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The authors describe a case of intra-articular fracture of the left mandibular condyle, successfully treated by the pumping technique in the upper and lower joint cavities, and show arthroscopic findings in these cavities. The patient was a 15-year-old boy whose maximum mouth opening was 30 mm. Computed tomography revealed a left intra-articular sagittal fracture of the condylar head. Aspiration of the hematoma in the upper and lower joint spaces was performed with ten pumping actions. In the upper and lower joint spaces, arthroscopic examination revealed the disappearance of the hematoma. The patient continued opening, protrusive, and lateral excursive exercises. One month after the surgery, the maximal interincisal distance was improved to 45 mm with straight opening. In the case presented, mouth-opening exercises, along with the pumping technique for treatment of an intraarticular fracture of the mandibular condyle, allowed satisfactory and stable results in the improvement of limited mouth movement. [ABSTRACT FROM AUTHOR]

The global prevalence of oral squamous cell carcinoma (OSCC) has been increasing. OSCC at the advanced stage tends to resist conventional treatment and causes local recurrence and distant metastasis, resulting in poor prognosis. Therefore, detecting this cancer at an early stage and performing early intervention are important. Promising biomarkers to detect OSCC have yet to be established; however, microRNAs (miRNAs/miRs) serve a crucial role in OSCC tumorigenesis and may be potential biomarkers. In the present case report, the availability of salivary miRNAs as a therapeutic and prognostic marker for patients with OSCC was assessed. The patient was a 33-year-old woman who was diagnosed with advanced OSCC of the tongue, and their miRNA profile isolated from a saliva sample at each clinical course was evaluated. Microarray analysis of the salivary samples revealed changes in the levels of four miRNAs (hsa-miR-6798-5p, miR-6803-5p, miR-6805-5p and miR-6845-5p) in accordance with the clinical course. Neoadjuvant chemotherapy and surgical procedure decreased the levels, whereas the levels increased when the patient was diagnosed with lung metastasis. Furthermore, tongue and lung metastatic lesion specimens exhibited expression of the vascular endothelial growth factor receptor-2, which is regulated by the four miRNAs. Accordingly, the present report proposed that salivary miRNAs could be a therapeutic and prognostic biomarker for OSCC., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Kimura et al.)

Background: The activity level of alkaline phosphatase, a zinc-requiring enzyme in the serum, is used to indicate zinc nutritional status; however, it does not correlate with serum zinc levels or subjective symptoms of taste disorder in many cases. Hence, this study focused on the total activity of alkaline phosphatase, a zinc-requiring enzyme. The total alkaline phosphatasa activity level in the saliva was measured before and after zinc supplementation, and the results were compared with serum zinc levels., Case Presentation: This study included patients with hypozincemia, specifically a patient with zinc-deficient taste disorder (patient 1: a 69-year-old Japanese woman) and a patient with glossodynia with zinc deficiency (patient 2: an 82-year-old Japanese woman). Saliva samples were collected, and blood tests were performed before and after zinc supplementation. Subjective symptoms and serum zinc levels were simultaneously evaluated. Zinc supplementation was performed using zinc acetate hydrate or Polaprezinc., Conclusions: Total alkaline phosphatase activity levels were found to be associated with serum zinc levels and subjective symptoms. A further study with a higher number of patients is necessary to confirm whether total alkaline phosphatase activity levels more accurately reflect the amounts of zinc in the body than serum zinc levels., (© 2024. The Author(s).)

