Significant Decrease In Microvessel Density Research Articles

Effects of Glutamine Synthetase on Neovascularization in Glioma: In Vivo MR Vessel Size Imaging and Histology.

Glutamine Synthetase (GS) could induce vascular sprouting through the improvement of endothelial cell migration in inflammatory diseases. MR vessel-size imaging has been proposed as a valuable approach for visualizing the underlying angiogenic processes in the brain. This study aims to investigate the role of GS in the neovascularization of gliomas through the utilization of MR vessel-size imaging and histopathological techniques. In this exploratory animal study, we randomly divided the C6 glioma rat models into a control group and an L-methionine sulfoximine (MSO) treatment group. Daily intraperitoneal injections were administered for three consecutive days, starting from day 10 following the implantation of C6 glioma cells in rats. Subsequently, MR vessel size imaging was conducted using a BRUKER 7 T/200 mm MRI scanner, and the MRI results were validated through histopathological examination. A significant decrease in microvessel density was observed in both the tumor periphery and center areas in the MSO treatment group compared to that in the control group. The mean vessel diameter (mVD) and vessel size index (VSI) did not exhibit significant changes compared to the control group. Moreover, the staining intensity of platelet endothelial cell adhesion molecule-1 (CD31) and GS in the tumor periphery was significantly decreased in the MSO treatment group. Additionally, the MSO treatment demonstrated a substantial inhibition of tumor growth. GS inhibitors significantly reduced angiogenesis in the periphery area of C6 glioma, exerting an inhibitory effect on tumor progression. Thus, GS inhibitors could be potential therapeutic agents for treating glioma. Additionally, in vivo MR vessel size imaging detects changes in vascularrelated parameters after tumor treatment, making it a promising method for detecting neovascularization in glioma.</p>.

Xanthine oxidase mediates stress-induced Alzheimer's Disease progression in triple transgenic mice

Alzheimer’s disease (AD) is a progressive disorder associated with oxidative stress and neuroinflammation promoting the deposition of β-amyloid (Aβ) plaques and neurofibrillary tau tangles. Xanthine oxidoreductase (XOR) is a molybdoenzyme responsible for the metabolism of hypoxanthine and xanthine to uric acid. Under inflammatory conditions, high plasma uric acid levels upregulate XOR leading to increased free radical production and promoting cerebrovascular dysfunction. Chronic stress induces cerebrovascular dysfunction and cognitive decline in C57BL/6 mice mediated by XOR, suggesting a link between chronic stress and dementia. However, there is no published data that link XOR and stress to AD pathology and progression. Thus, we tested the hypothesis that chronic stress accelerates AD pathology via the XOR pathway.At 4 months of age, male and female WT (B6129) and triple transgenic (3xTg) AD mice underwent the Unpredictable Chronic Mild Stress (UCMS) model paradigm for 8 weeks. Mice were euthanized at two different time points at 6 and 9 months of age for both control and UCMS groups, and the tissues were harvested. Brain tissue lysates were homogenized, and the total tau and XOR protein was quantified using Western blot and normalized to β-actin. Aβ (1-40) levels in mouse brain homogenates using Aβ ELISA kit. Immunohistochemical staining was performed to study the colocalization of XO and CD-31 in WT and 3xTg coronal sections and analyzed using Fiji ImageJ.UCMS accelerated Aβ(1-40) expression levels (p&lt;0.05) at both 6 and 9 months of age in the 3xTg mice. Tau levels were significantly increased at 6 months of age (p&lt;0.05), but showed no significant differences in protein expression at 9 months of age. XOR levels were significantly upregulated (p&lt;0.05) in the UCMS group at 6 months of age. At 9 months of age, no significant differences were seen in XOR expression across groups indicating that mice taken off from the UCMS protocol had normal levels of XOR due to reduced oxidative stress buildup in the brain. At 6 months of age, a significant decrease in microvessel density (p&lt;0.05) was seen in 3xTg-UCMS mice vs 3xTg controls, with no significant differences seen at 9 months of age, indicating the role of chronic stress on the decline in cerebrovascular functions. Thus, the study establishes the premise that chronic stress mediated by XOR accelerates AD pathology in our early-onset AD model. NIH, NINDS BINP R01 NS117754, Psychosocial Stress-Induced Vascular Contributions to Cognitive Impairment and Alzheimer's Disease: The Role of Xanthine Oxidase This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Active metronomic vinorelbine schedules decrease plasma interleukin-2 levels in mice with Lewis lung carcinoma

The aim of our study was to investigate the effects of metronomic vinorelbine (mVNR) in a tumor model of Lewis Lung (LL) cancer in immunocompetent C57BL/6 mice, looking at the plasma levels of interleukin-2 (IL-2) and interleukin-8 (IL-8). mVNR caused a concentration-dependent antiproliferative effect in vitro on LL/2 cells. The in vivo experiment showed the significant antitumor effects of mVNR at the dose of 4 mg/Kg and 5 mg/Kg, 3 times/week, and the significant dose-dependent decrease of IL-2 concentrations in plasma samples. Conversely, such an effect was not observed for IL-8. A significant decrease in microvessel density was also found at both the active mVNR doses. In conclusion, our study confirmed the activity of mVNR in an immunocompetent model of lung carcinoma and suggest multiple mechanisms of action, including the modulation of IL-2 levels.

