dox encapsulated in HccFn-Dox was selectively delivered into HCC tumor cells, and effectively killed subcutaneous and lung metastatic HCC tumors. In addition, HccFn-Dox significantly reduced drug exposure to healthy organs and improved the maximum tolerated dose by six-fold compared with free Dox. Conclusion: In conclusion, our findings clearly demonstrate that GRP78 is an effective biomarker for HCC therapy, and GRP78-targeted HccFn nanocage is effective in delivering anti-HCC drug without damage to healthy tissue.

The use of natural substance to ward off microbial infections has a long history. However, the large-scale production of natural extracts often reduces antibacterial potency, thus limiting practical applications. Here we present a strategy for converting natural organosulfur compounds into nano-iron sulfides that exhibit enhanced antibacterial activity. We show that compared to garlic-derived organosulfur compounds nano-iron sulfides exhibit an over 500-fold increase in antibacterial efficacy to kill several pathogenic and drug-resistant bacteria. Furthermore, our analysis reveals that hydrogen polysulfanes released from nano-iron sulfides possess potent bactericidal activity and the release of polysulfanes can be accelerated by the enzyme-like activity of nano-iron sulfides. Finally, we demonstrate that topical applications of nano-iron sulfides can effectively disrupt pathogenic biofilms on human teeth and accelerate infected-wound healing. Together, our approach to convert organosulfur compounds into inorganic polysulfides potentially provides an antibacterial alternative to combat bacterial infections.

Inflammation and calcification concomitantly drive atherosclerotic plaque progression and rupture and are the compelling targets for identifying plaque vulnerability. However, current imaging modalities for vulnerable atherosclerotic plaques are often limited by inadequate specificity and sensitivity. Here, we show that natural H-ferritin nanocages radiolabeled with technetium-99m (99mTc-HFn) can identify and accurately localize macrophage-rich, atherosclerotic plaques in living mice using combined SPECT and CT. Focal 99mTc-HFn uptake was observed in the atherosclerotic plaques with multiple high-risk features of macrophage infiltration, active calcification, positive remodeling, and necrosis on histology and in early active ongoing lesions with intense macrophage infiltration. The uptake of 99mTc-HFn in plaques enabled quantitative measuring of the dynamic changes of inflammation during plaque progression and anti-inflammation treatment. This strategy lays the foundation of using bioengineered endogenous human ferritin nanocages for the identification of vulnerable and early active plaques as well as potential assessment of anti-inflammation therapy.

Nanobodies consist of a single domain variable fragment of a camelid heavy-chain antibody. Nanobodies have potential applications in biomedical fields because of their simple production procedures and low cost. Occasionally, nanobody clones of interest exhibit low affinities for their target antigens, which, together with their short half-life limit bioanalytical or therapeutic applications. Here, we developed a novel platform we named fenobody, in which a nanobody developed against H5N1 virus is displayed on the surface of ferritin in the form of a 24mer. We constructed a fenobody by substituting the fifth helix of ferritin with the nanobody. TEM analysis showed that nanobodies were displayed on the surface of ferritin in the form of 6 × 4 bundles, and that these clustered nanobodies are flexible for antigen binding in spatial structure. Comparing fenobodies with conventional nanobodies currently used revealed that the antigen binding apparent affinity of anti-H5N1 fenobody was dramatically increased (∼360-fold). Crucially, their half-life extension in a murine model was 10-fold longer than anti-H5N1 nanobody. In addition, we found that our fenobodies are highly expressed in Escherichia coli, and are both soluble and thermo-stable nanocages that self-assemble as 24-polymers. In conclusion, our results demonstrate that fenobodies have unique advantages over currently available systems for apparent affinity enhancement and half-life extension of nanobodies. Our fenobody system presents a suitable platform for various large-scale biotechnological processes and should greatly facilitate the application of nanobody technology in these areas.

