Immature Vasculature Research Articles

TNFSF15 facilitates the differentiation of CD11b+ myeloid cells into vascular pericytes in tumors.

Immature vasculature lacking pericyte coverage substantially contributes to tumor growth, drug resistance, and cancer cell dissemination. We previously demonstrated that tumor necrosis factor superfamily 15 (TNFSF15) is a cytokine with important roles in modulating hematopoiesis and vascular homeostasis. The main purpose of this study was to explore whether TNFSF15 might promote freshly isolated myeloid cells to differentiate into CD11b+ cells and further into pericytes. A model of Lewis lung cancer was established in mice with red fluorescent bone marrow. After TNFSF15 treatment, CD11b+ myeloid cells and vascular pericytes in the tumors, and the co-localization of pericytes and vascular endothelial cells, were assessed. Additionally, CD11b+ cells were isolated from wild-type mice and treated with TNFSF15 to determine the effects on the differentiation of these cells. We observed elevated percentages of bone marrow-derived CD11b+ myeloid cells and vascular pericytes in TNFSF15-treated tumors, and the latter cells co-localized with vascular endothelial cells. TNFSF15 protected against CD11b+ cell apoptosis and facilitated the differentiation of these cells into pericytes by down-regulating Wnt3a-VEGFR1 and up-regulating CD49e-FN signaling pathways. TNFSF15 facilitates the production of CD11b+ cells in the bone marrow and promotes the differentiation of these cells into pericytes, which may stabilize the tumor neovasculature.

Does sevoflurane sedation in pediatric patients lead to "pseudo" leptomeningeal enhancement in the brain on 3 Tesla magnetic resonance imaging?

Prominent leptomeningeal contrast enhancement (LMCE) in the brain is observed in some pediatric patients during sedation for imaging. However, based on clinical history and cerebrospinal fluid analysis, the patients are not acutely ill and do not exhibit meningeal signs. Our study determined whether sevoflurane inhalation in pediatric patients led to this pattern of 'pseudo' LMCE (pLMCE) on 3 Tesla magnetic resonance imaging (MRI). To highlight the significance of pLMCE in pediatric patients undergoing enhanced brain MRI under sedation to avoid misinterpretation in reports. A retrospective cross-sectional evaluation of pediatric patients between 0-8 years of age was conducted. The patients underwent enhanced brain MRI under inhaled sevoflurane. The LMCE grade was determined by two radiologists, and interobserver variability of the grade was calculated using Cohen's kappa. The LMCE grade was correlated with duration of sedation, age and weight using the Spearman rho rank correlation. A total of 63 patients were included. Fourteen (22.2%) cases showed mild LMCE, 48 (76.1%) cases showed moderate LMCE, and 1 case (1.6%) showed severe LMCE. We found substantial agreement between the two radiologists in detection of pLMCE on post-contrast T1 imaging (kappa value = 0.61; P < 0.001). Additionally, we found statistically significant inverse and moderate correlations between patient weight and age. There was no correlation between duration of sedation and pLMCE. pLMCE is relatively common on post-contrast spin echo T1-weighted MRI of pediatric patients sedated by sevoflurane due to their fragile and immature vasculature. It should not be misinterpreted for meningeal pathology. Knowing pertinent clinical history of the child is an essential prerequisite to avoid radiological overcalling and the subsequent burden of additional investigations.

Lung Microvascular Niche, Repair, and Engineering

Biomaterials have been used for a long time in the field of medicine. Since the success of “tissue engineering” pioneered by Langer and Vacanti in 1993, tissue engineering studies have advanced from simple tissue generation to whole organ generation with three-dimensional reconstruction. Decellularized scaffolds have been widely used in the field of reconstructive surgery because the tissues used to generate decellularized scaffolds can be easily harvested from animals or humans. When a patient’s own cells can be seeded onto decellularized biomaterials, theoretically this will create immunocompatible organs generated from allo- or xeno-organs. The most important aspect of lung tissue engineering is that the delicate three-dimensional structure of the organ is maintained during the tissue engineering process. Therefore, organ decellularization has special advantages for lung tissue engineering where it is essential to maintain the extremely thin basement membrane in the alveoli. Since 2010, there have been many methodological developments in the decellularization and recellularization of lung scaffolds, which includes improvements in the decellularization protocols and the selection and preparation of seeding cells. However, early transplanted engineered lungs terminated in organ failure in a short period. Immature vasculature reconstruction is considered to be the main cause of engineered organ failure. Immature vasculature causes thrombus formation in the engineered lung. Successful reconstruction of a mature vasculature network would be a major breakthrough in achieving success in lung engineering. In order to regenerate the mature vasculature network, we need to remodel the vascular niche, especially the microvasculature, in the organ scaffold. This review highlights the reconstruction of the vascular niche in a decellularized lung scaffold. Because the vascular niche consists of endothelial cells (ECs), pericytes, extracellular matrix (ECM), and the epithelial–endothelial interface, all of which might affect the vascular tight junction (TJ), we discuss ECM composition and reconstruction, the contribution of ECs and perivascular cells, the air–blood barrier (ABB) function, and the effects of physiological factors during the lung microvasculature repair and engineering process. The goal of the present review is to confirm the possibility of success in lung microvascular engineering in whole organ engineering and explore the future direction of the current methodology.

