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Angiogenesis is one of crucial processes associated with tumor growth and development, and consequently a prime target for cancer therapy. Although tumor endothelial cells (TECs) play a key role in pathological angiogenesis, investigating phenotypical changes in neovessels when a gene expression in TEC is suppressed is a difficult task. Small interfering RNA (siRNA) represents a potential agent due to its ability to silence a gene of interest. We previously developed a system for in vivo siRNA delivery to cancer cells that involves a liposomal-delivery system, a MEND that contains a unique pH-sensitive cationic lipid, YSK05 (YSK-MEND). In the present study, we report on the development of a system that permits the delivery of siRNA to TECs by combining the YSK-MEND and a ligand that is specific to TECs. Cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD) is a well-known ligand to αVβ3 integrin, which is selectively expressed at high levels in TECs. We incorporated cRGD into the YSK-MEND (RGD-MEND) to achieve an efficient gene silencing in TECs. Quantitative RT-PCR and the 5' rapid amplification of cDNA ends PCR indicated that the intravenous injection of RGD-MEND at a dose of 4.0mg/kg induced a significant RNAi-mediated gene reduction in TEC but not in endothelial cells of other organs. Finally, we evaluated the therapeutic potency of the RGD-MEND encapsulating siRNA against vascular endothelial growth factor receptor 2. A substantial delay in tumor growth was observed after three sequential RGD-MEND injections on alternate days. In conclusion, the RGD-MEND represents a new approach for the characterization of TECs and for us in anti-angiogenic therapy., (© 2013.)

We describe herein the development of a high affinity and specific DNA aptamer as a new ligand for use in liposomal nanoparticles to target cultured mouse tumor endothelial cells (mTECs). Active targeted nanotechnology based drug delivery systems are currently of great interest, due to their potential for reducing side effects and facilitating the delivery of cytotoxic drugs or genes in a site specific manner. In this study, we report on a promising aptamer candidate AraHH036 that shows selective binding towards mTECs. The aptamer does not bind to normal cells, normal endothelial cells or tumor cells. Therefore, we synthesized an aptamer-polyethylene glycol (PEG) lipid conjugate and prepared aptamer based liposomes (ALPs) by the standard lipid hydration method. First, we quantified the higher capacity of ALPs to internalize into mTECs by incubating ALPs containing 1 mol%, 5 mol% and 10 mol% aptamer of total lipids and compared the results to those for unmodified PEGylated liposomes (PLPs). A confocal laser scanning microscope (CLSM) uptake study indicated that the ALPs were taken up more efficiently than PLPs. The measured Kd value of the ALPs was 142 nM. An intracellular trafficking study confirmed that most of the rhodamine labeled ALPs were taken up and co-localized with the green lysotracker, thus confirming that they were located in lysosomes. Finally, using an aptamer based proteomics approach, the molecular target protein of the aptamer was identified as heat shock protein 70 (HSP70). The results suggest that these ALPs offer promise as a new carrier molecule for delivering anti-angiogenesis drugs to tumor vasculature.

There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment.

Liver dysfunction is associated with a variety of liver diseases, including viral or alcoholic hepatitis, fibrosis, cirrhosis, and portal hypertension. A targeted drug delivery system would be very useful in the treatment of these diseases. We herein describe the development of a system comprised of a new peptide-lipid conjugate for the efficient delivery of molecules to LEC. The RLTRKRGLK sequence (3359-3367), which mediates the association of LDL with arterial CSPG and an LDL receptor, was utilized as a ligand for achieving this goal. The peptide modified PEG-LPs (RLTR-PEG-LPs) were efficiently taken up by primary liver endothelial cells (liver ECs) and other types of cells. In vivo biodistribution and confocal microscopy analysis showed that RLTR-PEG-LPs became widely accumulated in LECs within a short time. Distribution of RLTR-PEG-LPs was greatly reduced with a pretreatment of unlabeled RLTR-PEG-LPs, not cationic LPs, indicating that the sequence is important for LECs. The findings indicate that a reverse sequence of RLTR (KLGR) modified PEG-LPs (KLGR-PEG-LP) did the same pattern compared with RLTR-PEG-LPs, suggesting that the RKR or RXXR sequence might be essential for LECs targeting. Collectively RLTR-PEG-LPs and KLGR-PEG-LPs have the potential for delivering drugs to LECs., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Tumor blood vessels play important roles in tumor progression and metastasis. Thus, targeting tumor blood vessels is an important strategy for cancer therapy. Tumor endothelial cells (TECs) are the main targets of anti-angiogenic therapy. Although tumor blood vessels generally sprout from pre-existing vessels and have been thought to be genetically normal, they display a markedly abnormal phenotype, including morphological changes. The degree of angiogenesis is determined by the balance between the positive and negative regulating molecules that are released by tumor and host cells in the microenvironment. Reportedly, tumor blood vessels are heterogeneous with TECs differing from normal endothelial cells (in contrast to the conventional view). We recently compared characteristics of different TECs isolated from highly and low metastatic tumors. We found TECs from highly metastatic tumors had more proangiogenic phenotypes than those from low metastatic tumors. Elucidating the variety of TEC phenotypes and identifying TEC molecular signatures should lead to more complete understanding of the mechanisms of tumor progression, discovery of new therapeutic targets, and development of biomarkers. This review considers current studies on TEC heterogeneity and discusses the therapeutic implications of these findings., (© 2013 Japanese Cancer Association.)