Background/purpose: Delayed healing of the extraction socket is not uncommon when tooth extraction is performed on patients taking prednisolone. This study aimed to identify specific dosage of prednisolone and factors associated with delayed healing of the extraction socket in patients taking prednisolone., Materials and Methods: This single-center retrospective study included 80 patients who underwent tooth extraction under local anesthesia and were taking prednisolone orally. Patients were divided into the nondelayed healing group ( n  = 50) and delayed healing group ( n  = 30), and their background and dosage of prednisolone were compared., Results: The dosage of prednisolone was significantly higher in the delayed healing group than in the nondelayed healing group. A receiver operating characteristics curve analysis resulted in moderate accuracy when the cutoff value was set at 8.0, with 67% sensitivity, 76% specificity, and 0.765 area under the curve. The multivariate logistic regression analysis revealed that prednisolone dosage >8.0 mg/day (odds ratio [OR], 10.8; 95% confidence interval [CI], 2.79-41.6) and osteosclerotic changes beyond the alveolar bone around the tooth to be extracted (OR, 10.3; 95% CI, 2.81-37.8) in X-ray imaging had significant effects on delayed healing., Conclusion: The results of this study suggested that delayed healing following tooth extractions in patients taking prednisolone was related to a dosage of 8.0 mg/day or higher and osteosclerotic changes., Competing Interests: The authors have no conflicts of interest relevant to this article., (© 2022 Association for Dental Sciences of the Republic of China. Publishing services by Elsevier B.V.)

Background: Ethyl loflazepate (EL) is a benzodiazepine derivative that has been reported to activate the gustatory cortex. Our department routinely uses EL as a first-line treatment for idiopathic and psychogenic taste disorders, although little has been reported in the literature with respect to patient outcomes, so we conducted a retrospective study examining its safety and efficacy., Methods: Between 2008 and 2020, 49 patients (14 males and 35 females; mean age, 62.1 years) were diagnosed with taste disorders and received EL as their only treatment for > 14 days. Severity of taste disorder was evaluated using the paper disc method by Sakai et al., and treatment efficacy was evaluated using the Visual Analog Scale, wherein patients gave subjective ratings for their symptoms (reductions by > 50% after administration of EL for 4 weeks were defined as improvements)., Results: Results showed that the improvement rates for patients with idiopathic and psychogenic taste disorders were 55 and 70%, respectively. Additionally, the majority (78%) improved within 2 weeks, and side effects were mild (seven cases with drowsiness and one case with dizziness)., Conclusions: We conclude that EL is an appropriate first-line medication for patients with idiopathic and psychogenic taste disorders., (© 2022. The Author(s).)

Tumor blood vessels play important roles in tumor progression and metastasis. Targeting tumor endothelial cells (TECs) is one of the strategies for cancer therapy. We previously reported that biglycan, a small leucine-rich proteoglycan, is highly expressed in TECs. TECs utilize biglycan in an autocrine manner for migration and angiogenesis. Furthermore, TEC-derived biglycan stimulates tumor cell migration in a paracrine manner leading to tumor cell intravasation and metastasis. In this study, we explored the therapeutic effect of biglycan inhibition in the TECs of renal cell carcinoma using an in vivo siRNA delivery system known as a multifunctional envelope-type nanodevice (MEND), which contains a unique pH-sensitive cationic lipid. To specifically deliver MEND into TECs, we incorporated cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD) into MEND because α V β 3 integrin, a receptor for cRGD, is selective and highly expressed in TECs. We developed RGD-MEND-encapsulating siRNA against biglycan. First, we confirmed that MEND was delivered into OS-RC-2 tumor-derived TECs and induced in vitro RNAi-mediated gene silencing. MEND was then injected intravenously into OS-RC-2 tumor-bearing mice. Flow cytometry analysis demonstrated that MEND was specifically delivered into TECs. Quantitative RT-PCR indicated that biglycan was knocked down by biglycan siRNA-containing MEND. Finally, we analyzed the therapeutic effect of biglycan silencing by MEND in TECs. Tumor growth was inhibited by biglycan siRNA-containing MEND. Tumor microenvironmental factors such as fibrosis were also normalized using biglycan inhibition in TECs. Biglycan in TECs can be a novel target for cancer treatment., (© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Basal cell carcinoma (BCC) is the most common cancer worldwide. Most of BCCs can be detected in the early stages and are generally well controlled with local resection. Despite the high incidence of BCC, metastasis is rarely observed. Metastatic BCCs generally have an aggressive phenotype and are refractory to conventional treatment., Case Presentation: We describe a rare case of BCC in which a series of local relapses culminated in metastasis into the oral cavity 10 years after the first diagnosis of cutaneous BCC. We performed surgical resection and postoperative radiotherapy in this patient; 11 months after the final course of radiotherapy, the BCC remains stable, and the patient continues to be monitored regularly., Conclusions: Because metastatic BCC is refractory to current treatment and difficult to control, his treatment history and the pathohistological features of BCC had to be considered in posttreatment planning., (© 2022. The Author(s).)