Efficacy of pEgr-1-endostatin combined with ionizing radiation on hypoxic conditions in nude mice bearing SKOV3 ovarian carcinoma

Hypoxia occurs in a wide range of solid tumors, and is strongly associated with radio-resistance of malignant tumors. The aim of the present study was to investigate the effect of endostatin combined with ionizing radiation (IR) on hypoxic conditions. A total of 24 mice bearing SKOV3 ovarian carcinoma were divided into three groups. Following injection with pEgr-1-endostatin plasmid for 12 h, the mice in the endostatin-IR-treated group were exposed to 300 cGy/min X-ray for 48 h, and the IR-treated group was exposed to the same condition. Then, the expression of endostatin, hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) was detected by reverse transcription-polymerase chain reaction, ELISA, immunohistochemistry and western blotting. In addition, the tumor microvessel density (MVD) was examined by immunohistochemistry analysis of cluster of differentiation 31-positive cells. The results revealed that pEgr-1-endostatin was successfully induced by IR. The level of endostatin messenger RNA in the endostatin-IR-treated group was significantly higher than that in the control and IR-treated groups (F=380.078, P<0.001). Statistical differences were also examined at the protein level by western blotting and ELISA. An obvious increase in MVD was observed in the IR-treated group compared with that in the control group (t=7.040, P<0.001), and a significant decrease in MVD was observed in the endostatin-IR-treated group compared with that in the control group (t=18.153, P<0.001). By comparing the morphology of the tumor vasculature in the three groups, it was noticed that the microvessels in the endostatin-IR-treated group were more regularly distributed and had fewer giant branches than those in the IR-treated group. Further investigation revealed that the expression levels of HIF-1α and VEGF in the endostatin-IR-treated group were lower compared with those in the control (t=5.339, P=0.001; and t=13.880, P<0.001, respectively) and the IR-treated groups (t=12.930, P<0.001; and t=14.050, P<0.001, respectively). Our findings suggested that endostatin decreased the number of microvessels via the HIF-1/VEGF signaling pathway, and that pEgr-1-endostatin combined with IR may improve hypoxic conditions and may be a novel approach for treating solid tumors.

Regulation of proliferation, angiogenesis and apoptosis in hepatocellular carcinoma by miR-26b-5p.

MicroRNAs (miRNAs) play vital roles in cell proliferation, differentiation and apoptosis in hepatocellular carcinoma (HCC). miR-26b has been confirmed as an important regulator in carcinogenesis and other pathological processes. miR-26b-5p is one member of the mature miR-26 family, and its functional role in proliferation, angiogenesis and apoptosis in HCC remains unknown. Here, we demonstrate that miR-26b-5p expression was significantly decreased in HCC tissues and HCC cell lines compared with normal liver tissues and liver cells by quantitative real-time polymerase chain reaction (qRT-PCR). The relationships between miR-26b-5p and the clinical characteristics of HCC patients were further analysed, and miR-26b-5p was positively correlated with the differentiation of HCC cells. Computational searches were further used to identify the downstream targets and signalling pathways of miR-26b-5p in HCC cells. Cell viability, proliferation and tube formation abilities were assessed by scrape, 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and three-dimensional culture assays to confirm that miR-26b-5p inhibited HCC cell growth and impaired the tube formation ability of the HCC cells. Both in vitro and in vivo studies showed that miR-26b-5p could suppress vascular mimicry (VM) and angiogenesis by down-regulating the expression of VE-cadherin, Snail and MMP2 and could inhibit the apoptosis of HCC cells. Using mouse models, we revealed that tumours derived from miR-26b-5p-expressing HCC cells displayed a significant decrease in microvessel density compared with those derived from control cells. Therefore, our data provide further insight into the role of miR-26b-5p as a negative regulator of proliferation, angiogenesis, and apoptosis in HCC.

Abstract B11: A dual PI3K/mTOR inhibitor, BEZ235 blocks tumor-induced angiogenesis: Evidence for an effect on the tumor and endothelial compartment