Radial Glia-endothelial Cells' Bidirectional Interactions Control Vascular Maturation and Astrocyte Differentiation: Impact for Blood-brain Barrier Formation.

In the developing cerebral cortex, Radial Glia (RG) multipotent neural stem cell, among other functions, differentiate into astrocytes and serve as a scaffold for blood vessel development. After some time, blood vessel Endothelial Cells (ECs) become associated with astrocytes to form the neurovascular Blood-Brain Barrier (BBB) unit. Since little is known about the mechanisms underlying bidirectional RG-ECs interactions in both vascular development and astrocyte differentiation, this study investigated the impact of interactions between RG and ECs mediated by secreted factors on EC maturation and gliogenesis control. First, we demonstrated that immature vasculature in the murine embryonic cerebral cortex physically interacts with Nestin positive RG neural stem cells in vivo. Isolated Microcapillary Brain Endothelial Cells (MBEC) treated with the conditioned medium from RG cultures (RG-CM) displayed decreased proliferation, reduction in the protein levels of the endothelial tip cell marker Delta-like 4 (Dll4), and decreased expression levels of the vascular permeability associated gene, plasmalemma vesicle-associated protein-1 (PLVAP1). These events were also accompanied by increased levels of the tight junction protein expression, zonula occludens-1 (ZO-1). Finally, we demonstrated that isolated RG cells cultures treated with MBEC conditioned medium promoted the differentiation of astrocytes in a Vascular Endothelial Growth Factor-A (VEGF-A) dependent manner. These results suggest that the bidirectional interaction between RG and ECs is essential to induce vascular maturation and astrocyte generation, which may be an essential cell-cell communication mechanism to promote BBB establishment.

Targeted Delivery to Tumors: Multidirectional Strategies to Improve Treatment Efficiency.

Malignant tumors are characterized by structural and molecular peculiarities providing a possibility to directionally deliver antitumor drugs with minimal impact on healthy tissues and reduced side effects. Newly formed blood vessels in malignant lesions exhibit chaotic growth, disordered structure, irregular shape and diameter, protrusions, and blind ends, resulting in immature vasculature; the newly formed lymphatic vessels also have aberrant structure. Structural features of the tumor vasculature determine relatively easy penetration of large molecules as well as nanometer-sized particles through a blood–tissue barrier and their accumulation in a tumor tissue. Also, malignant cells have altered molecular profile due to significant changes in tumor cell metabolism at every level from the genome to metabolome. Recently, the tumor interaction with cells of immune system becomes the focus of particular attention, that among others findings resulted in extensive study of cells with preferential tropism to tumor. In this review we summarize the information on the diversity of currently existing approaches to targeted drug delivery to tumor, including (i) passive targeting based on the specific features of tumor vasculature, (ii) active targeting which implies a specific binding of the antitumor agent with its molecular target, and (iii) cell-mediated tumor targeting.

Effect of hypoxia on progesterone production by cultured bovine early and mid luteal cells.