Fluorescent and luminescent tools are commonly used to study the dynamics of cancer progression and metastases in real-time. Fluorophores have become essential tools to study biological events. However, few can sustain fluorescence long enough during long-term studies. In the present study, we focused on a series of new amphiphilic fluorophores known as POLARIC™, which emit strong fluorescence in lipid bilayers and can be readily modified using the Suzuki-Miyaura cross-coupling reaction. Appropriate chemical modifications of substituent groups can improve target-site specificity, reduce cytotoxicity and prolong emission. Therefore, in contrast to conventional fluorescent probes, these fluorophores show promise for long-term monitoring of biological processes. In the present study, we conducted long-term observations of tumor growth and metastasis using a POLARIC derivative as a novel fluorescent probe. For this purpose, we studied the metastatic melanoma cell line A375-SM, which proliferates at a high rate. We compared the characteristics of the POLARIC probe with the commercially available fluorescent dye PKH26 and fluorescent protein mRFP1. A375-SM cells were labeled with these fluorescent probes and orthotopically implanted into nude mice. The fluorescence emitted by POLARIC was detected more than five weeks after implantation without causing detectable harmful effects on tumor growth. By contrast, fluorescence of cells labeled with PKH26 could not be detected at this same time. Furthermore, POLARIC-, but not PKH26-labeled cells, were also detected in lung metastases. These results indicate that labeling cells with POLARIC fluorophores can significantly extend the time course of in vivo studies on tumor cell growth.

Dysfunctional sinusoidal liver endothelial cells (LECs) are associated with liver diseases, such as liver fibrosis, cirrhosis, and portal hypertension. Because of this, gene therapy targeted to LECs would be a useful and productive strategy for directly treating these diseases at the level of genes. Here, we report on the development of a transgene vector that specifically targets LECs. The vector is a liposome-based gene vector coated with hyaluronic acid (HA). HA is a natural ligand for LECs and confers desirable properties on particles, rendering them biodegradable, biocompatible, and nonimmunogenic. In this study, we constructed HA-modified carriers, and evaluated cellular uptake and transfection activity using cultured LECs from KSN nude mice (KSN-LECs). Cellular uptake analyses showed that KSN-LECs recognized the HA-modified carriers more effectively than skin endothelial cells. The transfection assay indicated that the efficient gene expression in KSN-LECs, using the HA-modified carriers, required an adequate lipid composition and a functional device to control intracellular trafficking. This finding contributes to our overall knowledge of transgene expression targeted to LECs., (Copyright © 2013 Wiley Periodicals, Inc.)

Size of the liposomes (LPs) specially governs its biodistribution. In this study, LPs were developed with controlled sizes, where variation in LP size dictates the ligand-receptor interaction, cellular internalization and its distribution within the tumor microenvironment. The therapeutic efficacies of doxorubicin (DOX)-loaded RGD modified small size (~100 nm in diameter, dnm) and large size (~300 dnm) PEGylated LPs (RGD-PEG-LPs) were compared to that of Doxil (a clinically used DOX-loaded PEG-LP, ~100 dnm) in DOX resistant OSRC-2 (Renal cell carcinoma, RCC) tumor xenografts. Doxil, which accumulated in tumor tissue via the enhanced permeability and retention (EPR) effect, failed to suppress tumor growth. Small size RGD-PEG-LP, that targets the tumor endothelial cells (TECs) and extravasates to tumor cells, failed to provide anti-tumor effect. Large size RGD-PEG-LP preferentially targets the TECs via minimization of the EPR effect, and significantly reduced the tumor growth, which was exerted through its strong anti-angiogenic activity on the tumor vasculature rather than having a direct effect on DOX resistant RCC. The prepared large size RGD-PEG-LP that targets the TECs via interacting with Integrin αvβ3, is a potentially effective and alternate therapeutic strategy for the treatment of DOX resistant tumor cells by utilizing DOX, in cases where Doxil is ineffective., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Tumor angiogenesis is necessary for tumor progression and metastasis; therefore, tumor blood vessels are potential therapeutic targets in anticancer therapy. We previously reported that tumor endothelial cells (TECs) exhibit different phenotypes compared with normal endothelial cells (NECs), and microarray analyses of mouse TECs and NECs have shown that several genes are upregulated in TECs compared with NECs. Among these genes, the expression levels of prostaglandin F receptor (PTGFR) mRNA, which encodes the prostaglandin F receptor (FP), were higher in TECs than in NECs. It has been reported that FP and its ligand, prostaglandin F(2α) , are involved in tumor angiogenesis. However, there have been no reports of the expression of PTGFR in the tumor vessels of renal cell carcinoma (RCC). Thus, we isolated human TECs (hTECs) from RCCs. The expression levels of PTGFR mRNA were also upregulated in hTECs. In addition, immunostaining showed that the PTGFR was expressed in human tumor blood vessels in vivo. These findings suggested that PTGFR is a novel TEC marker and that it may be a novel target for antiangiogenic therapy for RCC., (© 2013 The Authors. Pathology International © 2013 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.)

