Decreased Vessel Density Research Articles

Adipose Stromal Cell Contact with Endothelial Cells Results in Loss of Complementary Vasculogenic Activity Mediated by Induction of Activin A.

Adipose stem/stromal cells (ASCs) after isolation produce numerous angiogenic growth factors. This justifies their use to promote angiogenesis per transplantation. In parallel, local coimplantation of ASC with endothelial cells (ECs) leading to formation of functional vessels by the donor cells suggests the existence of a mechanism responsible for fine-tuning ASC paracrine activity essential for vasculogenesis. As expected, conditioned media (CM) from ASC promoted ECs survival, proliferation, migration, and vasculogenesis. In contrast, media from EC-ASC cocultures had neutral effects upon EC responses. Media from cocultures exhibited lower levels of vascular endothelial growth factor (VEGF), hepatic growth factor, angiopoietin-1, and stromal cell-derived factor-1 compared with those in ASC CM. Activin A was induced in ASC in response to EC exposure and was responsible for overall antivasculogenic activity of EC-ASC CM. Except for VEGF, activin A diminished secretion of all tested factors by ASC. Activin A mediated induction of VEGF expression in ASC, but also upregulated expression of VEGF scavenger receptor FLT-1 in EC in EC-ASC cocultures. Blocking the FLT-1 expression in EC led to an increase in VEGF concentration in CM. In vitro pre-exposure of ASC to low number of EC before subcutaneous coimplantation with EC resulted in decrease in vessel density in the implants. In vitro tests suggested that activin A was partially responsible for this diminished ASC activity. This study shows that neovessel formation is associated with induction of activin A expression in ASC; this factor, by affecting the bioactivity of both ASC and EC, directs the crosstalk between these complementary cell types to establish stable vessels.

S100A4-neutralizing antibody suppresses spontaneous tumor progression, pre-metastatic niche formation and alters T-cell polarization balance.

BackgroundThe tumor microenvironment plays a determinative role in stimulating tumor progression and metastasis. Notably, tumor-stroma signals affect the pattern of infiltrated immune cells and the profile of tumor-released cytokines. Among the known molecules that are engaged in stimulating the metastatic spread of tumor cells is the S100A4 protein. S100A4 is known as an inducer of inflammatory processes and has been shown to attract T-cells to the primary tumor and to the pre-metastatic niche. The present study aims to examine the immunomodulatory role of S100A4 in vivo and in vitro and assess the mode of action of 6B12, a S100A4 neutralizing antibody.MethodsThe therapeutic effect of the 6B12 antibody was evaluated in two different mouse models. First, in a model of spontaneous breast cancer we assessed the dynamics of tumor growth and metastasis. Second, in a model of metastatic niche formation we determined the expression of metastatic niche markers. The levels of cytokine expression were assessed using antibody as well as PCR arrays and the results confirmed by qRT-PCR and ELISA. T-cell phenotyping and in vitro differentiation analyses were performed by flow cytometry.ResultsWe show that the S100A4 protein alters the expression of transcription factor and signal transduction pathway genes involved in the T-cell lineage differentiation. T-cells challenged with S100A4 demonstrated reduced proportion of Th1-polarized cells shifting the Th1/Th2 balance towards the Th2 pro-tumorigenic phenotype. The 6B12 antibody restored the Th1/Th2 balance. Furthermore, we provide evidence that the 6B12 antibody deploys its anti-metastatic effect, by suppressing the attraction of T-cells to the site of primary tumor and pre-metastatic niche. This was associated with delayed primary tumor growth, decreased vessel density and inhibition of metastases.ConclusionThe S100A4 blocking antibody (6B12) reduces tumor growth and metastasis in a model of spontaneous breast cancer. The 6B12 antibody treatment inhibits T cell accumulation at the primary and pre-metastatic tumor sites. The 6B12 antibody acts as an immunomodulatory agent and thus supports the view that the 6B12 antibody is a promising therapeutic candidate to fight cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1034-2) contains supplementary material, which is available to authorized users.

Abstract A01: Pharmacological approaches to metastatic disease using an Inflammatory Breast Cancer model