A major role of the corpus luteum (CL) is to produce progesterone (P4). The CL has immature vasculature shortly after ovulation, suggesting it exists under hypoxic conditions. To elucidate the mechanism involved in regulation of luteal cell function during CL development, we compared the effect of hypoxia on P4 production by cultured bovine early and mid luteal cells. Luteal cells obtained from early and mid CL were incubated under different O2 concentrations (20% and 3%) with or without hCG (1 U/ml) for 6 h and 24 h. After 6 h of culture in the presence of hCG, P4 production was not affected by hypoxia whereas decrease in its production by mid luteal cells was observed. After 24 h of culture, P4 production was significantly decreased by hypoxia in both stages of luteal cells regardless of the use of hCG. At 6 h of culture, hypoxia increased mRNA expression of hydroxyl-Δ-5-steroid dehydrogenase, 3β- and steroid Δ-isomerase 1 (HSD3B1) in early luteal cells, and decreased mRNA expression of cytochrome P450 cholesterol side chain cleavage (CYP11A1) enzyme in mid luteal cells. At 24 h of culture, mRNA expressions of steroidogenic acute regulatory protein (STAR), CYP11A1, and HSD3B1 were not affected by hypoxia in both stages of luteal cells. The overall results suggest that early luteal cells maintain P4 production under hypoxic conditions, and hypoxia-induced HSD3B1 may support this P4 production in the bovine early CL.

Modulation of Angiopoietin 2 release from endothelial cells and angiogenesis by the synaptic protein Neuroligin 2

The synaptic protein Neuroligin 2, similarly to its isoform Neuroligin 1, is produced by endothelial cells, but its activity in the vascular context remains unknown. This study aimed at verifying the hypothesis that Neuroligin 2, in parallel with its extraneuronal involvement in pancreatic beta cells exocytosis, modulated cytokine release from endothelial cells and consequently angiogenesis.We used in vitro approaches to modulate Neuroligin 2 expression and Neuroligin 2 null mice to test our hypotheses. In vitro, upon VEGF stimulation, Neuroligin 2 silencing strongly reduces Angiopoietin 2 release in the medium and increases the endothelial cell retention of Weibel Palade Bodies, the specialized organelles that store Angiopoietin 2 and various other cytokines. On the contrary, Neuroligin 2 overexpression almost depletes cells of Weibel Palade Bodies, independent of VEGF. In vivo, both the retina and tumor xenografts grown in NLGN2- null mice display an immature vasculature, with lower pericyte coverage and lower Tie2 phosphorylation. At the molecular level NLGN2 colocalizes with its neuronal partner collibystin, a CDC42 guanine nucleotide exchange factor, which is also expressed by endothelial cells and in turn modulates Angiopoietin 2 release.Neuroligin 2, an inhibitory synaptic protein, modulates a peculiar aspect of vascular function and could represent a novel target of therapy in various fields, from tumor angiogenesis to vascular diseases.

Abstract B24: De-clotting tumor to improve the perfusion, distribution and efficacy of chemotherapy and nanotherapeutics