Anti-angiogenic therapy is a potential chemotherapeutic strategy for the treatment of drug resistant cancers. However, a method for delivering such drugs to tumor endothelial cells remains to be a major impediment to the success of anti-angiogenesis therapy. We designed liposomes (LPs) with controlled diameter of around 300 nm, and modified them with a specific ligand and a cell penetrating peptide (CPP) (a dual-ligand LP) for targeting CD13-expressing neovasculature in a renal cell carcinoma (RCC). We modified the LPs with an NGR motif peptide on the top of poly(ethylene glycol) and tetra-arginine (R4) on the surface of the liposome membrane as a specific and CPP ligand, respectively. The large size prevented extravasation of the dual-ligand LP, which allowed it to associate with target vasculature. While a single modification with either the specific or CPP ligand showed no increase in targetability, the dual-ligand enhanced the amount of delivered liposomes after systemic administration to OS-RC-2 xenograft mice. The anti-tumor activity of a dual-ligand LP encapsulating doxorubicin was evaluated and the results were compared with Doxil, which is clinically used to target tumor cells. Even though Doxil showed no anti-tumor activity, the dual-ligand LP suppressed tumor growth because the disruption of tumor vessels was efficiently induced. The comparison showed that tumor endothelial cells (TECs) were more sensitive to doxorubicin by 2 orders than RCC tumor cells, and the disruption of tumor vessels was efficiently induced. Collectively, the dual-ligand LP is promising carrier for the treatment of drug resistant RCC via the disruption of TECs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Molecules highly expressed in tumor endothelial cells (TEC) are important for specific targeting of these cells. Previously, using DNA microarray analysis, we found that the prostacyclin receptor (IP receptor) gene was upregulated in TEC compared with normal endothelial cells (NEC). Although prostacyclin is implicated in re-endothelialization and angiogenesis, its role remains largely unknown in TEC. Moreover, the effect of the IP receptor on TEC has not been reported. In the present study we investigated the function of the IP receptor in TEC. The TEC were isolated from two types of human tumor xenografts in nude mice, while NEC were isolated from normal counterparts. Prostacyclin secretion levels in TEC were significantly higher than those in NEC, as shown using ELISA. Real-time RT-PCR showed that the IP receptor was upregulated in TEC compared with NEC. Furthermore, migration and tube formation of TEC were suppressed by the IP receptor antagonist RO1138452. Immunohistostaining showed that the IP receptor was specifically expressed in blood vessels of renal cell carcinoma specimens, but not in glomerular vessels of normal renal tissue. These findings suggest that the IP receptor is a TEC-specific marker and might be a useful therapeutic target., (© 2012 Japanese Cancer Association.)

Tumor angiogenesis is necessary for progression and metastasis of solid tumor. Tumor blood vessels are morphologically different from their normal counterparts. In this study, we isolated tumor endothelial cells (TECs) and revealed their abnormalities. We have compared the gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and found that several genes were upregulated in TECs. Expression of the chemokine receptor CXCR7 mRNA was higher in TECs than in NECs. However, information regarding the expression of CXCR7 in the tumor vessels of renal cell carcinoma is limited. CXCR7 and its ligand CXCL12 have been implicated in tumor cell survival. In this study, the expression of CXCR7 in the tumor vessels of renal cell carcinoma (RCC) was investigated. Real-time PCR revealed higher expression level of CXCR7 in cultured TECs than in cultured NECs. Furthermore, similar to mouse TECs, immunostaining revealed strong expression of CXCR7 in vivo in human tumor vessels. These findings suggest that CXCR7 is a novel TEC marker and a target for antiangiogenic therapy for RCC., (© 2012 The Authors. Pathology International © 2012 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.)