Abstract Background: Tumor progression including HER2+ breast tumor, particularly in aggressive and malignant tumors, is associated with the angiogenesis. To investigate the effects of BEZ235 on angiogenesis, we evaluated its effects on the hypoxic stabilization or the PI3K-AKT-mTOR pathway-mediated synthesis of hypoxia-inducible factor -1α (HIF-1α) and tumor-induced angiogenesis. Methodology: Here we have studied the in vitro and in vivo effects of BEZ235 in HER2+/T-sensitive (BT474), HER2+/T-resistant (BT474HerR) and HER2+/PIK3CA (HCC1954 &amp; UACC893) mutated models. We assessed in vitro activation status of the PI3K-AKT-mTOR signaling pathway following BEZ235 treatment in HER2+ BC cell lines. We next evaluated the impact of BEZ235 on hypoxia-induced or the PI3K-AKT-mTOR pathway ⊠mediated HIF1α stabilization/synthesis which is a master regulator of angiogenesis and also tumor-induced angiogenesis using xenograft models. Results: The results show that 1) BEZ235 inhibited downstream activation of the PI3K-AKT-mTOR signaling pathway effectors, p-AKT (Ser473, The308), p-P70S6K, p-S6RP and p-4EBP1, 2) unlike other pan-PI3Kinase inhibitor (e.g. LY294002, SF1126), BEZ235 has no effect on hypoxia-mediated stabilization of HIF1α however, heregulin-induced HIF1α synthesis which is an important angiogenic modulator in breast cancer cells, was significantly decreased following the treatment of BEZ235 in HER2+ and HER2+/PIK3CA mutated breast cancer cells, 3) interestingly, BEZ235 mediated abrogation of HIF1α synthesis is more pronounced than mTORC1 inhibition alone, 4) integrin-directed endothelial cell migration is one of the critical steps for tumor-induced angiogenesis. BEZ235 abrogates endothelial cells functions especially integrin-directed HUVEC cells migration on vitronectin (avb3/ avb5) and significantly abrogates endothelial cells tube formation on Matrigel and 5) furthermore, we evaluated the effects of BEZ235 on the capacity of HER2+ and HER2+/PIK3CA mutated breast tumor cells to recruit a blood supply in vivo during thrice weekly treatments with BEZ235. A microvessel density (MVD) in control versus BEZ235-treated tumors showed a significant decrease in MVD (CD31+) and also VEGFR immunostaining in BEZ235 treated tumors. Conclusion: Our data suggest that BEZ235 has potent antiangiogenic activity in three different xenograft models probably via mTOR-HIF1α-VEGF signaling axis. Citation Format: Pradip De, Yuliang Sun, Jennifer H. Carlson, Xiaoqian Lin, Nandini Dey, Brian Leyland-Jones. A dual PI3K/mTOR inhibitor, BEZ235 blocks tumor-induced angiogenesis: Evidence for an effect on the tumor and endothelial compartment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B11.

Abstract 1492: Trastuzumab-induced normalization in a HER2+ murine model of breast cancer

Abstract Introduction: The primary goal of this study is to quantitatively map the vascular and cellular response to trastuzumab treatment in a murine model of HER2+ breast cancer. We report longitudinal dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) and immunohistochemical studies hypothesized to identify timing windows of trastuzumab-induced vascular normalization. Experimental Design: Mice were subcutaneously implanted with BT474 breast cancer cells and randomly assigned to treated (10 mg/kg trastuzumab) and control (saline) groups. After tumors reached ∼225 mm3, animals (n = 20) were imaged (7.0 T MRI) before treatment (day 0), and 24 hours after each treatment (day 1 and day 4). Subgroups were sacrificed for histology between days 0 through 10 (n = 45). DCE-MRI analysis yielded parametric maps of blood vessel perfusion and permeability (Ktrans) and the extravascular extracellular volume fraction (ve). Tumor sections were paraffin-embedded and stained with CD31, α-SMA, Ki67 and H&amp;E. Slides were scanned in high resolution (20×) and quantitatively analyzed with Leica SCN400 software. Results: Compared to controls, treated tumors exhibited a significant increase in Ktrans (p = 0.03) on day 4, indicative of enhanced vessel perfusion and/or permeability. Additionally at day 4, treated tumors exhibited a significant increase in extravascular extracellular volume fraction measured via ve (p = 0.01), indicating increased cell death. These significant changes occurred prior to a significant change in tumor size at day 4 (p = 0.11). Decreased Ki67 proliferation staining (p = 0.02) at day 3 and a significant decrease in tumor volume at day 7 (p = 0.04) in the treated group confirmed tumor response to trastuzumab. The increase in Ktrans suggests properties of vessel normalization with potential to improve delivery of systemic therapies. Immunohistochemical analyses showed treated tumors have a significant decrease in microvessel density (CD31 staining, p &amp;lt; 0.01), with a simultaneous significant increase in α-SMA staining (pericyte coverage, p = 0.05) on day 4; thus, there was an overall increase in the “vessel maturation index” (ratio of α-SMA to CD31 staining) compared to controls (p = 0.01). Conclusion: These concordant (though preliminary) imaging and histological data suggest that trastuzumab treatment promotes vascular normalization, providing motivation for future studies designed to improve the efficacy of combination therapies in HER2+ breast cancer. For example, DCE-MRI has the potential to identify windows of vascular normalization thereby indicating optimal timing for treatment dosing with a cytotoxic therapy. As trastuzumab is currently given clinically in combination with various chemotherapies for HER2+ cancer, trastuzumab-induced vascular normalization could be utilized to improve drug delivery efficiency and enhance overall patient response without increasing dose or systemic toxicity. Citation Format: Anna G. Sorace, Jennifer G. Whisenant, J. Oliver McIntyre, Thomas E. Yankeelov. Trastuzumab-induced normalization in a HER2+ murine model of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1492. doi:10.1158/1538-7445.AM2015-1492

Abstract 491: Tumor response to cabozantinib in the TH-MYCN GEM model of neuroblastoma