Over the last decades, considerable efforts have been put into developing active nanocarrier systems that cross the blood brain barrier (BBB) to treat brain-related diseases such as glioma tumors. However, to date none have been approved for clinical usage. Here, we show that a human H-ferritin (HFn) nanocarrier both successfully crosses the BBB and kills glioma tumor cells. Its principle point of entry is the HFn receptor (transferrin receptor 1), which is overexpressed in both BBB endothelial cells (ECs) and glioma cells. Importantly, we found that HFn enters and exits the BBB via the endosome compartment. In contrast, upon specifically targeting and entering glioma cells, nearly all of the HFn accumulated in the lysosomal compartment, resulting in the killing of glioma tumor cells, with no HFn accumulation in the surrounding healthy brain tissue. Thus, HFn is an ideal nanocarrier for glioma therapy and possesses the potential to serve as a therapeutic approach against a broad range of central nervous system diseases.

Cadherin switch is an initiating factor of epithelial-mesenchymal transition (EMT) and is intimately correlated with cancer metastatic potential; however, its underlying mechanisms remain unclear. Here, using a transforming growth factor-β (TGF-β)-induced EMT model, we provide explicit evidence that CD146, with elevated expression and activity in a variety of cancers, is a key factor involved in the cadherin switch. We show that CD146 can be induced by TGF-β signaling. Moreover, CD146 expression is positively correlated with the activation levels of STAT3/Twist and ERK pathways. Transcriptional response of the CD146/STAT3/Twist cascade inhibits E-cadherin expression, whereas the CD146/ERK cascade enhances N-cadherin expression. CD146 overexpression also significantly promotes EMT in both mouse embryonic fibroblasts (MEFs) and ovarian cancer cells. Clinically, ovarian cancer patients with detectable CD146 expression had a significantly lower survival rate than that of patients without CD146 expression. Furthermore, CD146-deficient MEFs exhibited decreased motility as a result of reversion in this cadherin switch, strongly suggesting that targeting CD146 is a potential strategy for cancer treatment. Therefore, CD146-mediated regulation of the E-cadherin-to-N-cadherin switch provides an insight into the general mechanisms of EMT as well as cancer metastasis.

The blood–brain barrier (BBB) establishes a protective interface between the central neuronal system and peripheral blood circulation and is crucial for homeostasis of the CNS. BBB formation starts when the endothelial cells (ECs) invade the CNS and pericytes are recruited to the nascent vessels during embryogenesis. Despite the essential function of pericyte–EC interaction during BBB development, the molecular mechanisms coordinating the pericyte–EC behavior and communication remain incompletely understood. Here, we report a single cell receptor, CD146, that presents dynamic expression patterns in the cerebrovasculature at the stages of BBB induction and maturation, coordinates the interplay of ECs and pericytes, and orchestrates BBB development spatiotemporally. In mouse brain, CD146 is first expressed in the cerebrovascular ECs of immature capillaries without pericyte coverage; with increased coverage of pericytes, CD146 could only be detected in pericytes, but not in cerebrovascular ECs. Specific deletion of Cd146 in mice ECs resulted in reduced brain endothelial claudin-5 expression and BBB breakdown. By analyzing mice with specific deletion of Cd146 in pericytes, which have defects in pericyte coverage and BBB integrity, we demonstrate that CD146 functions as a coreceptor of PDGF receptor-β to mediate pericyte recruitment to cerebrovascular ECs. Moreover, we found that the attached pericytes in turn down-regulate endothelial CD146 by secreting TGF-β1 to promote further BBB maturation. These results reveal that the dynamic expression of CD146 controls the behavior of ECs and pericytes, thereby coordinating the formation of a mature and stable BBB.

VEGF-C is essential for lymphangiogenesis during development and tumor progression. VEGFR-3 is the well-known cognate receptor of VEGF-C to regulate lymphatic migration and proliferation, but the receptor of VEGF-C in regulating lymphatic sprouting, the initiating step of lymphangiogenesis, still remains elusive. Here we use both in vitro and in vivo methods to demonstrate CD146 as a receptor of VEGF-C to regulate lymphangiogenesis, especially at the sprouting step. Mechanistically, CD146 selectively activates the downstream p38 kinase, upon VEGF-C stimulation, to regulate lymphatic sprouting. Moreover, CD146 can also activate ERK to mediate VEGF-C regulation of the subsequent proliferation and migration of lymphatic endothelial cells. In zebrafish embryos, knockdown or dysfunction of CD146 results in similar developmental defects in lymphatic sprouting, capillary network, parachordal lymphangioblast (PL), and thoracic duct (TD) similar to down-regulation of VEGF-C. Altogether, our data reveals a critical role of CD146 to mediate VEGF-C signaling pathway in lymphangiogenesis.