An important concept in tumor angiogenesis is that tumor endothelial cells (TECs) are genetically normal and homogeneous. However, we previously reported that TECs differ from normal ECs. Whether the characteristics of TECs derived from different tumors differ remains unknown. To elucidate this, in this study, we isolated two types of TECs from high-metastatic (HM) and low-metastatic (LM) tumors and compared their characteristics. HM tumor-derived TECs (HM-TECs) showed higher proliferative activity and invasive activity than LM tumor-derived TECs (LM-TECs). Moreover, the mRNA expression levels of pro-angiogenic genes, such as vascular endothelial growth factor (VEGF) receptors 1 and 2, VEGF, and hypoxia-inducible factor-1α, were higher in HM-TECs than in LM-TECs. The tumor blood vessels themselves and the surrounding area in HM tumors were exposed to hypoxia. Furthermore, HM-TECs showed higher mRNA expression levels of the stemness-related gene stem cell antigen and the mesenchymal marker CD90 compared with LM-TECs. HM-TECs were spheroid, with a smoother surface and higher circularity in the stem cell spheroid assay. HM-TECs differentiated into osteogenic cells, expressing activated alkaline phosphatase in an osteogenic medium at a higher rate than either LM-TECs or normal ECs. Furthermore, HM-TECs contained more aneuploid cells than LM-TECs. These results indicate that TECs from HM tumors have a more pro-angiogenic phenotype than those from LM tumors., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Tumor endothelial cells (TECs) are therapeutic targets in anti-angiogenic therapy. Contrary to the traditional assumption, TECs can be genetically abnormal and might also acquire drug resistance. In this study, mouse TECs and normal ECs were isolated to investigate the drug resistance of TECs and the mechanism by which it is acquired. TECs were more resistant to paclitaxel with the up-regulation of multidrug resistance (MDR) 1 mRNA, which encodes the P-glycoprotein, compared with normal ECs. Normal human microvascular ECs were cultured in tumor-conditioned medium (CM) and became more resistant to paclitaxel through MDR1 mRNA up-regulation and nuclear translocation of Y-box-binding protein 1, which is an MDR1 transcription factor. Vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and Akt were activated in human microvascular ECs by tumor CM. We observed that tumor CM contained a significantly high level of VEGF. A VEGFR kinase inhibitor, Ki8751, and a phosphatidylinositol 3-kinase-Akt inhibitor, LY294002, blocked tumor CM-induced MDR1 up-regulation. MDR1 up-regulation, via the VEGF-VEGFR pathway in the tumor microenvironment, is one of the mechanisms of drug resistance acquired by TECs. We observed that VEGF secreted from tumors up-regulated MDR1 through the activation of VEGFR2 and Akt. This process is a novel mechanism of the acquisition of drug resistance by TECs in the tumor microenvironment., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The present study used a spontaneous cell-based SELEX method (Systemic Evolution of Ligands by EXponential Enrichment) to produce DNA aptamers that specifically bind to cell surface proteins or biomarkers produced by primary cultured mouse tumor endothelial cells (mTECs). In solid tumors, new blood vessels are formed through an angiogenesis process, and this plays a critical role in cancer development as well as metastasis. To combat angiogenesis, an appropriate diagnosis and a molecular-level understanding of the different cancer types are now a high priority. The novel DNA aptamer AraHH001, developed in this study, binds specifically to mTECs with high affinity in the nano-molar range, but does not bind to normal skin endothelial cells (skin-ECs). The selected DNA aptamer was also found to bind to cultured human tumor endothelial cells (hTECs), isolated from a clinical patient with a renal carcinoma. The aptamer AraHH001 showed significant anti-angiogenesis activity by inhibiting tube formation by mTECs on matrigel. Interestingly, a confocal laser scanning microscopy examination of in vitro cellular uptake revealed that AraHH001 was assimilated by mTECs, and became co-localized in acidic compartments, as detected by labeling with Lysotracker Red. Therefore, the development of a specific DNA aptamer that binds to mTECs, as reported here for the first time, holds great promise not only as a therapeutic aptamer but also as a targeted molecular probe that appears to play a major role in angiogenesis, and for the development of a targeted new drug delivery system.

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Cyclooxygenase (COX)-2 is known to play an important role in cancer growth and invasion, and it activates the signaling pathways controlling cell proliferation, migration, apoptosis, and angiogenesis. COX-2 is reported to be expressed in many cancer cells. Several studies have reported successful treatment of cancer cells with COX-2 inhibitors (COX-2is). However, the effect of COX-2 inhibition on the tumor endothelium remains to be elucidated. Our study shows that COX-2 is expressed in the vasculature of surgically resected human tumors. To investigate the effects of COX-2 inhibition on the tumor endothelium in vitro, we isolated tumor endothelial cells (TECs) from human melanoma and oral carcinoma xenografts in mice, in which we confirmed that tumor growth was suppressed by inhibiting angiogenesis with the COX-2is NS398. COX-2 mRNA was upregulated in TECs compared to normal endothelial cells (NECs). Cell migration and proliferation were suppressed by NS398 in TECs but not in NECs. The effects of NS398 in vivo were consistent with the in vitro results. The number of CD133+ /vascular endothelial growth factor receptor-2+ cells in circulation was significantly suppressed by COX-2 inhibition. In addition, the number of progenitor marker-positive cells decreased in the tumor blood vessels after COX-2i treatment, which suggests that the homing of progenitor cells into the tumor was also blocked. We conclude that NS398 specifically targets both TECs and vascular progenitor cells without affecting NECs., (Copyright © 2011 UICC.)

Background: Increasing evidence indicates that tumor endothelial cells (TEC) differ from normal endothelial cells (NEC). Our previous reports also showed that TEC were different from NEC. For example, TEC have chromosomal abnormality and proangiogenic properties such as high motility and proliferative activity. However, the mechanism by which TEC acquire a specific character remains unclear. To investigate this mechanism, we focused on tumor-derived microvesicles (TMV). Recent studies have shown that TMV contain numerous types of bioactive molecules and affect normal stromal cells in the tumor microenvironment. However, most of the functional mechanisms of TMV remain unclear., Methodology/principal Findings: Here we showed that TMV isolated from tumor cells were taken up by NEC through endocytosis. In addition, we found that TMV promoted random motility and tube formation through the activation of the phosphoinositide 3-kinase/Akt pathway in NEC. Moreover, the effects induced by TMV were inhibited by the endocytosis inhibitor dynasore. Our results indicate that TMV could confer proangiogenic properties to NEC partly via endocytosis., Conclusion: We for the first time showed that endocytosis of TMV contributes to tumor angiogenesis. These findings offer new insights into cancer therapies and the crosstalk between tumor and endothelial cells mediated by TMV in the tumor microenvironment.