Abstract Neuroblastoma (NB) is the most common extra-cranial solid tumor in childhood. The proto-oncogene MYCN is amplified in 25% of NB, and is associated with enhanced angiogenesis and poor survival. Vascular endothelial growth factor (VEGF) is the most potent angiogenic growth factor and, with the hepatocyte growth factor (HGF)/c-MET signalling pathway, is implicated in the progression of human NB. Cabozantinib (CBZ) is a small-molecule kinase inhibitor with potent activity against VEGFR2 and MET, currently in clinical trials against NB. In this study, the efficacy of CBZ was evaluated in tumors spontaneously arising in the TH-MYCN GEM model of high-risk NB, previously shown to faithfully recapitulate the radiological and hypervascular characteristics of childhood disease. TH-MYCN mice with spontaneous abdominal tumors were treated daily with either vehicle or 30mg/kg CBZ. Non-invasive T2-weighted anatomical MRI revealed that CBZ induced significant (p&amp;lt;0.001) tumor growth arrest after 48 hours compared to control. Quantitative functional MRI showed this volumetric response was associated with significant decreases in both the native longitudinal MRI relaxation time T1 (p&amp;lt;0.01) a measure of the ratio of bound to free water in tissue reflecting the increased extracellular space in tumors, and transverse MRI relaxation rate R2* (p&amp;lt;0.05), sensitive to paramagnetic deoxyhemoglobin. Histopathological analysis revealed a significant increase in necrosis (p&amp;lt;0.01), and significant decrease in microvessel density (p&amp;lt;0.01) and VEGFR2 scoring (p&amp;lt;0.02) in CBZ-treated tumors compared to control. Significant (p&amp;lt;0.01) tumor growth delay was maintained after 7 days treatment with CBZ compared to control, which translated into a significant improvement in survival rate (p&amp;lt;0.0001). This study demonstrates that CBZ is efficacious against, and conveys a survival benefit for the TH-MYCN GEM model of NB. The data reinforces native T1 as a biomarker of response to anti-vascular therapy due to its sensitivity to therapy-induced damage to erythrocytes and/or cell death-mediated increase in macromolecules into the extracellular space. Relatively fast R2* rates in NB arising in the TH-MYCN model are strongly associated with vascular instability, characterised by numerous regions of aggregated paramagnetic erythrocytes (“blood lakes”), and treatment-induced thrombosis with the pan-VEGFR inhibitor cediranib which increased tumor R2*. The CBZ-induced decrease in R2* is clearly associated with vascular disruption, hence precluding the extravasation of deoxygenated red blood cells. Citation Format: Gilberto S. Almeida, Philippa King, Yann Jamin, Albert Hallsworth, Hannah Webber, Sergey Popov, Andrew D.J. Pearson, Louis Chesler, Simon P. Robinson. Tumor response to cabozantinib in the TH-MYCN GEM model of neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 491. doi:10.1158/1538-7445.AM2015-491

Combining bevacizumab and chemoradiation in rectal cancer. Translational results of the AXEBeam trial.

Background:This study characterises molecular effect of bevacizumab, and explores the relation of molecular and genetic markers with response to bevacizumab combined with chemoradiotherapy (CRT).Methods:From a subset of 59 patients of 84 rectal cancer patients included in a phase II study combining bevacizumab with CRT, tumour and blood samples were collected before and during treatment, offering the possibility to evaluate changes induced by one dose of bevacizumab. We performed cDNA microarrays, stains for CD31/CD34 combined with α-SMA and CA-IX, as well as enzyme-linked immunosorbent assay (ELISA) for circulating angiogenic proteins. Markers were related with the pathological response of patients.Results:One dose of bevacizumab changed the expression of 14 genes and led to a significant decrease in microvessel density and in the proportion of pericyte-covered blood vessels, and a small but nonsignificant increase in hypoxia. Alterations in angiogenic processes after bevacizumab delivery were only detected in responding tumours. Lower PDGFA expression and PDGF-BB levels, less pericyte-covered blood vessels and higher CA-IX expression were found after bevacizumab treatment only in patients with pathological complete response.Conclusions:We could not support the ‘normalization hypothesis' and suggest a role for PDGFA, PDGF-BB, CA-IX and α-SMA. Validation in larger patient groups is needed.

Abstract 1009: Tumor-specific expression of fatty acid synthase promotes angiogenesis in colorectal cancer