Background: Burning mouth syndrome (BMS) is a chronic condition characterized by pain in the oral cavity. Kampo medicine is a traditional Japanese medical system that has its roots partly in ancient Chinese medicine. The purpose of this study is to evaluate the efficacy of rikkosan-a traditional Japanese herbal medicine (Kampo)-in the treatment of primary BMS., Main Body: A single-center retrospective study was conducted in 32 patients who were diagnosed with primary BMS and treated with rikkosan alone through gargling (2.5 g rikkosan dissolved in 50 mL hot water) three times daily. Patients were asked to evaluate their pain using a numerical rating scale (NRS) at first visit and after 1 month. One patient had stomatitis as a side effect after gargling with rikkosan, however, no side effects were observed in other patients. Overall NRS scores decreased significantly between the first visit (7.6 ± 2.7) and the 1-month visit (5.6 ± 2.8)., Conclusions: Rikkosan may be an effective treatment for primary BMS., (© 2021. The Author(s).)

Development of quantitative analysis software has enabled application of several standardised uptake values (SUV) for bone analysis in single photon emission computed tomography (SPECT). The present retrospective study aimed to develop a reliable method of monitoring bone inflammatory activity in antiresorptive agent-related osteonecrosis of the jaw (ARONJ) using SPECT quantitative analysis software. Fifteen ARONJ patients underwent SPECT before and after anti-inflammatory therapy. We calculated the mean maximum SUV (SUVmax) of the bilateral cranial bones using quantitative analysis software and used this as the control [C]. We attempted to adjust the SUVmax of the lesion [L] as follows: adjusted SUVmax (aSUVmax) = [L] - [C]. The optimum threshold to calculate the metabolic bone volume (MBV) (cm 3 ) was [C] + 3. The threshold values obtained for each case were input to calculate MBV at each osteomyelitis site. Retrospectively, we compared aSUVmax and MBV of each patient's ARONJ before and after anti-inflammatory therapy. The patients' high aSUVmax or large MBV of the ARONJ reduced rapidly, reflecting individual clinical findings after treatment. Application of SPECT quantitative analysis software to monitor bone inflammatory activity in ARONJ could improve the prognosis-deciding abilities of clinicians and enable them to treat ARONJ effectively.

Medication-related osteonecrosis of jaws (MRONJ) is one of the most complicated inflammatory conditions in oral and maxillofacial region. It is very difficult to correctly evaluate the degree and extent of necrosis and infection. This refractory osteonecrosis often needs extended surgery, leading to impaired quality-of-life. We have performed hyperbaric oxygen therapy (HBO) combined with conservative surgery for advanced cases. We have appraised the value of FDG-PET and 3-phase bone scintigraphy in the diagnosis and management of this condition. MRONJ showed significantly higher SUV max on FDG-PET than the others. Although the 3 phase pool bone images did not change significantly, perfusion and static bone image as well as PET showed remarkable response to HBO for MRONJ. SUV max after HBO was significantly lower than those of before HBO. These preliminary results indicate that FDG-PET is useful for monitoring the effect of HBO for MRONJ.

We report a case of osteonecrosis of the jaw (ONJ) associated with denosumab therapy in a 62-year-old female patient being treated for bone metastases from breast cancer. Upon initial presentation at the Department of Oral Medicine, Hokkaido University Hospital, the patient's mandibular molar teeth were extracted because of severe periodontal disease. Two months later, epithelialization of the sockets was observed and treatment with anti-resorptive drugs was started for bone metastases. One year after tooth extraction, bone exposure in the right lower first molar region was observed, and stage 2 medication-related ONJ (MRONJ) was diagnosed. Up to this time, the patient had received zoledronic acid twice and denosumab 22 times. Denosumab was discontinued by the oncologist, and oral antibiotics with rinsing of the exposed bone area were prescribed. By 36 weeks after discontinuation of denosumab, a sequestrum in the posterior part of the mandible was naturally shed, and the site was healed. Bisphosphonate is deposited in bones, whereas denosumab functions extracellularly and circulates in the blood. The effect of denosumab on bone remodeling is reversed shortly after the drug has been discontinued.