Abstract Goal of Study: The lymphovascular embolus is a marker of breast cancer aggressiveness, recurrence, metastatic progression and therapeutic failure. This is especially reflective in inflammatory breast cancer (IBC) which presents as florid intravasation in situ of the lymphovasculature by tumor emboli. This study utilizes a novel 3-dimensional (3D) in vitro and in vivo model of IBC to systematically and strategically develop pharmacological approaches in the treatment of both IBC and metastatic disease. Background: Intravasation, the rate-limiting step of metastasis, is extremely important in tumor progression and expansion, yet the mechanism underlying intravasation is still poorly understood. The consequence of intravasation is the resultant lymphovascular embolus – the key defining feature of metastasis. This is most prevalent in IBC, the most lethal form of breast cancer. Clinical IBC features, erythema, peau d'orange (dimpling of thickened skin) and warmth are due to the IBC signature phenotype, namely extensive intravasation in situ of the lymphatic and blood vessels by tumor emboli. Nearly 100% of all women with IBC have lymph node involvement and approximately 25% of patients with IBC have distant metastases at diagnosis. The IBC xenograft model, MARY-X, has precisely captured the IBC phenotype, where the tumor emboli grow exclusively within the murine lymphatic and blood vessels and exhibit erythema of the overlying skin. MARY-X is an invaluable preclinical IBC model and an overall model of metastasis providing a means to validate and translate therapeutic strategies. MARY-X, in vitro, is a cellular derivative of primary tumor explants. These tumor cells form tight, compact aggregates of cells termed “MARY-X spheroids”. Comparable to human IBC emboli, a persistent, over-expression of an intact E-cadherin/α, β-catenin axis mediates the compaction of both in vitro and in vivo MARY-X spheroids and tumor emboli, respectively. These comparative architectural features (i.e. mimics the in vivo metastasis) of the spheroid of MARY-X provide an in vitro model with tractable in vivo applications. Research Strategy: By exploiting the MARY-X in vitro 3-D model, high throughput (HTP) screening is performed to systematically probe the factors involved in maintenance of tumor emboli architecture as it pertains to sensitivity/resistance to therapeutics in an overall effort to develop efficacious therapeutics for treatment of IBC and metastatic disease. Additionally, MARY-X spheroids co-cultured with bone marrow derived cells (BMDCs) in matrigel overlays are employed to assess targeting of mediators of crosstalk between tumor spheroid/embolus soluble factors (e.g. cytokines, exosomes) and the microenvironment. Soluble factors identified as mediators (i.e. targets) of metastatic progression, that showed the most promise in the HTP screen, are further validated in the in vivo preclinical model, MARY-X. A significant decrease in vessel density of the MARY-X tumor will be indicative of successful blocking of this ‘crosstalk’ i.e. lymphovasculogenesis or the formation of lymphovascular emboli (i.e. intravasation) – the defining feature of metastatic disease. Preliminary Data and Conclusions: Preliminary data show that dissolution of the spheroid/embolus augment apoptotic effects of chemo/irradiation therapy. Suggesting that apoptosis resistance of lymphovascular emboli may be independent of traditional apoptotic mechanisms. Therefore, therapy evasion, in part, is due to formation and maintenance of the architectural features of the lymphovascular embolus. In addition, abrogation of ‘crosstalk’ has proven to hinder the establishment of lymphovascular emboli. Citation Format: Mary L. Alpaugh, Emmanuel Theodorakis. Pharmacological approaches to metastatic disease using an Inflammatory Breast Cancer model. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A01.

Abstract 4492: Blockade of angiopoietin-2 or Tie2 is equally effective at inhibiting tumor growth and reducing tumor vessel density in most human tumor xenograft models

Abstract Angiopoietin-1 (Ang1) and -2 (Ang2) regulate angiogenesis via the endothelial cell-specific receptor tyrosine kinase Tie2. Blocking Ang2 binding to Tie2 decreases vessel density and inhibits tumor growth in various human xenograft models. However, not all tumors respond to Ang2 blockade, which could be due to high Ang1 levels acting as another ligand for Tie2, to Ang2 activities independent of Tie2, or to a weak role for Ang-2/Tie2 signaling in these tumors. To elucidate if Ang1 plays an integral role in tumor angiogenesis and growth, we compared the effects of blocking Ang2 or Tie2 in various tumor xenografts with differing levels of Ang1 expression. We found that in six out of seven tumor models, antibodies that specifically bind Ang2 (REGN910, 10 mg/kg 2x/wk) or that bind Tie2 and block binding of both Ang1 and Ang2 (REGN1376, 10 mg/kg 2x/wk) were equally efficacious at inhibiting tumor growth and decreasing vessel density. In only one tumor model (Calu-6), blockade of Tie2 with REGN1376 was more effective than blockade of Ang2 (REGN910) at reducing tumor growth and decreasing tumor vessel density. Although Calu-6 tumors express high Ang1 levels, other tumors tested (Lox, LS174T) have comparable or higher Ang1 levels and respond similarly to Ang2 and Tie2 blockade, thus the levels of Ang1 do not account for the differential response of Calu-6 tumors. Taken together, our data suggest that Ang2 is the dominant ligand for Tie2 in the microenvironment of most tumors, and further, that the effects of Ang2 are mediated primarily via Tie2. These findings support the approach to specifically block the binding of Ang2 to Tie2 as an anti-angiogenic therapy. Citation Format: Alexander P. Adler, Christopher Daly, Asma A. Parveen, Thomas Nevins, Jing Shan, Jeanette Fairhurst, Tammy Huang, Joel Martin, Nicholas Papadopoulos, George D. Yancopoulos, Gavin Thurston, Gavin Thurston, Alexandra Eichten. Blockade of angiopoietin-2 or Tie2 is equally effective at inhibiting tumor growth and reducing tumor vessel density in most human tumor xenograft models. [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 4492. doi:10.1158/1538-7445.AM2014-4492