The function of blood-brain barrier is often disrupted during the progression of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the molecular mechanism of blood-brain barrier modulation during neuroinflammation remains unclear. Herein, we show that the expression of interferon-γ (IFNγ) receptor on endothelial cells (ECs) protected mice from the brain inflammation during EAE. IFNγ stabilized the integrity of the cerebral endothelium and prevented the infiltration of leukocytes into the brain. Further analysis revealed that IFNγ increased the expression of tight junction proteins zonula occludens protein 1 and occludin, as well as membranous distribution of claudin-5, in brain ECs. Silencing claudin-5 abolished the IFNγ-mediated improvement of EC integrity. Taken together, our results show that IFNγ, a pleiotropic proinflammatory cytokine, stabilizes blood-brain barrier integrity and, therefore, prevents brain inflammation during EAE.

CD146, an endothelial biomarker, has been shown to be aberrantly upregulated during pathological angiogenesis and functions as a coreceptor for vascular endothelial growth factor receptor 2 (VEGFR-2) to promote disease progression. However, the regulatory mechanisms of CD146 expression during angiogenesis remain unclear. Using a microRNA screening approach, we identified a novel negative regulator of angiogenesis, microRNA 329 (miR-329), that directly targeted CD146 and inhibited CD146-mediated angiogenesis in vitro and in vivo. Endogenous miR-329 expression was downregulated by VEGF and tumor necrosis factor alpha (TNF-α), resulting in the elevation of CD146 in endothelial cells. Upregulation of CD146 facilitated an endothelial response to VEGF-induced SRC kinase family (SKF)/p38 mitogen-activated protein kinase (MAPK)/NF-κB activation and consequently promoted endothelial cell migration and tube formation. Our animal experiments showed that treatment with miR-329 repressed excessive CD146 expression on blood vessels and significantly attenuated neovascularization in a mouse model of pathological angiogenesis. Our findings provide the first evidence that CD146 expression in angiogenesis is regulated by miR-329 and suggest that miR-329 could present a potential therapeutic tool for the treatment of angiogenic diseases.

A nanoparticle-based immunoassay was developed for the rapid and sensitive detection of avian influenza virus (AIV). In this method, AIV-specific pentabody (pVHH3B) was conjugated to magnetic nanoparticles (MNPs) and used to capture AIV. Gold nanoparticles (GNPs), labelled with the anti-AIV mouse monoclonal antibody 3C8, were used as a detector. In the presence of target samples, the pentabody pVHH3B enriched AIV on the MNPs. Thereafter, mAb 3C8-labelled GNPs (GNPs-mAb3C8) bound to MNPs via AIV and were separated using a magnetic field. GNPs in the complex catalyzed the oxidation of hydroquinone to quinone, and the OD value of quinone was measured. The developed assay displayed substantial signal change after incubation in an AIV sample in a concentration-dependent manner. The detection limit was 10 ng/ml, which is 10 times more sensitive than conventional double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). In conclusion, by combining MNPs and a novel pentabody pVHH3B, this study provided a sensitive influenza viral detection assay that has the potential to become a rapid, sensitive and inexpensive diagnostic tool for infectious diseases.

The concept that mesoporous silica nanoparticles (MSNs) are regarded as ideal novel drug delivery carriers in tumor therapy has been introduced extensively, but the effects of MSNs on tumor growth have received little attention. Here a model of nude mice xenografted with human malignant melanoma cells (A375) was used to investigate the effect of MSNs on tumor growth. Surprisingly, we found that MSNs have no toxicity to human malignant melanoma but increasing tumor growth in vivo. It was also confirmed that MSNs significantly promoted A375 cell proliferation and accelerated cell cycle progression in vitro. Cellular uptake mechanism showed that MSNs may affect molecular behavior of A375 cells when they entered into cytoplasm. Then, a detailed mechanism indicated that the promotion effect induced by MSNs was due to the decreasing of endogenous reactive oxygen species (ROS) in cells. Further results demonstrated that the upregulation of anti-apoptotic molecules Bcl-2 expression and the inhibition of NF-kappaB activation by MSNs may promote cell proliferation in a redox-sensitive signal pathway. These results show that tumor growth can be regulated by nanocarriers themselves in a ROS-dependent manner and imply that nanocarriers are not necessarily suitable for all kinds of tumor therapy in development drug delivery system.