Tumor blood vessels play an important role in tumor progression and metastasis. Thus, targeting the tumor blood vessels is an important strategy in cancer therapy. Tumor blood vessels generally arise from pre-existing vessels and have been thought to be genetically normal. However, they have been shown to differ from their normal counterparts, e.g. with regard to the morphological changes. We isolated tumor endothelial cells (TEC) from mouse tumor xenografts and showed that they were abnormal. TEC up-regulate many genes, proliferate more rapidly and migrate more than normal endothelial cells (NEC). Furthermore, the TEC in our study were cytogenetically abnormal. We concluded that TEC can acquire cytogenetic abnormalities while in the tumor microenvironment. In order to develop ideal antiangiogenic therapies, understanding the crosstalk between blood vessels and the tumor microenvironment is important. This review considers the current studies on TEC abnormalities and discusses the possible mechanism by which the tumor microenvironment produces abnormal TEC., (© 2011 The Authors. Pathology International © 2011 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.)

The objective of this study was to develop an efficient dual-ligand based PEGylated liposomal delivery system that had target specificity as well as properties that would enhance cellular uptake. PEGylated liposomes (PEG-LP) were prepared by the lipid film hydration method by adding distearoyl phosphoethanolamine-polyethylene-glycol-2000 conjugate (DSPE-PEG2000) to a lipid mixture. The cyclic RGD (Arg-Gly-Asp) peptide, a specific ligand with affinity for Integrin α(v)β(3) was coupled to the distal end of the PEG on the PEG-LP (RGD-PEG-LP). Stearylated octaarginine (STR-R8) was incorporated on the surface of the RGD-PEG-LP as dual-ligand (R8/RGD-PEG-LP) that functions as a cell penetrating peptide (CPP). RGD-PEG-LP and R8/RGD-PEG-LP were preferentially taken up by caveolae-mediated and clathrin-mediated endocytosis pathways, respectively. Compared to PEG-LP, R8/RGD-PEG-LP showed an enhanced cellular uptake as well as a higher transfection efficiency in Integrin α(v)β(3) expressing cells. However, the amount of cellular uptake or gene expression by the single ligand versions was negligible, even in Integrin α(v)β(3) expressing cells. No remarkable difference in cellular uptake or gene expression was observed for cells in which the expression of targeted receptors was absent. It can be concluded that dual-ligand modified PEG-LP possesses a strong capability for the efficient internalization of PEG-LP and consequently would be an effective tool for the targeted delivery of macromolecules or chemotherapeutics through accelerated cellular uptake., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Tumor metastasis to lymph nodes occurs through the lymphatic vessels located in the tumor circumference. However, few studies have focused on the phenotypes of lymphatic vessels around these tumors. We investigated the characteristics of the lymph vessels of tongue squamous cell carcinoma (SCC) and compared them to clinicopathological characteristics. A total of 43 patients diagnosed as having tongue SCC consulted Hokkaido University Hospital were examined. The lymphatic vessels were identified by antibody D2-40 and the number and diameter of tumor lymphatic vessels were measured. The proliferative activity of lymphatic endothelial cells was also examined by immunostaining using antibody MIB-1. We then measured the DNA density of lymphatic endothelial cells in normal and tumor tissues. The number of tumor lymphatic vessels significantly increased in highly metastatic cases of tongue SCC, particularly in cases with a large number of micro lymphatic vessels. A significant correlation was found between the metastatic and proliferative activity of tumor lymphatic endothelial cells. Moreover, the DNA density of tumor lymphatic endothelial cells increased compared to normal tissues. These results suggest that the phenotypes of tumor lymphatic endothelial cells are an indicator of lymph node metastasis of tongue SCC.

In this study, a dual-ligand liposomal system comprised of a specific ligand and a cell penetrating peptide (CPP) is described to enhance selectivity and cellular uptake. Dual-ligand PEGylated liposomes were prepared by modifying the end of the PEG with an NGR motif peptide, followed by a surface coating of the liposomes with stearylated oligoarginine (STR-RX). The NGR motif recognizes CD13, a marker protein located on tumor endothelial cells. A suitable number of RX units was determined to be R4, since it can be masked by the PEG aqueous layer. Although no enhanced cellular uptake was observed when a single modification of PEGylated liposomes with either NGR- or STR-R4 was used, the dual-modification with NGR and STR-R4 stimulated uptake of PEGylated liposomes by CD13 positive cells, and this uptake was superior to that obtained by PEG-unmodified liposomes modified with STR-R4. The dual-ligand system shows a synergistic effect on cellular uptake. Collectively, the dual-ligand system promises to be useful in the development efficient and specific drug delivery systems., (Copyright 2010 Elsevier B.V. All rights reserved.)