Abstract 3945: Inhibition of MMP2 expression enhances the efficacy of radiation therapy for a murine astrocytoma

Abstract Radiation therapy (RT) remains the front-line treatment for high-grade gliomas; however, tumor recurrence remains the main obstacle for the clinical success of RT. Using a murine astrocytoma tumor cell line, ALTS1C1, we have previously shown that RT can effectively reduce primary tumor volume and increase mice surviving time, but the mice eventually died associated with increased tumor infiltration. In this study, we explore the role of MMP2 (Matrix metalloproteinase-2), a reported molecule associated with brain tumor invasion, on ALTS1C1 tumor response to RT. In this study, the MMP2 expression was inhibited by specific lentiviral-shRNA construct. Eight Gray of whole brain irradiation could only prolong the median survival days of brain tumor-bearing mice from 25 days to 28 days, but the inhibition of MMP2 expression by siRNA prolonged the median survival days of tumor bearing mice from 25 days to 42 days. The median survival days were further prolonged to 53 days when 8 Gy of radiation and MMP2 inhibition were concurrently applied. Immunohistochemical (IHC) results showed that the synergistic effect as a result of RT-caused vessel density decrease (inadequate vasculature) and the retardation of vessel maturation as a result of MMP2 inhibition-caused the decrease of pericyte-covered vessels (insufficient vessel function). This study provides a new treatment option for treating invasive brain tumors. (This study was supported by NHRI-EX103-10132BI and NSC 102-2314-B-007-003-MY3 grants) Citation Format: Ching-Fang Yu, Ying-Chieh Yang, Ji-Hong Hong, Chi-Shiun Chiang. Inhibition of MMP2 expression enhances the efficacy of radiation therapy for a murine astrocytoma. [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 3945. doi:10.1158/1538-7445.AM2014-3945

Alterations in the expression of vascular endothelial growth factor in the rat brain following gamma knife surgery.

Gamma knife surgery (GKS) is used for the treatment of various brain diseases. However, the mechanisms underlying brain injury following irradiation remain to be elucidated. Given that vascular endothelial growth factor (VEGF) is closely associated with pathological angiogenesis and the permeability of the blood brain barrier (BBB), the present study was designed to analyze temporal alterations in VEGF expression in the cerebral cortex and the effect of VEGF on cerebral edema in rats following GKS. Adult male Wistar rats were subjected to GKS at maximum doses of 60 Gy. Animals were sacrificed between 4 and 24 weeks after GKS. Immunohistochemistry, enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction (RT-PCR) were employed for detecting VEGF expression. The vessel density was measured by CD31+ cell count and vascular structures were examined using electron microscopy. Brain water content and BBB permeability were measured in the present study. VEGF expression in the irradiated cortex progressively increased until 16 weeks after GKS when the maximal expression was reached, and then gradually decreased to the control level 24 weeks after GKS. These findings were confirmed by RT-PCR. A mild decrease in vessel density was observed 4 weeks after GKS, followed by an increase in vessel density between 8 and 20 weeks later. Furthermore, previous studies also demonstrated vascular damage, opening of the BBB and an increase in brain water content occurring simultaneously. To the best of our knowledge, these data demonstrated for the first time dynamic changes in VEGF expression following GKS and also suggest the importance of VEGF expression in pathological angiogenesis and edema formation following GKS.

Ecophysiological implications of vascular differentiation and plant evolution

Key message Environmental cues regulate plant vascular differentiation and plant evolution through simple hor- monal mechanisms of a single or a few moving signals. Abstract Mechanisms regulating the responses of plants and their vascular tissues to environmental stimuli are mediated by continuously moving hormonal signals that enable continuous response to ecological cues. Auxin from young leaves is the primary hormonal signal that can induce vascular differentiation by itself. Its concentrations determine whether phloem or xylem is induced. Auxin produced in a parasitic plant induces continuous vessel system into its host with open perforation at their junction. Polar auxin gradients along trees regulate the gradual widening of vessel diameter and decrease in vessel density from leaves to roots. This basic mechanism also regulates vascular adaptation to the plant's environment. Gibberellin from mature leaves, in the presence of auxin, promotes cambial activity and woodiness, and is the specific signal inducing fibers. The evolutionary development of vessels and fibers from tracheids reflects their hormonal special- ization; from the combined mechanism of auxin and gib- berellin for tracheids in gymnosperms, to the specialized mechanisms of auxin inducing vessels, and gibberellin inducing fibers in angiosperms. Cytokinin from root tips promotes cambial activity and sensitivity enabling the extreme differentiation of ring-porous wood in temperate deciduous hardwood trees. These mechanisms are discussed for clarifying the role of the environment in vascular adaption and evolution.