Abstract Tumors exhibit several physical and physiological barriers that limit the distribution of molecules as small as oxygen; tumor barriers are compressed vessels due to solid stress, immature vasculature with geometric resistances, high interstitial fluid pressure, and the dense extracellular matrix. Tumors are also thrombotic in nature; pancreatic, colon, gastric, lung, ovarian cancers, melanoma, glioblastoma, lymphomas, etc. are associated with a high rate of thrombosis. Solid tumors obstruct venous return which cause static blood flow, endothelial cell injury, and coagulation activation. We hypothesize that coagulation/thrombosis act as a drug transport barrier inside the tumors and the use of anticoagulants or anti-thrombotics overcome this barrier. We posit that perfusion and penetration of drugs trap in thrombotic tumor vessels, whereas the small intra-thrombotic spacing within the vessel wall and interstitial space of tumor retards the movement of particles. In this work, we show that heparin or heparin-based anticoagulants prevent clot formation in tumors, enhances blood perfusion and nanoparticle distribution, and potentiates both chemo- and nano-therapeutic efficiency in tumor treatment. We compared the vessel perfusion and their thrombi fractions in tumors of P-selectin-/- vs wild-type (WT) mice. B16f10 melanoma cells were grown subcutaneously in P-selectin-/- and WT mice and Cy7 labeled dextran (Dex-Cy7; as a marker of perfused vessels) and Cy5.5 labeled fibrinoigen (Fib-Cy5.5; a marker of blood clots) were injected simultaneously through the tail vein. B16f10 tumors in WT mice grew with high level of thrombus, as Fib-Cy5.5 concentrated inside their vessels. However, B16f10 tumors, when grown in P-selectin-/- mice, had lower accumulation of Fib-Cy5.5 and higher fractions of perfused vessels; Dex-Cy7 concentrated and overlapped with tumor vessels. Studies with several subcutaneously developed tumors—squamous (SCC7), melanoma, pancreatic (AsPC-1), glioblastoma (U87)—and an orthotopically developed pancreatic (AsPC-1-Luc) cancer suggest that tumor vessel thrombosis fractions were inversely associated with perfused vessel fractions. SCC7 was the only tumor with high Dex-Cy7 perfusion and low abundance of Fib-Cy5.5. Intravenous administration of chitosan-based nanoparticles (CNPs) showed high perfusion and distribution in SCC7 tumors. Interestingly, thermal-heating to SCC7 tumors disrupted blood vessels, activated coagulation cascades, and produced enormous thrombus in their vessels, which blocked the intratumoral distribution of CNPs. Here we address that the use of anticoagulant, preferably with low molecular weight heparin (LMWH) or its orally available LMWH-based conjugate (LHe-tetraD) as an adjuvant, leads to easier passage of chemotherapy or nanoparticles into the deep recesses of a tumor. Subcutaneous LMWH or oral LHe-tetraD pretreatment for 7 days improved the CNPs accumulation and distribution in B16f10 tumors. Doppler ultrasound analysis revealed that LHe-tetraD treatment increased B16f10 tumor blood flow and perfusion. Orthotopic B16f10 melanoma (n = 5) and AsPC-1-Luc pancreatic tumors (n = 8~10) were treated with placebo, LHe-tetraD once daily, 3 cycles of oxaliplatin/gemcitabine once per three days, and combination after 14 days of tumor inoculation. LHe-tetraD alone did not regress tumor growth; however, LHe-tetraD/oxaliplatin/gemcitabine combination markedly reduced tumor volume than oxaliplatin/gemcitabine treated group. In another set of experiment, B16f10 tumors were treated with 4 cycles of doxorubicin loaded CNPs (CNP-Dox; 3 mg/kg per three days) with or without LHe-tetraD. Co-treatment with LHe-tetraD also enhanced the tumor-inhibition efficacy of CNP-Dox than CNP-Dox only. Our results demonstrate that anticoagulant prevents the generation of tumor thrombus and represents a new way to improve the penetration and drug efficacy into solid tumors. Citation Format: Taslim A. Al-Hilal, Jeong Uk Choi, In-San Kim, Youngro Byun, Fakhrul Ahsan. De-clotting tumor to improve the perfusion, distribution and efficacy of chemotherapy and nanotherapeutics. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B24.

New Insights into Phloem Unloading and Expression of Sucrose Transporters in Vegetative Sinks of the Parasitic Plant Phelipanche ramosa L. (Pomel).

The plant-parasitic plant interaction is a interesting model to study sink-source relationship and phloem unloading. The parasitic plants, such as the achlorophyllous plant Phelipanche ramosa, connect to the host phloem through the haustorium and act as supernumerary sinks for the host-derived photoassimilates, primarily sucrose. The application of the fluorescent symplastic tracer, carboxyfluorescein (CF) derived from carboxyfluorescein diacetate (CFDA), to the leaves of the host plant (Brassica napus) showed direct phloem connections at the host-parasite interface. These experiments also evidenced the dominant apoplastic pathway for phloem unloading in major vegetative sinks of the parasite, including tubercles and shoots, except the adventitious root apices. The CF experiments showed also the symplastic isolation of the phloem tissues from the sink tissues in tubercle and shoot of the parasite, then suggesting the pivotal role of sucrose transporters in sucrose unloading in P. ramosa sinks. Three cDNAs encoding sucrose transporters (PrSUT) were isolated from the parasitic plant. PrSUT1 transcripts accumulated at the same level in the tubercle throughout the parasite growth while a significant increase in transcript accumulation occurred after emergence in the flowering shoot, notably in the growing apical part. The in situ hybridization experiments revealed the PrSUT1 transcript accumulation in the mature phloem cells of both subterranean and flowering shoots, as well as in shoot terminal sinks corresponding to apical meristem, scale leaf primordia and immature vasculature. The transient expression experiments in Arabidopsis protoplasts showed that PrSUT1 was localized at the plasma membrane, suggesting its role in phloem functioning and sucrose uptake by the sink cells in P. ramosa. Conversely, the PrSUT2 transcript accumulation was constantly low in tubercles and shoots but PrSUT3 transcripts accumulated markedly in the subterranean and flowering shoots, in concordance with the PrSUT3 mRNA accumulation in multiple sink areas including apical meristem, scale-leaf primordia, immature vasculature and even storage parenchyma. However, the PrSUT3 transcripts did not accumulate in the mature phloem cells. The transient expression experiments in Arabidopsis protoplasts suggested a tonoplast localization of PrSUT3, for which nevertheless the involvement in intracellular sucrose transport needs clarification.