Unexplained pregnancy loss and recurrent miscarriage seriously impair human fecundity. However, the underlying molecular mechanisms remain elusive. Recent studies suggest that the adhesion molecule CD146 may be involved in unexplained recurrent miscarriage. Here, we investigate the effect of CD146 on early pregnancy. Using in situ hybridization and immunohistochemistry, we found that CD146 was specifically expressed in the receptive maternal uteri and invasive embryonic trophoblasts during the early stages of pregnancy, but it was completely absent in the non-pregnant uterus. Our in vitro studies demonstrated that blocking CD146 with a function-perturbation antibody AA98 significantly inhibited the attachment of blastocysts onto the receptive uterine luminal epithelial monolayer, the trophoblastic outgrowth of blastocysts and ectoplacental cones, and the secretion of matrix metalloproteinases. Animal experiments showed that applying this antibody before embryo implantation caused pregnancy failure in mice. Our data present direct evidence for the role of CD146 in mediating embryonic attachment and trophoblastic invasion, and provide new insight into the molecular mechanism underlying unexplained pregnancy loss and recurrent miscarriage. J. Cell. Physiol. 215: 621‐626, 2008. 2008 Wiley-Liss, Inc. Recurrent miscarriage, defined as three or more consecutive, spontaneous pregnancy losses, affects approximately 1‐2% of

Avidin layer was bound on the substrate surface of Silicon wafer modified with aldehyde. The interaction between avidin and biotin was adopted for the immobilization of mouse monoclonal biotin-anti-M13 (antibody GP3)-labeled biotin. The surface was incubated in a solution containing phage M13KO7, which was trapped by the antibody GP3 with the interaction between phage M13KO7 and antibody GP3, resulting in a variation of layer thickness that was detected by imaging ellipsometry. The results showed a saturated layer of antibody GP3 with a thickness about 6.9 nm on the surface of the silicon wafer. The specific interaction between phage M13KO7 and antibody GP3 resulted in a variation of the layer thickness. The layer of phage M13KO7 bound with antibody GP3 was 17.5 nm in the concentration of 1.1 x 10(10) pfu/mL. Each variation of layer thickness corresponded to a concentration of phage M13KO7 in the range of 0.1 x 10(10) approximately 2.5 x 10(10) pfu/mL, with the sensitivity of 10(9) pfu/mL. Compared with other methods, the optical protein-chip requires only short measurement time, is label free, is a quantitative test, and can be visualized. This study could be significant on the investigation of interactions between the antibody and virus, and shows potential in the early diagnosis of virosis.

The monoclonal antibody T2-2 was originally raised against the colorectal carcinoma cell line LS174T and was found to bind to several other human carcinomas, including hepatoma and ovarian cancer. The goal of this study was to investigate the antitumor activity of mAb T2-2 in human tumor models and further characterize the antigen. mAb T2-2 inhibited the growth of human hepatocellular cell line SMMC 7721 in vivo and in vitro. Western blot analysis revealed that this mAb recognizes an unique Mr 45,000 band from tissue extracts of human hepatocellular carcinoma (HCC), which localizes to the cell periphery. In vitro cell assays indicate that T2-2 decreases cell adhesion to laminin, implying the functional role of T2-2 antigen in cell-matrix interaction and cell migration.

The ability to selectively block the entry of leukocytes into the central nervous system (CNS) without compromising the immune system is an attractive therapeutic approach for treating multiple sclerosis (MS). Using endothelial CD146-deficienct mice as a MS model, we found that endothelial CD146 plays an active role in the CNS-directed extravasation of encephalitogenic T cells, including CD146(+) TH1 and TH17 lymphocytes. Moreover, treating both active and passive MS models with the anti-CD146 antibody AA98 significantly decreased the infiltrated lymphocytes in the CNS and decreased neuroinflammation. Interestingly, the ability of AA98 to inhibit the migration of CD146(+) lymphocytes was dependent on targeting endothelial CD146, but not lymphocytic CD146. These results suggest a key molecular target located on the blood-brain barrier endothelium that mediates the extravasation of inflammatory cells into the CNS. In addition, our data suggest that the AA98 is a promising candidate for treating MS and other CNS autoimmune diseases.