Therapeutic effects of artesunate in hepatocellular carcinoma: repurposing an ancient antimalarial agent.

Artemisinins are antimalarial drugs that exert potent anticancer activity. We evaluated the effects of artesunate, a semisynthetic derivative of artemisinin, on tumor growth, angiogenesis, the unfolded protein response, and chemoresistance in hepatocellular carcinoma. The effect of artesunate was examined in HepG2 and BWTG3 cells under normoxic and hypoxic conditions and in a diethylnitrosamine-induced mouse model. Histology was performed with hematoxylin/eosin and reticulin staining. The expression of chemoresistance-related transporters and angiogenic and unfolded protein response factors was determined. Cytotoxicity was assessed by alanine and aspartate transaminase, lactate dehydrogenase, water-soluble tetrazolium salt, and caspase-3 activity assays. Small animal imaging was performed using dynamic contrast-enhanced MRI and choline PET to assess tumor progression. Artesunate dose dependently reduced cell viability (from 50 μmol/l; P<0.05) and increased caspase-3 activity (P<0.05) in HepG2 and BWTG3 cells. These effects were enhanced by hypoxia (from 12.5 μmol/l; P<0.01). Moreover, artesunate downregulated vascular endothelial growth factor and placental growth factor expression in vitro (both P<0.05) and in vivo (both P<0.01). In mice, artesunate decreased vessel density and tumor burden (both P<0.05). These in-vivo effects were enhanced by combination with sorafenib (P<0.05 and P=0.07, respectively), without apparent hepatotoxicity. Furthermore, artesunate modulated the unfolded protein response in vitro and in vivo, increasing proapoptotic signaling, and did not induce doxorubicin chemoresistance. These findings indicate that artesunate could offer a new approach to the therapy of hepatocellular carcinoma. Clinical trials with artesunate as monotherapy or in combination with current hypoxia-inducing approaches are necessary.

Disrupted pulmonary artery cyclic guanosine monophosphate signaling in mice with hyperoxia-induced pulmonary hypertension.

Pulmonary hypertension (PH) occurs in 25 to 35% of premature infants with significant bronchopulmonary dysplasia (BPD). Neonatal mice exposed to 14 days of hyperoxia develop BPD-like lung injury and PH. To determinne the impact of hyperoxia on pulmonary artery (PA) cyclic guanosine monophosphate (cGMP) signaling in a murine model of lung injury and PH, neonatal C57BL/6 mice were placed in room air, 75% O2 for 14 days (chronic hyperoxia [CH]) or 75% O2 for 24 hours, followed by 13 days of room air (acute hyperoxia with recovery [AHR]) with or without sildenafil. At 14 days, mean alveolar area, PA medial wall thickness (MWT), right ventricular hypertrophy (RVH), and vessel density were assessed. PA protein was analyzed for cGMP, soluble guanylate cyclase, and PDE5 activity. CH and AHR mice had RVH, but only CH mice had increased alveolar area and MWT and decreased vessel density. In CH and AHR PAs, soluble guanylate cyclase activity was decreased, and PDE5 activity was increased. In CH mice, sildenafil attenuated MWT and RVH but did not improve mean alveolar area or vessel density. In CH and AHR PAs, sildenafil decreased PDE5 activity and increased cGMP. Our results indicate that prolonged hyperoxia leads to lung injury, PH, RVH, and disrupted PA cGMP signaling. Furthermore, 24 hours of hyperoxia causes RVH and disrupted PA cGMP signaling that persists for 13 days. Sildenafil reduced RVH and restored vascular cGMP signaling but did not attenuate lung injury. Thus, hyperoxia can rapidly disrupt PA cGMP signaling in vivo with sustained effects, and concurrent sildenafil therapy can be protective.

Contribution of ADAMTS1 as a tumor suppressor gene in human breast carcinoma. Linking its tumor inhibitory properties to its proteolytic activity on nidogen-1 and nidogen-2.

The extracellular protease ADAMTS1 (A disintegrin and metalloprotease with thrombospondin repeats 1) has been described as an anti-angiogenic molecule and its role as a putative tumor protective molecule has also been suggested. Here, we have used a tumor xenograft model to determine the role of ADAMTS1 in tumor growth and angiogenesis. Increasing levels of the protease led to the complete inhibition of tumor growth. In an attempt to elucidate the mechanism of action of this protease, we focused our attention on its proteolytic activity on nidogens, one of the main components of the vascular basement membrane. The increased expression of ADAMTS1 was accompanied by increased proteolysis of nidogen-1 and -2 and their almost complete removal from vascular structures, together with major morphological alterations of tumor blood vessels and a decreased vessel density. The clinical relevance of this work is supported by our observations that ADAMTS1 expression is decreased in breast tumor specimens when compared with healthy tissue. Our studies also reveal that the cleavage of nidogen-1 and -2 is partially inhibited in human tumor samples. Moreover, the deposition of both nidogens surrounding vascular structures is drastically altered, implying a possible reduction in the maintenance of vessel integrity. Our studies reflect the requirement to explore the functional interactions between proteases and specific substrates in cancer biology.