Abstract Up regulation of fatty acid synthase (FASN), a key enzyme of de novo lipogenesis, is associated with metastasis in colorectal cancer (CRC). However, the mechanisms of regulation of metastasis by FASN are elusive. The induction of angiogenesis is crucial for metastasis. Therefore, the purpose of the present study was (i) to determine the role of FASN in regulation of secretion of angiogenic factors by CRC cells, (ii) to evaluate the effect of tumor-specific expression of FASN on endothelial cells, and (iii) to assess the role of FASN in regulation of tumor vasculature. METHODS: The effect of FASN inhibition on secretion of angiogenic factors was analyzed using the Human Angiogenesis Antibody Array 1 (RayBiotech). Co-culture of CRC and HMVEC-L cells was used to assess the effect of FASN expression on growth, migration, and tube formation of endothelial cells. To evaluate the effect of FASN on vasculature, we utilized murine colonoscopy, the orthotopic colon cancer model, the chick chorioallantoic membrane (CAM) model, and the Matrigel plug assay. RESULTS: We found that inhibition of FASN is associated with an evident change in the profile of secreted angiogenic factors by CRC cells and this change is associated with a significant decrease in microvessel density as determined by analysis of CD31 staining (Aperio Image Scope) in orthotopic colon tumors. A decrease in expression of FASN is also associated with “normalization” of tumor vasculature in the CAM model. The mechanisms of inhibition of angiogenesis by knockdown of FASN included down regulation of VEGF189, an isoform associated with aggressive CRC, up regulation of anti-angiogenic isoform VEGF165b, and a decrease in expression and activity of MMP-9. Furthermore, medium conditioned on FASN-knockdown CRC cells inhibited proliferation, migration, and tube formation of endothelial cells via inhibition of VEGFR-2 activation and its downstream signaling as compared to control medium. CONCLUSIONS: This study suggests that FASN promotes angiogenesis via alteration of the profile of secreted angiogenic factors by CRC cell and stimulation of endothelial cell proliferation. Furthermore, the regulation of angiogenesis by FASN, at least in part, is mediated by up-regulation of VEGF189, a VEGF-A isoform that tightly correlates with poor prognosis in CRC, and by an increase in activity of MMP-9. Together, these findings suggest that inhibition of FASN upstream of VEGF-A and other angiogenic pathways can be a novel therapeutic strategy to prevent or inhibit metastasis in CRC. Citation Format: Yekaterina Y. Zaytseva, Victoria Elliott, Piotr Rychahou, William Mustain, Ji Tae Kim, Joseph Valentino, Tianyan Gao, Kathleen O'Connor, Janna Neltner, Heidi Weiss, B. Mark Evers. Tumor-specific expression of fatty acid synthase promotes angiogenesis in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1009. doi:10.1158/1538-7445.AM2014-1009

Antiangiogenic Therapy with Human Apolipoprotein(a) Kringle V and Paclitaxel in a Human Ovarian Cancer Mouse Model

INTRODUCTION: The present study compared the effect of combination therapy using human apolipoprotein(a) kringle V (rhLK8) to conventional chemotherapy with paclitaxel for human ovarian carcinoma producing high or low levels of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: Human ovarian carcinoma cells producing high (SKOV3ip1) or low (HeyA8) levels of VEGF were implanted into the peritoneal cavity of female nude mice. Seven days later, mice were randomized into four groups: control (vehicle), paclitaxel [5 mg/kg, weekly intraperitoneal (i.p.) injection], rhLK8 (50 mg/kg, daily i.p. injection), or the combination of paclitaxel and rhLK8. Mice were treated for 4 weeks and examined by necropsy. RESULTS: In mice implanted with SKOV3ip1 cells, rhLK8 treatment had no significant effect on tumor incidence or the volume of ascites but induced a significant decrease in tumor weight compared with control mice. Paclitaxel significantly reduced tumor weight and ascites volume, and combination treatment with paclitaxel and rhLK8 had an additive therapeutic effect. Similarly, in HeyA8 mice, the effect of combination treatment on tumor weight and tumor incidence was statistically significantly greater than that of paclitaxel or rhLK8 alone. Immunohistochemical analysis showed a significant decrease in microvessel density and a marked increase of apoptosis in tumor and tumor-associated endothelial cells in response to combination treatment with paclitaxel and rhLK8. CONCLUSION: Collectively, these results suggest that antiangiogenic therapy with rhLK8 in combination with taxane-based conventional chemotherapy could be effective for the treatment of ovarian carcinomas, regardless of VEGF status.

Axitinib (AG-013736), an oral specific VEGFR TKI, shows potential therapeutic utility against cholangiocarcinoma.

Cholangiocarcinoma is a refractory cancer whose incidence has been increasing worldwide in recent years. Neoangiogenesis plays an important role in the growth of various solid cancers, including cholangiocarcinoma. Vascular endothelial growth factor plays an important role in tumor-induced angiogenesis and its expression is associated with the progression and prognosis of cholangiocarcinoma. This study examined whether axitinib (AG-013736, INLYTA(®)), a potent and selective second-generation inhibitor of vascular endothelial growth factor receptors 1, 2 and 3, could be a potentially useful therapeutic agent for cholangiocarcinoma. We performed expression profiling of angiogenesis-related molecules in eight cholangiocarcinoma cell lines and found that three of them showed high vascular endothelial growth factor expression. Among them, we examined the in vivo anti-tumor effect of axitinib on NCC-BD1 (a gemcitabine-sensitive extra-hepatic cholangiocarcinoma cell line) and TKKK (a gemcitabine-resistant intra-hepatic cholangiocarcinoma cell line) using subcutaneous xenograft models. Oral administration of axitinib significantly inhibited the growth of TKKK xenografts at a dose of 6 mg kg(-1) day(-1) (P<0.05), and the growth of NCC-BD1 xenografts at 30 mg kg(-1)day(-1) (P<0.05). Treated tumors showed a significant decrease of microvessel density and the tumor cell proliferation index and a mild but significant increase of the apoptotic index in comparison with untreated tumors. Our results suggest that axitinib should be a promising therapy for vascular endothelial growth factor-expressing cholangiocarcinoma, irrespective of tumor origin and gemcitabine sensitivity.