Currently in clinic, people use hematoxylin and eosin stain (H&E stain) and immunohistochemistry methods to identify the generation and genre of cancers for human pathological samples. Since these methods are inaccurate and time consuming, developing a rapid and accurate method to detect cancer is urgently demanded. In our study, binding peptides for lung cancer cell line A549 were identified using bacteria surface display method. With those binding peptides for A549 cells on the surface, the fluorescent bacteria (Escherichia coli with stably expressed green fluorescent protein) were served as specific detecting reagents for the diagnosis of cancers. The binding activity of peptide-fluorescent bacteria complex was confirmed by detached cancer cells, attached cancer cells and mice tumor xenograft samples. A unique fixation method was developed for peptide-bacteria complex in order to make this complex more feasible for the clinic use. This peptide-fluorescent bacteria complex has great potential to become a new diagnostic tool for clinical application.

Cell adhesion molecules (CAMs) are crucial effectors for the development and maintenance of the nervous system. Mutations in human CAM genes are linked to brain disorders and psychological diseases, and CAM knockout mice always exhibit similar behavioral abnormalities. CD146 is a CAM of the immunoglobulin superfamily that interacts with Neurite Outgrowth Factor and involved in neurite extension in vitro. However, little is known about its in vivo function in the nervous system. In this study, we used a murine CD146 nervous system knockout (CD146(ns-ko)) model. We found that the brains of some CD146(ns-ko) mice were malformed with small olfactory bulbs. CD146(ns-ko) mice exhibited lower body weights and smaller food intake when compared with wild type littermates. Importantly, behavior tests revealed that CD146(ns-ko) mice exhibited significant decreased locomotor activity and impaired capacity for spatial learning and memory. Our results demonstrate that CD146 is important for mammalian nervous system development and proper behavior patterns.

Summary: The aim of this study is to raise tumor-suppressing monoclonal antibodies (mAbs) against colorectal carcinomas. Here, we generated a novel mAb 3P9, targeted a cancer-associated carbohydrate antigen sialyl-Tn (sTn), which showed significant inhibitory effect on proliferation and migration of sTn + cells and tumor growth by inducing apoptosis. We also demonstrated that mAb 3P9 showed higher sensitivity and specificity in immunohistochemistry assay on colonic adenocarcinoma than the broadly used commercial anti-sTn antibody B72.3. These results provide the first evidence that mAb 3P9 has potential applications, not only for diagnosis but also for antibody-based tumor therapy.

CD146 is a novel endothelial biomarker and plays an essential role in angiogenesis; however, its role in the molecular mechanism underlying angiogenesis remains poorly understood. In the present study, we show that CD146 interacts directly with VEGFR-2 on endothelial cells and at the molecular level and identify the structural basis of CD146 binding to VEGFR-2. In addition, we show that CD146 is required in VEGF-induced VEGFR-2 phosphorylation, AKT/p38 MAPKs/NF-κB activation, and thus promotion of endothelial cell migration and microvascular formation. Furthermore, we show that anti-CD146 AA98 or CD146 siRNA abrogates all VEGFR-2 activation induced by VEGF. An in vivo angiogenesis assay showed that VEGF-promoted microvascular formation was impaired in the endothelial conditional knockout of CD146 (CD146EC-KO). Our animal experiments demonstrated that anti-CD146 (AA98) and anti-VEGF (bevacizumab) have an additive inhibitory effect on xenografted human pancreatic and melanoma tumors. The results of the present study suggest that CD146 is a new coreceptor for VEGFR-2 and is therefore a promising target for blocking tumor-related angiogenesis.