Synchrotron microbeam radiation therapy induces hypoxia in intracerebral gliosarcoma but not in the normal brain

PurposeSynchrotron microbeam radiation therapy (MRT) is an innovative irradiation modality based on spatial fractionation of a high-dose X-ray beam into lattices of microbeams. The increase in lifespan of brain tumor-bearing rats is associated with vascular damage but the physiological consequences of MRT on blood vessels have not been described. In this manuscript, we evaluate the oxygenation changes induced by MRT in an intracerebral 9L gliosarcoma model. MethodsTissue responses to MRT (two orthogonal arrays (2×400Gy)) were studied using magnetic resonance-based measurements of local blood oxygen saturation (MR_SO2) and quantitative immunohistology of RECA-1, Type-IV collagen and GLUT-1, marker of hypoxia. ResultsIn tumors, MR_SO2 decreased by a factor of 2 in tumor between day 8 and day 45 after MRT. This correlated with tumor vascular remodeling, i.e. decrease in vessel density, increases in half-vessel distances (×5) and GLUT-1 immunoreactivity. Conversely, MRT did not change normal brain MR_SO2, although vessel inter-distances increased slightly. ConclusionWe provide new evidence for the differential effect of MRT on tumor vasculature, an effect that leads to tumor hypoxia. As hypothesized formerly, the vasculature of the normal brain exposed to MRT remains sufficiently perfused to prevent any hypoxia.

Abstract 5092: Ziv-aflibercept treatment reduces intra-hepatic tumor growth of a preclinical colorectal carcinoma liver metastasis model, monitored using non-invasive micro-ultrasound and bioluminescent imaging.

Abstract The liver is a frequent metastatic site for different types of cancers, including colorectal carcinomas (CRC). Therapies that inhibit the angiogenic growth factor VEGF, such as bevacizumab and ziv-aflibercept, are used in combination with chemotherapies to treat metastatic CRC. Ziv-aflibercept (also known as VEGF Trap) is being developed as a part of a collaboration between Sanofi and Regeneron Pharmaceuticals, Inc. and was recently approved by the FDA in metastatic colorectal cancer that is resistant to or has progressed following an oxaliplatin-containing regimen. To better understand the role of VEGF in metastatic CRC, we aimed to assess the effects of ziv-aflibercept on intrahepatic CRC tumor growth, invasion and angiogenesis in a preclinical model. For that we developed and characterized a CRC liver metastasis model that would allow us to longitudinally assess the effects of ziv-aflibercept on CRC growth in the liver parenchyma using non-invasive high frequency micro-ultrasound and bioluminescent imaging modalities. Briefly, luciferase-expressing HCT116 (human colorectal carcinoma) tumor cells were implanted into the left liver lobe of male CB17 SCID mice using image-guided micro-ultrasound. Tumor growth was monitored using both micro-ultrasound and bioluminescent imaging. When tumors were established (approximately 70 mm3), mice were treated with either control protein (hFc, 25 mg/kg, 2x/wk) or ziv-aflibercept (25 mg/kg, 2x/wk) for up to 13 days. Ziv-aflibercept treatment inhibited tumor growth by 95% compared to control treatment. This inhibition of tumor growth was accompanied by significantly decreased cell proliferation (50-70%), as measured by Ki67 immunohistochemistry, and markedly decreased vessel density (approximately 50%), as revealed by CD31 immunohistochemistry. Ziv-aflibercept did not seem to increase tumor invasiveness, as H&amp;E histology did not reveal dramatic differences at the tumor/stroma border between the ziv-aflibercept and control treated tumors. These experiments confirm that ziv-aflibercept reduces growth and vascularity of colorectal tumors growing in the liver in a preclinical model. Citation Format: Alexander P. Adler, Asma Parveen, Gavin Thurston, Alexandra Eichten. Ziv-aflibercept treatment reduces intra-hepatic tumor growth of a preclinical colorectal carcinoma liver metastasis model, monitored using non-invasive micro-ultrasound and bioluminescent imaging. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5092. doi:10.1158/1538-7445.AM2013-5092

HDAC7 mediated activation of STAT3 in human PTEN deficient glioma cells suppresses angiogenesis and tumor growth in vivo