Tumor blood vessels play an important role in tumor progression and metastasis. We previously reported that tumor endothelial cells (TEC) exhibit several altered phenotypes compared with normal endothelial cells (NEC). For example, TEC have chromosomal abnormalities and are resistant to several anticancer drugs. Furthermore, TEC contain stem cell-like populations with high aldehyde dehydrogenase (ALDH) activity (ALDH high TEC). ALDH high TEC have proangiogenic properties compared with ALDH low TEC. However, the association between ALDH high TEC and drug resistance remains unclear. In the present study, we found that ALDH mRNA expression and activity were higher in both human and mouse TEC than in NEC. Human NEC:human microvascular endothelial cells (HMVEC) were treated with tumor-conditioned medium (tumor CM). The ALDH high population increased along with upregulation of stem-related genes such as multidrug resistance 1, CD90, ALP, and Oct-4. Tumor CM also induced sphere-forming ability in HMVEC. Platelet-derived growth factor (PDGF)-A in tumor CM was shown to induce ALDH expression in HMVEC. Finally, ALDH high TEC were resistant to fluorouracil (5-FU) in vitro and in vivo. ALDH high TEC showed a higher grade of aneuploidy compared with that in ALDH low TEC. These results suggested that tumor-secreting factor increases ALDH high TEC populations that are resistant to 5-FU. Therefore, ALDH high TEC in tumor blood vessels might be an important target to overcome or prevent drug resistance., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Reactive oxygen species (ROS) are unstable molecules that activate oxidative stress. Because of the insufficient blood flow in tumors, the tumor microenvironment is often exposed to hypoxic condition and nutrient deprivation, which induces ROS accumulation. We isolated tumor endothelial cells (TECs) and found that they have various abnormalities, although the underlying mechanisms are not fully understood. Here we showed that ROS were accumulated in tumor blood vessels and ROS enhanced TEC migration with upregulation of several angiogenesis related gene expressions. It was also demonstrated that these genes were upregulated by regulation of Nuclear factor erythroid 2-related factor 2 (NRF2). Among these genes, we focused on Biglycan, a small leucine-rich proteoglycan. Inhibition of Toll-like receptors 2 and 4, known BIGLYCAN (BGN) receptors, cancelled the TEC motility stimulated by ROS. ROS inhibited NRF2 expression in TECs but not in NECs, and NRF2 inhibited phosphorylation of SMAD2/3, which activates transcription of BGN. These results indicated that ROS-induced BGN caused the pro-angiogenic phenotype in TECs via NRF2 dysregulation.

Aim: We evaluated the prognostic value of vasohibin-1 (VASH1) expression in head and neck squamous cell carcinoma., Materials and Methods: Immunohistochemistry for VASH1 and cluster of differentiation 34 (CD34) was performed on 61 head and neck squamous cell carcinoma specimens. The association between VASH1 expression in the tumour and clinical outcomes was analyzed statistically., Results: VASH1 staining in normal tissue adjacent to cancerous tissue was negative, whereas it was positive in tumour blood vessels and AE1/AE3 and Ki67-positive tumour cells. Therefore, we examined the association between VASH1 expression in the tumour and clinical outcomes. Patients with high VASH1 expression in tumour had significantly shorter disease-free survival and more frequently had lymph node recurrence than those with low VASH1 expression., Conclusion: These results suggest that VASH1 expression is associated with tumour progression and may be useful as a prognostic marker of head and neck squamous cell carcinoma., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Tumour blood vessels are gateways for distant metastasis. Recent studies have revealed that tumour endothelial cells (TECs) demonstrate distinct phenotypes from their normal counterparts. We have demonstrated that features of TECs are different depending on tumour malignancy, suggesting that TECs communicate with surrounding tumour cells. However, the contribution of TECs to metastasis has not been elucidated. Here, we show that TECs actively promote tumour metastasis through a bidirectional interaction between tumour cells and TECs. Co-implantation of TECs isolated from highly metastatic tumours accelerated lung metastases of low metastatic tumours. Biglycan, a small leucine-rich repeat proteoglycan secreted from TECs, activated tumour cell migration via nuclear factor-κB and extracellular signal-regulated kinase 1/2. Biglycan expression was upregulated by DNA demethylation in TECs. Collectively, our results demonstrate that TECs are altered in their microenvironment and, in turn, instigate tumour cells to metastasize, which is a novel mechanism for tumour metastasis.