99 A COMPARISON OF URETHRAL STRICTURE FORMATION IN A PORCINE LARGE ANIMAL MODEL WITH HUMAN STRICTURE DEVELOPMENT

You have accessJournal of UrologyTrauma/Reconstruction: Trauma & Reconstructive Surgery1 Apr 201199 A COMPARISON OF URETHRAL STRICTURE FORMATION IN A PORCINE LARGE ANIMAL MODEL WITH HUMAN STRICTURE DEVELOPMENT Jörg Seibold, Christin Selent-Stier, Julia Wiedemann, Tim-Oliver Greiner, Elisabeth Gustafsson, Brigitte Schuhmacher, Gerhard Feil, Arnulf Stenzl, and Karl-Dietrich Sievert Jörg SeiboldJörg Seibold Tübingen, Germany More articles by this author , Christin Selent-StierChristin Selent-Stier Tübingen, Germany More articles by this author , Julia WiedemannJulia Wiedemann Tübingen, Germany More articles by this author , Tim-Oliver GreinerTim-Oliver Greiner Tübingen, Germany More articles by this author , Elisabeth GustafssonElisabeth Gustafsson Tübingen, Germany More articles by this author , Brigitte SchuhmacherBrigitte Schuhmacher Tübingen, Germany More articles by this author , Gerhard FeilGerhard Feil Tübingen, Germany More articles by this author , Arnulf StenzlArnulf Stenzl Tübingen, Germany More articles by this author , and Karl-Dietrich SievertKarl-Dietrich Sievert Tübingen, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.164AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urethral stricture has been described as a fibrotic process in scarred tissue that reduces the urethral lumen caliber. Urethral stricture development can be caused by indirect and direct trauma-like side effects of urethral slings for the urinary incontinence treatment and post transurethral prostate resection. The aim of the study was to establish a large animal model for the investigation of urethral stricture formation comparable to human stricture development. METHODS In 12 male Göttingen minipigs (mean body weight 20 kg) the stricture formation was evaluated with urethrography at 1, 8 and 12 weeks after stricture induction by ligation (L), urethrotomy (U), or thermocoagulation (T). The effect of urethral damage was investigated for the microvessel density (anti-von Willebrand factor antibody) and calculation of the collagen I/III ratio after western blot analysis. Normal human urethral tissue of 3 patients and stricture tissue of another 3 patients were compared to those of animal specimens. RESULTS Postoperatively all 12 animals developed a stricture in the injured area. The animal urethral stricture formation was characterized by the loss of tissue integrity in early phases of stricture development compared to normal tissue. However, over time an increase of connective tissue, a narrowing of urethral lumen, a significant decrease of microvessel density (p<0.001), and a significantly increased collagen I/III ratio (p<0.001) was noted. The three methods for stricture induction resulted in different severities of stricture formation in the animal model (T>L>U). In normal human urethral tissue a 1.9 fold higher expression of collagen type I compared to collagen type III was observed. In human stricture tissue samples the collagen type I/III ratio showed a mean ratio of 4.8:1. Microvessel density was significantly reduced in human urethral stricture tissue compared to normal human urethral tissue (p<0.001). CONCLUSIONS A large animal model to study urethral stricture formation after iatrogenic surgical interventions was successfully developed. The findings demonstrated a direct clinical context to human urethral strictures. The animal model enables investigations on new therapeutic approaches for urethral stricture therapy, e. g. based on tissue engineering. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e42 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jörg Seibold Tübingen, Germany More articles by this author Christin Selent-Stier Tübingen, Germany More articles by this author Julia Wiedemann Tübingen, Germany More articles by this author Tim-Oliver Greiner Tübingen, Germany More articles by this author Elisabeth Gustafsson Tübingen, Germany More articles by this author Brigitte Schuhmacher Tübingen, Germany More articles by this author Gerhard Feil Tübingen, Germany More articles by this author Arnulf Stenzl Tübingen, Germany More articles by this author Karl-Dietrich Sievert Tübingen, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Anti-tumor effects of a novel chimeric peptide on S180 and H22 xenografts bearing nude mice

In recent years, many endogenous peptides have been identified by screening combinatory phage display peptide library, which play important roles in the process of angiogenesis. A heptapeptide, ATWLPPR, binds specifically to NRP-1 and selectively inhibits VEGF165 binding to VEGFR-2. Another heptapeptide, NLLMAAS, blocks both Ang-1 and Ang-2 binding to Tie-2 in a dose-dependent manner. In the present study, we aimed to connect ATWLPPR ( V1) with NLLMAAS ( V2) via a flexible linker, Ala-Ala, to reconstruct a novel peptide ATWLPPRAANLLMAAS ( V3). We firstly investigated the anti-tumor and anti-angiogenic effects of peptide V3 on sarcoma S180 and hepatoma H22 bearing BALB/c nude mice. Mice were continuously subcutaneously administrated with normal saline, V1 (320 μg/kg/d), V2 (320 μg/kg/d), V1 + V2 (320 μg/kg/d), and V3 (160, 320 and 480 μg/kg/d), for 7 days. Treatment with peptide V3 could significantly reduce the tumor weight and volume. Pathological examination showed that the tumors treated with peptide V3 had a larger region of necrosis than that of peptide V1, V2, and V1 + V2 at the same dose. A significant decrease of microvessel density (MVD) in a dose-dependent manner was observed in each group of peptide V3. The results of pathological examination on normal tissue, lung, heart, liver, spleen, kidney and white blood cells showed that peptide V3 might have no significant toxicity. In conclusion, our results demonstrated that peptide V3 could be more effective on inhibiting tumor growth and angiogenesis than that of V1, V2, and V1 + V2. Peptide V3 could be considered as a novel chimeric peptide with potent anti-tumor activity.