Phosphatase and tensin homolog (PTEN) is a key tumor suppressor whose loss is commonly found in glioblastoma multiforme (GBM). In GBM, loss of PTEN leads to inactivation of signal transducer and activator of transcription 3 (STAT3). In contrast to the pro‐oncogenic function of STAT3 in other tumors, STAT3 has emerged as a key tumor suppressor in PTEN−/− GBM.We report for the first time a histone deacetylase 7 (HDAC7) mediated mechanism of STAT3 activation in PTEN−/− GBM. SiRNA induced depletion of HDAC7 in human U87 glioma cells triggered (both in vitro and in vivo) phosphorylation of STAT3 on Y705. In accordance to our previous data, we show that this event is dependent of A‐kinase anchor protein 12 (AKAP12), which is rapidly induced upon HDAC7 silencing. In vivo, siRNA mediated HDAC7 repression abrogated tumor growth in U87‐glioma chick‐chorioallantoic membrane animal model. This effect was reversible when AKAP12 was co‐supressed. Intrigued by the macroscopical appearance of U87 tumors upon HDAC7 silencing, exhibiting small, pale nodules with prominent hemorrhage, we verified if defects in angiogenesis may explain the lack of tumor growth. Indeed, lectin‐based staining of the tumor vasculature revealed a significantly decreased vessel density. We demonstrate that several angiogenesis relevant genes (e.g. angiopoietin 1, angiogenin, interleukin 8) were significantly down‐regulated at transcriptional level.The present study highlights a STAT3 dependent pathway for suppressing the growth PTEN−/− GBM with potential for clinical translation.This work is sponsored by FNRS, Belgium.

Anti‐angiogenic and anti‐tumor effects of TAK‐593, a potent and selective inhibitor of vascular endothelial growth factor and platelet‐derived growth factor receptor tyrosine kinase

We recently reported that TAK-593, a novel imidazo[1,2-b]pyridazine derivative, is a highly potent and selective inhibitor of the vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptor tyrosine kinase families. Moreover, TAK-593 exhibits a uniquely long-acting inhibitory profile towards VEGF receptor 2 (VEGFR2) and PDGF receptor β (PDGFRβ). In this study, we demonstrated that TAK-593 potently inhibits VEGF- and PDGF-stimulated cellular phosphorylation and proliferation of human umbilical vein endothelial cells and human coronary artery smooth muscle cells. TAK-593 also potently inhibits VEGF-induced tube formation of endothelial cells co-cultured with fibroblasts. Oral administration of TAK-593 exhibited strong anti-tumor effects against various human cancer xenografts along with good tolerability despite a low level of plasma exposure. Even after the blood and tissue concentrations of TAK-593 decreased below the detectable limit, a pharmacodynamic marker (phospho VEGFR2) was almost completely suppressed, indicating that its long duration of enzyme inhibition might contribute to the potent activity of TAK-593. Immunohistochemical staining indicated that TAK-593 showed anti-proliferative and pro-apoptotic effects on tumors along with a decrease of vessel density and inhibition of pericyte recruitment to microvessels in vivo. Furthermore, dynamic contrast-enhanced magnetic resonance imaging revealed that TAK-593 reduced tumor vessel permeability prior to the onset of anti-tumor activity. In conclusion, TAK-593 is an extremely potent VEGFR/PDGFR kinase inhibitor whose potent anti-angiogenic activity suggests therapeutic potential for the treatment of solid tumors.

Diverse Responses to Vascular Disrupting Agent Combretastatin A4 Phosphate: A Comparative Study in Rats with Hepatic and Subcutaneous Tumor Allografts Using MRI Biomarkers, Microangiography, and Histopathology

OBJECTIVE: Differently located tumors of the same origin may exhibit diverse responses to the same therapeutics. To test this hypothesis, we compared the responses of rodent hepatic and subcutaneous engrafts of rhabdomyosarcoma-1 (R1) to a vascular disrupting agent Combretastatin A4 phosphate (CA4P). METHODS: Twelve WAG/Rij rats, each bearing three R1 implanted in the right and left hepatic lobes and subcutaneously in the thoracic region, received CA4P intravenously at 5 mg/kg (n = 6) or solvent (n = 6). Therapeutic responses were compared interindividually and intraindividually among tumors of different sites till 48 hours after injection using in vivo MRI, postmortem digital microangiography, and histopathology. RESULTS: MRI revealed that the subcutaneous tumors (STs) significantly increased in volume than hepatic tumors (HTs) 48 hours after CA4P (P < .05). Relative to vehicle controls and treated group at baseline, necrosis ratio, apparent diffusion coefficient, and enhancement ratio changed slightly with the STs but significantly with HTs (P < .05) after CA4P treatment. Vessel density derived from microangiography was significantly lower in STs compared to HTs without CA4P treatment. CA4P treatment resulted in decreased vessel density in HTs, while it did not affect vessel density in STs. MRI and microangiography outcomes were supported by histopathologic findings. CONCLUSIONS: MRI and microangiography allowed quantitative comparison of therapeutic responses to CA4P in rats with multifocal tumors. The discovered diverse effects of the same drug on tumors of the same origin but different locations emphasize the presence of cancer heterogeneity and the importance of individualization of drug delivery.