Tumor angiogenesis plays an important role in tumor progression and metastasis. Tumor endothelial cells (TECs) are a therapeutic target of antiangiogenic chemotherapy that was recently developed and is currently being investigated in the clinic with promising results. Low-dose chemotherapy, which is the long-term administration of relatively low doses of chemotherapeutic agents, has been proposed for targeting tumor angiogenesis in various types of cancers. Although the efficacy of low-dose chemotherapy has been confirmed in several clinical models, some studies show insufficient therapeutic effect for malignant cancers. As a possible mechanism of the treatment failure, it has been considered that tumor cells may acquire resistance to this therapy. However, drug resistance by TECs may also be due to another mechanism for resistance of tumor cells to low-dose chemotherapy. We reported elsewhere that TECs were resistant to the anticancer drug paclitaxel, which is a mitotic inhibitor, concomitant with P-glycoprotein up-regulation. Verapamil, a P-glycoprotein inhibitor, abrogated TEC resistance in vitro. Herein, we demonstrated that verapamil coadministration enhanced the effects of low-dose paclitaxel concomitant with inhibiting tumor angiogenesis in a preclinical in vivo mouse melanoma xenograft model. Furthermore, verapamil coadministration reduced lung metastasis. These results suggest that inhibiting P-glycoprotein in TECs may be a novel strategy for low-dose chemotherapy targeting TECs., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Tumor blood vessels play an important role in tumor progression and metastasis. It has been reported that tumor endothelial cells (TECs) exhibit highly angiogenic phenotypes compared with those of normal endothelial cells (NECs). TECs show higher proliferative and migratory abilities than those NECs, together with upregulation of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). Furthermore, compared with NECs, stem cell markers such as Sca-1, CD90, and multidrug resistance 1 are upregulated in TECs, suggesting that stem-like cells exist in tumor blood vessels. In this study, to reveal the biological role of stem-like TECs, we analyzed expression of the stem cell marker aldehyde dehydrogenase (ALDH) in TECs and characterized ALDHhigh TECs. TECs and NECs were isolated from melanoma-xenografted nude mice and normal dermis, respectively. ALDH mRNA expression and activity were higher in TECs than those in NECs. Next, ALDHhigh/low TECs were isolated by fluorescence-activated cell sorting to compare their characteristics. Compared with ALDHlow TECs, ALDHhigh TECs formed more tubes on Matrigel-coated plates and sustained the tubular networks longer. Furthermore, VEGFR2 expression was higher in ALDHhigh TECs than that in ALDHlow TECs. In addition, ALDH was expressed in the tumor blood vessels of in vivo mouse models of melanoma and oral carcinoma, but not in normal blood vessels. These findings indicate that ALDHhigh TECs exhibit an angiogenic phenotype. Stem-like TECs may have an essential role in tumor angiogenesis.

Recent studies have reported that stromal cells contribute to tumor progression. We previously demonstrated that tumor endothelial cells (TEC) characteristics were different from those of normal endothelial cells (NEC). Furthermore, we performed gene profile analysis in TEC and NEC, revealing that suprabasin (SBSN) was upregulated in TEC compared with NEC. However, its role in TEC is still unknown. Here we showed that SBSN expression was higher in isolated human and mouse TEC than in NEC. SBSN knockdown inhibited the migration and tube formation ability of TEC. We also showed that the AKT pathway was a downstream factor of SBSN. These findings suggest that SBSN is involved in the angiogenic potential of TEC and may be a novel TEC marker., (© 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