Adrenomedullin (AM) is a multifunctional 52-amino acid peptide. AM has several effects and acts as a growth factor in several types of cancer cells. Our previous study revealed that an AM antagonist (AMA) suppressed the growth of pancreatic tumors in mice, although its mechanism was not elucidated. In this study, we constructed an AMA expression vector and used it to treat renal cell carcinoma (RCC) in mice. This AMA expression vector significantly reduced tumor growth in mice. In addition, microvessel density was decreased in AMA-treated tumors. To analyze the effect of AMA on tumor angiogenesis in this model, tumor endothelial cells (TECs) were isolated from RCC xenografts. TEC proliferation was stimulated by AM and it was inhibited by AMA significantly. AM induced migration of TECs and it was also blocked by AMA. However, normal ECs (NECs) were not affected by either AM or AMA. These results demonstrate that AMA has inhibitory effects on TECs specifically, not on NEC, thereby inhibiting tumor angiogenesis. Furthermore, we showed that vascular endothelial growth factor-induced mobilization of endothelial progenitor cell (EPC) into circulation was inhibited by AMA. These results suggest that AMA can be considered a good anti-angiogenic reagent that selectively targets TECs and EPC in renal cancer.

Adipose tissue has long been considered to be a simple tissue that contains adipocytes. Because of this, the isolation and characterization of microvascular endothelical cells, which are also present in adipose tissue, have been neglected, even though they are components of a capillary network that surrounds each individual adipocyte. Here we report on a protocol for producing highly purified murine microvascular endothelial cells (MECs) from diverse sites of murine adipose tissues including inguinal and epididymal adipose tissues. The method is based on a combination of negative and positive immunomagnetic selection. The protocol involves the preparation of a single cell suspension (digestion, filtration and density gradient centrifugation), immunomagnetic enrichment of the CD45(-) cell population and the purification of the MECs by a combination of common specific markers CD31, CD102 and isolectin B4. The isolated MECs can be successively cultured for 10 to 12 passages without any detectable changes in morphology and phenotype. Therefore, the method described herein represents a protocol for the isolation and long-term maintenance of highly pure mouse MECs in high yields from adipose tissues.

Members of the bone morphogenetic protein (BMP) family have been implicated in the development and maintenance of vascular systems. Whereas members of the BMP-2/4 and osteogenic protein-1 groups signal via activin receptor-like kinase (ALK)-2, ALK-3 and ALK-6, BMP-9 and BMP-10 have been reported to bind to ALK-1 in endothelial cells. However, the roles of BMP-9-ALK-1 signaling in the regulation of endothelial cells have not yet been fully elucidated. Here, using various systems, we examined the effects of BMP-9 on the proliferation of endothelial cells. Vascular-tube formation from ex vivo allantoic explants of mouse embryos was promoted by BMP-9. BMP-9, as well as BMP-4 and BMP-6, also induced the proliferation of in-vitro-cultured mouse embryonic-stem-cell-derived endothelial cells (MESECs) by inducing the expression of vascular endothelial growth factor receptor 2 and Tie2, a receptor for angiopoietin-1. A decrease in ALK-1 expression or expression of constitutively active ALK-1 in MESECs abrogated and mimicked the effects of BMP-9 on the proliferation of MESECs, respectively, suggesting that BMP-9 promotes the proliferation of these cells via ALK-1. Furthermore, in vivo angiogenesis was promoted by BMP-9 in a Matrigel plug assay and in a BxPC3 xenograft model of human pancreatic cancer. Consistent with these in vivo findings, BMP-9 enhanced the proliferation of in-vitro-cultured normal endothelial cells from dermal tissues of adult mice and of tumor-associated endothelial cells isolated from tumor xenografts in host mice. These findings suggest that BMP-9 signaling activates the endothelium tested in the present study via ALK-1.

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells. In this study, we have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research., (2010 Elsevier Inc. All rights reserved.)

Tumor blood vessels are thought to contain genetically normal and stable endothelial cells (ECs), unlike tumor cells, which typically display genetic instability. Yet, chromosomal aberration in human tumor-associated ECs (hTECs) in carcinoma has not yet been investigated. Here we isolated TECs from 20 human renal cell carcinomas and analyzed their cytogenetic abnormalities. The degree of aneuploidy was analyzed by fluorescence in situ hybridization using chromosome 7 and chromosome 8 DNA probes in isolated hTECs. In human renal cell carcinomas, 22-58% (median, 33%) of uncultured hTECs were aneuploid, whereas normal ECs were diploid. The mechanisms governing TEC aneuploidy were then studied using mouse TECs (mTECs) isolated from xenografts of human epithelial tumors. To investigate the contribution of progenitor cells to aneuploidy in mTECs, CD133(+) and CD133(-) mTECs were compared for aneuploidy. CD133(+) mTECs showed aneuploidy more frequently than CD133(-) mTECs. This is the first report showing cytogenetic abnormality of hTECs in carcinoma, contrary to traditional belief. Cytogenetic alterations in tumor vessels of carcinoma therefore can occur and may play a significant role in modifying tumor- stromal interactions.