Image-based 3D modeling study of the influence of vessel density and blood hemoglobin concentration on tumor oxygenation and response to irradiation

Hypoxia is one of the most important factors influencing clinical outcome after radiotherapy. Improved knowledge of factors affecting the levels and distribution of oxygen within a tumor is needed. The authors constructed a theoretical 3D model based on histological images to analyze the influence of vessel density and hemoglobin (Hb) concentration on the response to irradiation. The pancreases of a Rip-Tag2 mouse, a model of malignant insulinoma, were excised, cryosectioned, immunostained, and photographed. Vessels were identified by image thresholding and a 3D vessel matrix assembled. The matrix was reduced to functional vessel segments and enlarged by replication. The steady-state oxygen tension field of the tumor was calculated by iteratively employing Green's function method for diffusion and the Michaelis-Menten model for consumption. The impact of vessel density on the radiation response was studied by removing a number of randomly selected vessels. The impact of Hb concentration was studied by independently changing vessel oxygen partial pressure (pO(2)). For each oxygen distribution, the oxygen enhancement ratio (OER) was calculated and the mean absorbed dose at which the tumor control probability (TCP) was 0.99 (D(99)) was determined using the linear-quadratic cell survival model (LQ model). Decreased pO(2) shifted the oxygen distribution to lower values, whereas decreased vessel density caused the distribution to widen and shift to lower values. Combined scenarios caused lower-shifted distributions, emphasising log-normal characteristics. Vessel reduction combined with increased blood pO(2) caused the distribution to widen due to a lack of vessels. The most pronounced radiation effect of increased pO(2) occurred with tumor tissue with 50% of the maximum vessel density used in the simulations. A 51% decrease in D(99), from 123 to 60 Gy, was found between the lowest and highest pO(2) concentrations. Our results indicate that an intermediate vascular density region exists where enhanced blood oxygen concentration may be beneficial for radiation response. The results also suggest that it is possible to distinguish between diffusion-limited and anemic hypoxia from the characteristics of the pO(2) distribution.

Xylem Adjustment in Erica Arborea to Temperature and Moisture Availability in Contrasting Climates

Plasticity of xylem architecture can be a species specific strategy to reduce vulnerability to climate change. To study how the evergreen shrub Erica arborea regulates its xylem at different time scales as a response to climatic variability, we compared time series of annual xylem traits such as ring-width (growth), vessel size (hydraulic diameter and mean vessel area), vessel density, and potential conductivity (Kh) at two sites characterized by contrasting Mediterranean climates in Italy and Spain. Shrubs regulated their xylem in response to major differences in climate by modifying mostly their growth and vessel density. The different adjustment of xylem observed at the two sites was partly explained by the nonlinear nature of the relationship between the studied traits and temperature. Xylem development was mostly limited by low winter temperature at the cold and moist site, where plants produced more vessels per unit area of xylem and reduced growth in comparison with the warm and dry site. The responses of vessel size and density to climate were opposite. Vessel size and Kh were similar at the two sites and exhibited less sensitivity to climate than vessel density and growth. Humid conditions in spring increased growth and vessel size but decreased vessel density at the cold site, whereas the effect on xylem adjustment of high temperatures during the vegetation period was generally contrary to that of high moisture availability. These results likely express within species adaptations of the hydraulic function and the safety-efficiency trade-off in response to climate. A decline in growth in response to a recent decrease in precipitation at the dry site could be interpreted as a first sign of vulnerability to increasing drought severity. Similar to other species, climate change may have contrasting effects for E. arborea at its cold and dry distributional limits.

A Functional Requirement for Astroglia in Promoting Blood Vessel Development in the Early Postnatal Brain

Astroglia are a major cell type in the brain and play a key role in many aspects of brain development and function. In the adult brain, astrocytes are known to intimately ensheath blood vessels and actively coordinate local neural activity and blood flow. During development of the neural retina, blood vessel growth follows a meshwork of astrocytic processes. Several genes have also been implicated in retinal astrocytes for regulating vessel development. This suggests a role of astrocytes in promoting angiogenesis throughout the central nervous system. To determine the roles that astrocytes may play during brain angiogenesis, we employ genetic approaches to inhibit astrogliogenesis during perinatal corticogenesis and examine its effects on brain vessel development. We find that conditional deletion from glial progenitors of orc3, a gene required for DNA replication, dramatically reduces glial progenitor cell number in the subventricular zone and astrocytes in the early postnatal cerebral cortex. This, in turn, results in severe reductions in both the density and branching frequency of cortical blood vessels. Consistent with a delayed growth but not regression of vessels, we find neither significant net decreases in vessel density between different stages after normalizing for cortical expansion nor obvious apoptosis of endothelial cells in these mutants. Furthermore, concomitant with loss of astroglial interactions, we find increased endothelial cell proliferation, enlarged vessel luminal size as well as enhanced cytoskeletal gene expression in pericytes, which suggests compensatory changes in vascular cells. Lastly, we find that blood vessel morphology in mutant cortices recovers substantially at later stages, following astrogliosis. These results thus implicate a functional requirement for astroglia in promoting blood vessel growth during brain development.