The identification of a specific biomarker involves the development of new clinical diagnostic tools, and an in-depth understanding of the disease at the molecular level. When new blood vessels form in tumor cells, endothelial cell production is induced, a process that plays a key role in disease progression and metastasis to distinct organs for solid tumor types. The present study reports on the identification of a new biomarker on primary cultured mouse tumor endothelial cells (mTECs) using our recently developed high-affinity DNA aptamer AraHH001 (Kd = 43 nmol/L) assisted proteomics approach. We applied a strategy involving aptamer-facilitated biomarker discovery. Biotin-tagged AraHH001 was incubated with lysates of mTECs and the aptamer-proteins were then conjugated with streptavidin magnetic beads. Finally, the bound proteins were separated by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. We identified troponin T via matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, the molecular target of aptamer AraHH001, and its presence was confirmed by measuring mRNA, protein levels, western blot, immunostaining, a gel shift assay of AraHH001 with troponin T. We first report here on the discovery of troponin T on mTECs, a promising and interesting diagnostic tool in the development of antiangiogenic therapy techniques the involves the targeting of the tumor vasculature., (© 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

The objective of this study was to construct our recently developed aptamer-modified targeted liposome nano-carrier (Apt-PEG-LPs) system to target primary cultured mouse tumor endothelial cells (mTEC), both in vitro and in vivo. We first synthesized an aptamer-polyethylene glycol 2000-distearoyl phosphoethanolamine (Apt-PEG2000-DSPE). The conjugation of the Apt-PEG2000-DSPE was confirmed by MALDI-TOF mass spectroscopy. A lipid hydration method was used to prepare Apt-PEG-LPs, in which the outer surface of the PEG-spacer was decorated with the aptamer. Apt-PEG-LPs were significantly taken up by mTECs. Cellular uptake capacity was observed both quantitatively and qualitatively using spectrofluorometry, and confocal laser scanning microscopy (CLSM), respectively. In examining the extent of localization of aptamer-modified liposomes that entered the cells, approximately 39% of the Apt-PEG-LPs were not co-localized with lysotracker, indicating that they had escaped from endosomes. The uptake route involved a receptor mediated pathway, followed by clathrin mediated endocytosis. This Apt-PEG-LP was also applied for in vivo research whether this system could target tumor endothelial cells. Apt-PEG-LP and PEG5000-DSPE modified Apt-PEG-LP (Apt/PEG5000-LP) were investigated by human renal cell carcinoma (OS-RC-2 cells) inoculating mice using CLSM. Apt-PEG-LP and Apt/PEG5000-LP showed higher accumulation on tumor vasculature compared to PEG-LP and the co-localization efficacy of Apt-PEG-LP and Apt/PEG5000-LP on TEC were quantified 16% and 25% respectively, which was also better than PEG-LP (3%). The findings suggest that this system is considerable promise for targeting tumor endothelial cells to deliver drugs or genes in vitro and in vivo., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Targeting tumor angiogenesis is an established strategy for cancer therapy. Because angiogenesis is not limited to pathological conditions such as cancer, molecular markers that can distinguish between physiological and pathological angiogenesis are required to develop more effective and safer approaches for cancer treatment. To identify such molecules, we determined the gene expression profiles of murine tumor endothelial cells (mTEC) and murine normal endothelial cells using DNA microarray analysis followed by quantitative reverse transcription-polymerase chain reaction analysis. We identified 131 genes that were differentially upregulated in mTEC. Functional analysis using siRNA-mediated gene silencing revealed five novel tumor endothelial cell markers that were involved in the proliferation or migration of mTEC. The expression of DEF6 and TMEM176B was upregulated in tumor vessels of human renal cell carcinoma specimens, suggesting that they are potential targets for antiangiogenic intervention for renal cell carcinoma. Comparative gene expression analysis revealed molecular differences between tumor endothelial cells and normal endothelial cells and identified novel tumor endothelial cell markers that may be exploited to target tumor angiogenesis for cancer treatment., (© 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

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.

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.)

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.)

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.)

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.

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 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.

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 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.

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.