The polyphenol epigallocatechin-3 gallate (EGCG) in green tea suppresses tumor growth by direct action on tumor cells and by inhibition of angiogenesis, but it is not known whether it specifically inhibits tumor angiogenesis. We examined the anti-angiogenic effect of EGCG on tumor-associated endothelial cells (TEC), endothelial progenitor cells (EPC), and normal endothelial cells (NEC). EGCG suppressed the migration of TEC and EPC but not NEC. EGCG also inhibited the phosphorylation of Akt in TEC but not in NEC. Furthermore, vascular endothelial growth factor-induced mobilization of EPC into circulation was inhibited by EGCG. MMP-9 in the bone marrow plasma plays key roles in EPC mobilization into circulation. We observed that expression of MMP-9 mRNA was downregulated by EGCG in mouse bone marrow stromal cells. In an in vivo model, EGCG suppressed growth of melanoma and reduced microvessel density. Our study showed that EGCG has selective anti-angiogenic effects on TEC and EPC. It is suggested that EGCG could be a promising angiogenesis inhibitor for cancer therapy.

A case of unilateral coronoid hyperplasia successfully treated by coronoidotomy with prolonged postoperative physiotherapy and reveal the postoperative radiographic changes between the sectioned part of the coronoid process and the mandibular ascending ramus is described. The patient was a 28-year-old man whose maximum mouth opening was 30 mm. A coronoidotomy of the left coronoid process was performed. Nine days after surgery, the patient started physiotherapy with a HU-OS(r) appliance. After coronoidotomy and physiotherapy, the maximum mouth opening had increased to 43 mm. Radiographic follow-up showed that the coronoid process apparently united with the mandibular ascending ramus, with moderate dislocation and inclination posteriorly. In the case presented, an intraoral coronoidotomy with postoperative physiotherapy for treatment of coronoid process hyperplasia allowed satisfactory and stable results in the correction of coronoid-malar interference.

We describe here a procedure of endoscopic removal of a gutta-percha point under the maxillary sinus mucosa by using an ultrathin arthroscope. In this study, a 1.2-mm-diameter ultrathin arthroscope was introduced for observation into the maxillary sinus. The surgical technique is suggested to be a reliable and minimally invasive procedure that provides limited incision and bone removal and respects the integrity of the sinus.

A 54-year-old man presented with an 8-year history of a hard asymptomatic mass of the left submandibular area. Total excision of the left submandibular gland with radical neck dissection was performed under a diagnosis of a submandibular tumor, probably a malignant mixed tumor. The pathologic diagnosis was carcinosarcoma consisting of carcinomatous and sarcomatous elements. The epithelial component was composed of squamous cell carcinoma, undifferentiated carcinoma, and adenocarcinoma. The nonepithelial component was composed of chondrosarcoma, osteosarcoma, spindle cell sarcoma, rhabdomyosarcoma, and liposarcoma. In the central area of the tumor, a few remnants of benign pleomorphic adenoma were identifiable. The finding suggested that in our patient, the carcinosarcoma arose from a preexisting pleomorphic adenoma. In view of the expected aggressive nature of the tumor, the patient was treated with postoperative radiotherapy of 60 Gy total, in 30 daily fractions of 2 Gy, and chemotherapy. He currently remains well and free of disease 24 months after treatment.

O-Methylasparvenone (1) and asparvenone (2) were isolated from an Aspergillus parvulus Smith broth in a microbial screening for 5-HT2C ligands and found to be 5-HT2C antagonists. They represent the first nitrogen-free serotonin ligands. The absolute configuration of 1 was determined to be S by X-ray analysis of the corresponding Mosher-ester. A short and efficient synthesis of rac-1 was developed. This protocol was applied to the synthesis of derivatives of 1 and a structure-affinity relationship was established.

In 9 patients with the common type of atrial flutter (AF), a zone of slow conduction was observed proximal to the coronary sinus orifice. Class IA antiarrhythmic drugs were administrated intravenously in all 9 patients. AF was converted to sinus rhythm in only 1 patient. In the remaining 8 patients, although IA drugs suppressed conduction velocity, this effect was not preferential with regard to the zone of slow conduction. Conversion of AF to sinus rhythm by rapid pacing was achieved in these 8 patients. At a critical pacing cycle length, AF was interrupted just after conduction block developed in the zone of slow conduction. A progressive decrease in conduction velocity was observed in the zone of slow conduction for several beats just before the onset of conduction block. In conclusion, class IA drugs facilitate the interruption of AF by rapid atrial pacing by inducing conduction block in the zone of slow conduction.

To evaluate the significance of the signal-averaged P wave in various pathological conditions of the atrium, signal-averaged electrocardiograms and echocardiograms were studied in the following 4 groups: (1) 10 normal subjects (control group), (2) 24 patients with paroxysmal atrial fibrillation or flutter (AF) (AF group), (3) 12 patients with left atrial overload without AF (LA group), and (4) 10 patients with right atrial overload without AF (RA group). Original P wave durations showed no significant difference among the 4 groups. Filtered P wave durations (F-PDs) in the AF and LA groups were significantly longer than that in the control group. F-PD correlated significantly with left atrial dimension (LAD). F-PD in AF patients with LAD shorter than 40 mm was significantly longer than that in the control group, but there was no significant difference in F-PD between AF patients with LAD longer than 40 mm and the LA group. Root mean square voltages of original P waves were significantly higher in the LA and RA groups than that in the control group, but in AF patients it did not differ from that in the control group. In conclusion, the signal-averaged P wave is useful to predict atrial fibrillation, but prolongation of F-PD is seen not only in patients with AF but also in patients with LA overload.