Iron oxide magnetic nanoparticles (MNPs) are widely used as diagnostic and therapeutic agents for biomedical applications. Quantitatively analyzing biodistribution, pharmacokinetics and organ clearance of MNPs in mouse models is important for understanding their in vivo behavior. In this study, we developed a novel histochemical method for visualizing unlabeled MNPs in mouse tissues by employing their intrinsic peroxidase-mimicking activity, regarding which we reported previously that MNPs could catalyze the oxidation of peroxidase substrates to produce a color reaction at the site of MNPs (Gao et al. Nat. Nanotechnol.2007, 2, 577-583). Based on this MNPs-peroxidase approach, we determined the biodistribution and organ clearance of MNPs by visualizing and quantifying the localization of MNPs within the main organs. Compared to traditional Prussian blue assay, this novel MNPs-peroxidase approach has higher sensitivity. In conclusion, the developed MNPs-peroxidase approach based on intrinsic peroxidase activity of iron oxide nanoparticles was used effectively for quantitative detection of MNPs in mice by histochemical staining. Presumably, other nanoparticles having intrinsic peroxidase activity could also be considered.

The epithelial-mesenchymal transition (EMT) plays an important role in breast cancer metastasis, especially in the most aggressive and lethal subtype, “triple-negative breast cancer” (TNBC). Here, we report that CD146 is a unique activator of EMTs and significantly correlates with TNBC. In epithelial breast cancer cells, overexpression of CD146 down-regulated epithelial markers and up-regulated mesenchymal markers, significantly promoted cell migration and invasion, and induced cancer stem cell-like properties. We further found that RhoA pathways positively regulated CD146-induced EMTs via the key EMT transcriptional factor Slug. An orthotopic breast tumor model demonstrated that CD146-overexpressing breast tumors showed a poorly differentiated phenotype and displayed increased tumor invasion and metastasis. We confirmed these findings by conducting an immunohistochemical analysis of 505 human primary breast tumor tissues and found that CD146 expression was significantly associated with high tumor stage, poor prognosis, and TNBC. CD146 was expressed at abnormally high levels (68.9%), and was strongly associated with E-cadherin down-regulation in TNBC samples. Taken together, these findings provide unique evidence that CD146 promotes breast cancer progression by induction of EMTs via the activation of RhoA and up-regulation of Slug. Thus, CD146 could be a therapeutic target for breast cancer, especially for TNBC.

Purpose: Although histone deacetylase inhibitors (HDACi) are emerging as a new class of anticancer agents, one of the most significant concerns is that interactions with a wide array of substrates using these agents might initiate both therapeutic and undesired protective responses. Here, we sought to identify the potential protective reactions initiated by HDACi and determine whether targeting these reactions would enhance the antitumoral activity of HDACi. Experimental Design: Gene expression profiles were analyzed by cDNA microarray in Molt-4 cells before and after treatment of vorinostat. Induction of CD146 by vorinostat was examined in a wide range of tumors and nonmalignant cells. AA98, an anti-CD146 monoclonal antibody, was used to target CD146 function. Synergistic antitumoral and antiangiogenic effects between AA98 and vorinostat were examined both in vitro and in vivo. The potential effect of combined AA98 and vorinostat treatment on the AKT pathway was determined by Western blotting. Results: The induction of CD146 is a common phenomenon in vorinostat-treated cancer but not in nonmalignant cells. Targeting of CD146 with AA98 substantially enhanced vorinostat-induced killing via the suppression of activation of AKT pathways in cancer cells. Moreover, AA98 in combination with vorinostat significantly inhibited angiogenesis. In vivo, AA98 synergized with vorinostat to inhibit tumor growth and metastasis. Conclusion: The present study provided the first evidence that an undesired induction of CD146 could serve as a protective response to offset the antitumor efficacy of vorinostat. On the other hand, targeting CD146 in combination with vorinostat could be exploited as a novel strategy to more effectively kill cancer cells. Clin Cancer Res; 16(21); 5165–76. ©2010 AACR.