Abstract 101: Endothelial-Specific Fibroblast Growth Factor Receptor 1 and 2 Deletion Impairs Vascular Remodeling and Recovery in an In Vivo Closed-Chest Model of Cardiac Ischemia-Reperfusion Injury

Introduction: Fibroblast growth factor (FGF) signaling is cardioprotective in various models of myocardial infarction. FGF receptors (FGFRs) are expressed in multiple cell types in the adult heart, but the cell type-specific FGFR signaling which mediates different cardioprotective endpoints is currently unknown. Hypothesis: We hypothesize that endothelial FGFR signaling is essential for vascular remodeling after cardiac ischemia-reperfusion (IR) injury. Methods: We have generated mice with a conditional ablation of the Fgfr1 and Fgfr2 genes in endothelial cells ( Tie2-Cre, Fgfr1 f/f , Fgfr2 f/f ). These mice and littermate controls were subjected to a clinically relevant, in vivo , closed-chest model of cardiac IR injury which includes 90 minutes of occlusion of the left anterior descending artery followed by reperfusion for 7 days. Echocardiography was performed at baseline, day 1, and day 7 of reperfusion, and histological staining was performed on day 7. Results: Tie2-Cre, Fgfr1 f/f , Fgfr2 f/f mice had no baseline abnormalities in cardiac morphometry or function. When subjected to closed-chest, regional cardiac IR injury, Tie2-Cre, Fgfr1 f/f , Fgfr2 f/f mice showed significantly increased infarct size compared to controls at 7 days (37±3% vs. 15±5%, p&lt;0.05) but not 1 day after reperfusion. Tie2-Cre, Fgfr1 f/f , Fgfr2 f/f mice also showed significantly worsened cardiac function (ejection fraction, stroke volume, and fractional shortening) compared to controls at 7 days (p&lt;0.05) but not 1 day after reperfusion. Myocyte cross-sectional area measurement showed no impairment in the cardiac hypertrophic response in the Tie2-Cre, Fgfr1 f/f , Fgfr2 f/f mice. Tie2-Cre, Fgfr1 f/f , Fgfr2 f/f mice have normal vessel density in the non-injured state, but after cardiac IR injury, Tie2-Cre, Fgfr1 f/f , Fgfr2 f/f hearts showed significantly decreased vessel density (both capillaries and smooth muscle actin containing vessels, p&lt;0.05) compared to controls in the peri-infarct area. Conclusions: Ablation of FGFR1 and FGFR2 in endothelial cells results in impaired vascular remodeling, worsened cardiac functional recovery, and increased infarct size without affecting the cardiac hypertrophic response in an in vivo , closed-chest model of cardiac IR injury.

Upregulation of SATB1 is associated with the development and progression of glioma

BackgroundSpecial AT-rich sequence-binding protein-1 (SATB1) has been reported to be expressed in several human cancers and may have malignant potential. This study was aimed at investigating the expression and potential role of SATB1 in human glioma.MethodThe relationship between SATB1 expression, clinicopathological parameters, Ki67 expression and MGMT promoter methylation status was evaluated, and the prognostic value of SATB1 expression in patients with gliomas was analyzed. SATB1-specific shRNA sequences were synthesized, and U251 cells were transfected with SATB1 RNAi plasmids. Expression of SATB1 mRNA and protein was investigated by RT-PCR and immunofluoresence staining and western blotting. The expression of c-Met, SLC22A18, caspase-3 and bcl-2 protein was determined by western blotting. U251 cell growth and adherence was detected by methyl thiazole tetrazolium assay. The apoptosis of U251 cells was examined with a flow cytometer. The adherence, invasion, and in vitro angiogenesis assays of U251 cells were done. The growth and angiogenesis of SATB1 low expressing U251 cells was measured in an in vivo xenograft model.ResultsOf 70 tumors, 44 (62.9%) were positive for SATB1 expression. SATB1 expression was significantly associated with a high histological grade and with poor survival in univariate and multivariate analyses. SATB1 expression was also positively correlated with Ki67 expression but negatively with MGMT promoter methylation in glioma tissues. SATB1 shRNA expression vectors could efficiently induce the expression of SLC22A18 protein, increase the caspase-3 protein, inhibit the expression of SATB1, c-Met and bcl-2 protein, the growth, invasion, metastasis and angiogenesis of U251 cells, and induce apoptosis in vitro. Furthermore, the tumor growth of U251 cells expressing SATB1 shRNA were inhibited in vivo, and immunohistochemical analyses of tumor sections revealed a decreased vessel density in the animals where shRNA against SATB1 were expressed.ConclusionsSATB1 may have an important role as a positive regulator of glioma development and progression, and that SATB1 might be a useful molecular marker for predicting the prognosis of glioma.