Experimental Model of Ocular Hypertension in the Rat: Study of the Optic Nerve Capillaries and Action of Hypotensive Drugs

To investigate quantitatively the effect of elevated intraocular pressure (IOP) on the microvasculature of the optic nerve with and without topical treatment with two hypotensive drugs, timolol and latanoprost. Three groups of rats underwent cauterization of three episcleral veins to produce elevated IOP in the right eye. Two of these groups were treated with timolol or latanoprost for 3 months. Eyeballs were incubated with anti-GLUT-1 polyclonal antibody. GLUT-1-positive capillaries of the optic nerve head (ONH) and optic nerve exit (ON) were examined and analyzed for their number per square millimeter, volume fraction, length per unit volume, surface area per unit volume, and mean diameter. An increase in IOP resulted in a significant decrease in microvessel density in the laminar region (LR) and postlaminar region (PR) and ON compared with the control group. The other parameters fell significantly in all regions of the optic nerve. Topical treatment with timolol or latanoprost did not modify the density of the capillaries, although the other parameters increased significantly compared with the untreated experimental group. Additionally, the mean diameter of the capillaries in the LR and the PR recovered after treatment. The results indicate that the capillaries of the LR and the PR of the ONH are the most susceptible to IOP elevation. The authors suggest that timolol and latanoprost have a certain vascular action by increasing the available blood volume, surface area per unit volume, length per unit volume, and diameter of the capillaries of the ONH in these two regions.

TS‐1 enhances the effect of radiotherapy by suppressing radiation‐induced hypoxia‐inducible factor‐1 activation and inducing endothelial cell apoptosis

The therapeutic effect of concurrent chemoradiotherapy with TS-1 has been confirmed in various solid tumors; however, the detailed mechanism of action has not yet been fully elucidated. In the present study, we identified hypoxia-inducible factor-1 (HIF-1) as one of the targets of TS-1 in chemoradiotherapy. In growth delay assays using a tumor xenograft of non-small-cell lung carcinoma, H441, TS-1 treatment enhanced the therapeutic effect of single gamma-ray radiotherapy (14 Gy) and significantly delayed tumor growth by 1.58-fold compared to radiotherapy alone (P < 0.01). An optical in vivo imaging experiment using a HIF-1-dependent 5HRE-luc reporter gene revealed that TS-1 treatment suppressed radiation-induced activation of HIF-1 in the tumor xenografts. The suppression led to apoptosis of endothelial cells resulting in both a significant decrease in microvessel density (P < 0.05; vs radiation therapy alone) and a significant increase in apoptosis of tumor cells (P < 0.01; vs radiation therapy alone) in tumor xenografts. All of these results indicate that TS-1 enhances radiation-induced apoptosis of endothelial cells by suppressing HIF-1 activity, resulting in an increase in radiosensitivity of the tumor cells. Our findings strengthen the importance of both HIF-1 and its downstream gene, such as vascular endothelial cell growth factor, as therapeutic targets to enhance the effect of radiotherapy.

Vector‐based Ape1 small interfering RNA enhances the sensitivity of human osteosarcoma cells to endostatin in vivo

Osteosarcoma is a highly vascular and extremely destructive malignancy, and the survival of patients with osteosarcoma has not improved significantly in recent years. Antiangiogenic therapy currently holds great potential in conjunction with conventional treatment modalities for osteosarcoma. However, there are examples of gradual loss of response, and perhaps acquired resistance to antiangiogenic drugs. The acquired resistance of antiangiogenesis may be associated with a lot of hypoxia-response genes. The human apurinic/apyrimidinic endonuclease (Ape1) protein, a bifunctional redox factor and apurinic/apyrimidinic (AP) endonuclease, plays a crucial role in protecting against cell death due to hypoxia. We therefore hypothesized that Ape1 may contribute to the resistance of antiangiogenic therapy. To investigate the effect of Ape1 on the sensitivity of human osteosarcoma cells to endostatin, we constructed an Ape1 small interfering RNA expression vector, pSilenceApe1. Transfection of human osteosarcoma 9901 and HOS cells with pSilenceApe1 resulted in a dose-dependent loss of Ape1 protein. pSilenceApe1 also significantly suppressed the expression of vascular endothelial growth factor (VEGF) protein in the 9901 cells. Combined treatment with pSilenceApe1 and recombinant human endostatin (rhES) showed potent antiangiogenic effects in the transwell chamber invasion assay. Then, 20 nude mice bearing 9901 xenografts were divided into four groups: the phosphate-buffered saline treatment control group; the rhES treatment group (1.5 mg/kg, daily); the pSilenceApe1 treatment group (20 microg, once every 3 days); and the combination of rhES and pSilenceApe1 treatment group. pSilenceApe1 significantly suppressed the expression of Ape1 and VEGF protein in the 9901 xenografts. The tumor-inhibition rate of the pSilenceApe1, rhES, and combination of rhES and pSilenceApe1 treatment groups was 38.23, 35.29, and 62.18%, respectively. Furthermore, a significant decrease in microvessel density with an increase in apoptosis was observed following combined treatment with pSilenceApe1 and rhES, compared with control and either agent alone in 9901 xenografts. These results indicate that Ape1 small interfering RNA could enhance the sensitivity of osteosarcoma cells to endostatin.