Surface functionalization and water solubility of magnetic nanoparticles are crucial for bioapplication. Here, we describe a synthetic approach for direct preparation of a wide range of functionalized and hydrophilic magnetic polymer particles (MPPs) that is both simple and general and involves using different polymers as the source of functional groups. This simple strategy of changing the polymer used in the reaction can give rise to a wide variety of hydrophilic MPPs with a high number of functional groups. For the purpose of bioapplication, we synthesized three types of MPPs with typical functional groups, such as hydroxyl groups (-OH), amino groups (-NH2), and carboxyl groups (-COOH), and further characterized these MPPs by transmission electronic microscopy (TEM), scanning electronic microscopy (SEM), thermogravimetric analysis (TGA), X-ray powder diffraction (XRD), Raman spectroscopy, and Fourier transform infrared (FTIR) spectroscopy. The magnetic saturation of the MPPs was also studied and was adequate for most bioapplications. MPPs were shown to have good biocompatibility using cell proliferation and apoptosis assays. Two types of MPPs with different functional groups were used successfully for intracellular imaging and antibody purification. Our results demonstrate that this simple and general synthesis strategy has potential for designing hydrophilic magnetic nanoparticles with multifunctionalities that cater for a range of bioapplications.

We developed a novel silica coating magnetic nanoparticle-based silver enhancement immunoassay (SEIA) for ricin toxin (RT) rapid electrical detection using interdigitated array microelectrodes (IDAMs) as electrodes. This novel system was developed by taking advantage of the separation and enrichment properties of magnetic nanoparticles (MNPs) and the catalytic properties of gold nanoparticles (GNPs). In this system, MNPs labeled with anti-ricin A chain antibody 6A6 were used to capture ricin and GNPs labeled with anti-ricin B chain antibody 7G7 were used as detectors. To enhance the electrical signal, the catalytic properties of GNPs were used to promote silver reduction. In the presence of ricin, a sandwich structure was formed which could be separated by a magnetic field. The sandwich complex was then transferred to IDAMs. The silver particles bridged the IDAM gaps and gave rise to an enhancing electrical signal that was detected by conductivity measurements. The results showed that the sensitivity of the SEIA for ricin electrical detection was five times greater than that of conventional colorimetric sandwich ELISA. Once the antibody used for detection was coated on the plates or MNPs, our system was three times more rapid than colorimetric sandwich ELISA. This rapid and sensitive detection system provides promising new potential for ricin detection.

CD146 (MUC18, Mel-CAM/MCAM) is a transmembrane protein, originally identified as a biomarker of melanoma, and plays an important role in cancer invasion and metastasis. Further studies revealed that CD146 as a novel endothelial marker was also involved in angiogenesis. Previous studies reported several anti-CD146 antibodies, such as MUC18, A32, S-endo1, and P1H12, showing different binding patterns to the endothelium of various types of blood vessels. To examine the possibility that antibodies targeting different epitopes on CD146 could have different behaviors, we generated a panel of anti-human CD146 monoclonal antibodies, named AA1-5 and AA7, by immunizing mice with human CD146 protein purified from HUVEC. Their specificity and binding affinity were intensively characterized using Western blotting, flow cytometry, and immunohistochemical assay. On the basis of epitope mapping, we divided the six monoclonal antibodies (MAb) into two groups, groups V1 and C2-2, corresponding to the different extracellular domains harboring these epitopes, the first IgV and the second IgC2 domains, respectively. Furthermore, owing to different epitopes, the two groups of antibodies behaved differentially in cellular and histological levels. Therefore, these anti-CD146 MAbs targeting different domains should be useful tools in studying the expression and function of human CD146.

We used comparative proteomic techniques to identify aging-related brain proteins in normal mice from neonate to old age. By 2-dimensional electrophoresis (2-DE), matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and peptide mass fingerprint (PMF) analysis, 39 proteins were identified, among which 6 stayed unchanged since 3 months, 6 increased and 27 decreased in various manners during aging. They are mainly involved in processes usually with destructive changes during aging, such as metabolism, transport, signaling, stress response and apoptosis. The 27 proteins' decrease may be responsible for brain aging. In particular, decrease of proteasome alpha subunits 3/6, ubiquitin carboxyl-terminal esterase L3, valosin-containing protein and calreticulin may be responsible for the declination of protein quality control; glutamate dehydrogenase 1, isocitrate dehydrogenase 1 and ubiquinol cytochrome c reductase core protein 2 for the shortage of energy and reducing agent; ubiquitin-conjugating enzyme E2N and heterogeneous nuclear ribonucleoprotein A2/B1 for the increase of DNA damage and transcription detuning; calbindin 1 and amphiphysin for the disturbance of synaptic transport and ion signals. The six proteins' increase may be involved in anti-aging processes. In particular, transketolase, mitochondrial creatine kinase 1 and ribosomal protein L37 may help to enhance energy metabolism; triosephosphate isomerase 1 may help to resist oxidative stress. Moreover, most of these proteins were found for the first time to be involved in the natural senescence of brain, which would provide new clues about the mechanism of brain aging.