A novel regulatory protein for rho p21, a ras p21-like GTP-binding protein (G protein), was purified from the cytosol fraction of rabbit intestine. This protein, designated as rho GDP dissociation inhibitor (GDI), regulated the GDP/GTP exchange reaction of rho p21 by inhibiting the dissociation of GDP from and subsequent binding of GTP to it. rho GDI did not affect the GTPase activity of rho p21. rho GDI formed a complex with the GDP-bound form of rho p21 but not the GTP-bound form. rho GDI was inactive for other small G proteins including ras p21, smg p21 and smg p25A. These results indicate that rho GDI is a novel type of regulatory protein for the GDP/GTP exchange reaction of rho p21.

We have previously purified from bovine brain cytosol a novel regulatory protein for the GTP-binding proteins of the rho gene family. This regulatory protein, designated as rho GDP dissociation inhibitor (GDI), makes a complex stoichiometrically with the GDP-bound form of the rho gene products and thereby regulates the GDP/GTP exchange reaction by inhibiting the dissociation of GDP from and the subsequent binding of GTP to them. We show here that rho GDI also regulates the binding of rhoB p20, a member of the rho gene products, to various membranes including rat brain synaptic plasma membranes and human erythrocyte ghosts. Both the GDP- and GTP-bound forms of rhoB p20 bound to the membranes. This binding was not inhibited by prior treatment of the membranes with boiling or tryptic digestion, suggesting that a protein molecule of the membranes is not essential for the binding of rhoB p20 to the membranes. rho GDI inhibited the binding of the GDP-bound form of rhoB p20, but not that of the GTP-bound form, to the membranes. Moreover, rho GDI induced the dissociation of the GDP-bound form, but not that of the GTP-bound form, of rhoB p20 exogenously bound to the membranes from them. These results suggest that the rho gene products bind to the membranes, presumably in a reversible manner and that this binding is regulated by their specific GDI.

A protein stimulating the GTPase activity of rhoB p20, a ras p21-like GTP-binding protein (G protein), was partially purified from the cytosol fraction of bovine brain. This protein, designated as rhoB p20 GTPase-activating protein (GAP), did not stimulate the GTPase activity of other ras p21/ras p21-like G proteins including c-Ha-ras p21, smg p21 and smg p25A. The activities of c-Ha-ras p21 GAP and smg p21 GAP were also detected in the cytosol fraction of bovine brain and rhoB p20 GAP was separated from these GAPs. The activity of rhoB p20 GAP was eliminated by tryptic digestion or boiling. The Mr value of rhoB p20 GAP was estimated to be 150-200 x 10(3) and 37 x 10(3) by gel filtration and sucrose density gradient ultracentrifugation, respectively. These results indicate that there is rhoB p20 GAP in addition to c-Ha-ras p21 GAP and smg p21 GAP in bovine brain. In rat brain, about 50% of rhoB p20 GAP was found with the highest specific activity in the P2 fraction containing myelin, synaptosomes and mitochondria. In the P2 fraction, about 30% of rhoB p20 GAP was found in the P2C fraction containing mainly synaptosomes. rhoB p20 GAP was detected in the cytosol and particulate fractions of not only rat brain but also other rat tissues.

Two proteins stimulating the GTPase activity of the smg-21 GTP-binding protein (smg p21) having the same effector domain as the ras proteins (ras p21s) are partially purified from the cytosol fraction of human platelets. These proteins, designated as smg p21 GTPase activating protein (GAP) 1 and 2, do not stimulate the GTPase activity of c-Ha-ras p21. The GAP activity for c-Ha-ras p21 is also detected in the cytosol fraction of human platelets. smg p21 GAP1 and 2 are separated from c-Ha-ras p21 GAP by column chromatographies. The activity of smg p21 GAP1 and 2 is killed by tryptic digestion or heat boiling. The Mr values of smg p21 GAP1 and 2 are similar and are estimated to be 2.5-3.5 x 10(5) by gel filtration analysis. These results indicate that there are two GAPs for smg p21 in addition to a GAP for c-Ha-ras p21 in human platelets.

A novel regulatory protein for rhoB p20, a ras p21-like GTP-binding protein (G protein), was partially purified from the cytosol fraction of rabbit intestine. This protein, designated as rhoB p20 GDP dissociation inhibitor (GDI), inhibited the dissociation of GDP from rhoB p20. rhoB p20 GDI also inhibited the binding of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to the GDP-bound form of rhoB p20 but not of that to the guanine nucleotide-free form. GDI did not affect the GTPase activity of rhoB p20 and by itself showed no GTP gamma S-binding activity. GDI was inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, smg p21 and smg p25A. The Mr value of GDI was estimated to be about 27,000 from the S value. These results indicate that rabbit intestine contains a novel regulatory protein that inhibits the dissociation of GDP from and thereby the subsequent binding of GTP to rhoB p20.