Level of endothelial cell apoptosis required for a significant decrease in microvessel density

Endothelial cell apoptosis plays a critical role in the disruption of blood vessels mediated by natural inhibitors of angiogenesis and by anti-vascular drugs. However, the proportion of endothelial cells required to mediate a significant decrease in microvessel density is unknown. A system based on an inducible caspase (iCaspase-9) offers a unique opportunity to address this question. The dimerizer drug AP20187 induces apoptosis of human dermal microvascular endothelial cells stably transduced with iCaspase-9 (HDMEC–iCaspase-9), but not control cells (HDMEC–LXSN). Here, we generated blood vessels containing several HDMEC–iCaspase-9:HDMEC–LXSN ratios, and developed a mathematical modeling involving a system of differential equations to evaluate experimentally inaccessible ratios. A significant decrease in capillary sprouts was observed when at least 17% of the endothelial cells underwent apoptosis in vitro. Exposure to vascular endothelial growth factor (VEGF 165) did not prevent apoptosis of HDMEC–iCaspase-9, but increased the apoptotic requirement for sprout disruption. In vivo experiments showed the requirement of at least 22% apoptotic endothelial cells for a significant decrease in microvascular density. The combined use of biological experimentation with mathematical modeling allowed us to conclude that apoptosis of a relatively small proportion of endothelial cells is sufficient to mediate a significant decrease in microvessel density.

Pigment epithelium-derived factor overexpression inhibits orthotopic osteosarcoma growth, angiogenesis and metastasis

Despite significant improvements, the current management of primary osteosarcoma is still limited by the development of metastatic disease, which occurs in approximately 30% of patients despite aggressive multiagent chemotherapy and tumor-ablative surgery. Therefore, there is a need for the development of novel agents to improve the outcome of these patients. Pigment epithelium-derived factor (PEDF) has been shown to be one of the most potent inhibitors of angiogenesis, and more recently has demonstrated a functional role in tumor growth, invasion and metastasis. In this study we report, for the first time, the multitargeted role of PEDF in the inhibition of growth, angiogenesis and metastasis of two orthotopic models of osteosarcoma (rat UMR 106-01 and human SaOS-2). Through stable plasmid-mediated gene transfer of full-length human PEDF, we show that PEDF overexpression significantly reduced tumor cell proliferation (P<0.05) and Matrigel invasion (UMR(PEDF), P<0.001; SaOS(PEDF), P<0.05) and increased adhesion to collagen type-1 (P<0.01), in vitro. In vivo, PEDF overexpression dramatically suppressed orthotopic osteosarcoma growth (P<0.05) and the development of spontaneous pulmonary metastases (UMR(PEDF), P<0.05; SaOS(PEDF), P<0.001). Furthermore, PEDF-overexpressing tumors exhibited reduced intratumoral angiogenesis, evidenced by a significant decrease in microvessel density (P<0.05). Therefore, together these results suggest that PEDF may be a new and promising approach for the treatment of osteosarcoma.

Effects of radiotherapy and chemotherapy on angiogenesis and leukocyte infiltration in rectal cancer

We and others have shown that angiogenesis and leukocyte infiltration are important prognostic factors in rectal cancer. However, little is known about its possible changes in response to radiotherapy (RTX), which is frequently given to rectal tumors as a neoadjuvant treatment to improve the prognosis. We therefore investigated the biologic effects of RTX on these parameters using fresh-frozen biopsy samples of tumor and normal mucosa tissue before and after RTX. Biopsy samples were taken from a total of 34 patients before and after either a short course or long course of RTX combined with chemotherapy. The following parameters were analyzed by immunohistochemistry, flow cytometry, or quantitative real-time polymerase chain reaction: Microvessel density, leukocyte infiltration, proliferating epithelial and tumor cells, proliferating endothelial cells, adhesion molecule expression on endothelial cells, and the angiogenic mRNA profile. The tumor biopsy samples taken after RTX treatment demonstrated a significant decrease in microvessel density and the number of proliferating tumor cells and proliferating endothelial cells (p < 0.001). In contrast, the leukocyte infiltration, the levels of basic fibroblast growth factor in carcinoma tissue, and the adhesion molecule expression on endothelial cells in normal as well as carcinoma tissue increased significantly (p < 0.05). Our data show that together with an overall decrease in tumor cell and endothelial cell proliferation, RTX results in an increase in the expression of adhesion molecules that stimulate leukocyte infiltration. This suggests the possibility that, in addition to its direct cytotoxic effect, radiation may also stimulate an immunologic tumor response that could contribute to the documented improvement in local tumor control and distal failure rate of rectal cancers.