The goal of our study was to raise monoclonal antibodies (mAbs) against endothelial cell-surface proteins specific for tumor vasculature. Here, we describe the generation and intensive characterization of mAb AA98, including its functional properties and its antigen identification. In our study, an enhanced mAb AA98 immunoreactivity was observed on stimulated human umbilical vein endothelial cells (HUVECs). In addition, mAb AA98 showed remarkably restricted immunoreactivity against intratumoral neovasculature compared with blood vessels of normal tissues. We identified the AA98 antigen as human CD146, an adhesion molecule belonging to the immunoglobulin superfamily. Data from in vitro experiments imply structural and signaling functions for endothelial CD146; however, the role of CD146 in vivo is largely unknown. Here, we show that mAb AA98 displays antiangiogenic properties in vitro and in vivo. Proliferation and migration of HUVECs were inhibited by mAb AA98 as was angiogenesis in chicken chorioallantoic membrane (CAM) assays and tumor growth in 3 xenografted human tumor models in mice. Our data provide new insights into the function of CD146 on endothelial cells, validate CD146 as a novel target for antiangiogenic agents, and demonstrate that mAb AA98 has potential as a diagnostic and therapeutic agent in vascular and cancer biology.

Carcinoembryonic antigen (CEA, CEACAM5, and CD66e) has been found to be associated with various types of cancers, particularly colorectal carcinoma, and developed to be a molecular target for cancer diagnosis and therapy. In present study, we generated a novel anti-CEACAM5 monoclonal antibody, namely mAb CC4, by immunizing mice with living colorectal cancer LS174T cells. Immunohistochemical studies found that mAb CC4 specifically and strongly binds to tumor tissues, especially colorectal adenocarcinoma. In xenografted mice, mAb CC4 is specifically accumulated in tumor site and remarkably represses colorectal tumor growth. In vitro functional analysis showed that mAb CC4 significantly suppresses cell proliferation, migration and aggregation of colorectal cancer cells and also raises strong ADCC reaction. More interestingly, mAb CC4 is able to enhance NK cytotoxicity against MHC-I-deficient colorectal cancer cells by blocking intercellular interaction between epithelial CEACAM5 and NK inhibitory receptor CEACAM1. These data suggest that mAb CC4 has the potential to be developed as a novel tumor-targeting carrier and cancer therapeutic.

CD146 is a newly identified endothelial biomarker that has been implicated in angiogenesis. Though in vitro angiogenic function of CD146 has been extensively reported, in vivo evidence is still lacking. To address this issue, we generated endothelial-specific CD146 knockout (CD146EC-KO) mice using the Tg(Tek-cre) system. Surprisingly, these mice did not exhibit any apparent morphological defects in the development of normal retinal vasculature. To evaluate the role of CD146 in pathological angiogenesis, a xenograft tumor model was used. We found that both tumor volume and vascular density were significantly lower in CD146EC-KO mice when compared to WT littermates. Additionally, the ability for sprouting, migration and tube formation in response to VEGF treatment was impaired in endothelial cells (ECs) of CD146EC-KO mice. Mechanistic studies further confirmed that VEGF-induced VEGFR-2 phosphorylation and AKT/p38 MAPKs/NF-κB activation were inhibited in these CD146-null ECs, which might present the underlying cause for the observed inhibition of tumor angiogenesis in CD146EC-KO mice. These results suggest that CD146 plays a redundant role in physiological angiogenic processes, but becomes essential during pathological angiogenesis as observed in tumorigenesis. Electronic supplementary material The online version of this article (doi:10.1007/s13238-014-0047-y) contains supplementary material, which is available to authorized users.