Hypoxia increases glucose transporter 1 expression in bovine corpus luteum at the early luteal stage.

A major role of the corpus luteum (CL) is to produce progesterone (P4). The CL has immature vasculature shortly after ovulation, suggesting it exists under hypoxic conditions. Hypoxia-inducible factor-1 (HIF1) induces the expression of glucose transporter 1 (GLUT1). To clarify the physiological roles of GLUT1 in bovine CL, we examined GLUT1 mRNA expression in the CL under hypoxic conditions by quantitative RT-PCR. We also measured the effects of glucose (0–25 mM) and GLUT1 inhibitors (cytochalasin B, STF-31) on P4 production in bovine luteal cells. GLUT1 mRNA expression in bovine CL was higher at the early luteal stage compared to the other later stages. Hypoxia (3% O2) increased GLUT1 mRNA expression in early luteal cells, but not in mid luteal cells. Glucose (0–25 mM) increased P4 production in early luteal cells, but not in mid luteal cells. Both GLUT1 inhibitors decreased P4 production in early and mid luteal cells. Overall, the results suggest that GLUT1 (possibly induced by hypoxic conditions in the early CL) plays a role in the establishment and development of bovine CL, especially in supporting luteal P4 synthesis at the early luteal stage.

Remodeling of the Extracellular Matrix by Endothelial Cell-Targeting siRNA Improves the EPR-Based Delivery of 100 nm Particles.

A number of nano drug delivery systems have recently been developed for cancer treatment, most of which are based on the enhanced permeability and retention effect. The advantages of the enhanced permeability and retention effect can be attributed to immature vasculature. Herein we evaluated the intratumoral distribution of lipid nanoparticles when the VEGF receptor 2 on tumor endothelial cells was inhibited by liposomal siRNA. VEGF receptor 2 inhibition resulted in an increase in intratumoral distribution and therapeutic efficacy despite the maturation of the tumor vasculature. A small molecule inhibitor against matrix metalloproteinase and macrophage depletion cancelled the improvement in the distribution of the lipid nanoparticles, suggesting that remodeling of tumor microenvironment played a role in the facilitated intratumoral distribution via the down-regulation of VEGF receptor 2. Accordingly, our results suggest that the enhanced permeability and retention effect is dependent, not only on the structure of the tumor vasculature, but also on the dynamics of the tumor microenvironment including extracellular matrix remodeling. Regulating the tumor microenvironment and the extracellular matrix by delivering tumor endothelial cell-targeting siRNA could potentiate the enhanced permeability and retention effect-based strategy.

Changes in pericytic expression of NG2 and PDGFRB and vascular permeability in the sensory circumventricular organs of adult mouse by osmotic stimulation

The blood-brain barrier (BBB) is a barrier that prevents free access of blood-derived substances to the brain through the tight junctions and maintains a specialized brain environment. Circumventricular organs (CVOs) lack the typical BBB. The fenestrated vasculature of the sensory CVOs, including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP), allows parenchyma cells to sense a variety of blood-derived information, including osmotic ones. In the present study, we utilized immunohistochemistry to examine changes in the expression of NG2 and platelet-derived growth factor receptor beta (PDGFRB) in the OVLT, SFO and AP of adult mice during chronic osmotic stimulation. The expression of NG2 and PDGFRB was remarkably prominent in pericytes, although these angiogenesis-associated proteins are highly expressed at pericytes of developing immature vasculature. The chronic salt loading prominently increased the expression of NG2 in the OVLT and SFO and that of PDGFRB in the OVLT, SFO and AP. The vascular permeability of low-molecular-mass tracer fluorescein isothiocyanate was increased significantly by chronic salt loading in the OVLT and SFO but not AP. In conclusion, the present study demonstrates changes in pericyte expression of NG2 and PDGFRB and vascular permeability in the sensory CVOs by chronic osmotic stimulation, indicating active participation of the vascular system in osmotic homeostasis.

Abstract 2334: Anti-angiogenic activity of anthrax lethal toxin using the mouse retina as a model system

Abstract Our laboratory has previously shown that inhibition of MKK pathways by anthrax lethal toxin (LeTx) inhibits tumor growth and angiogenesis, reduces VEGF secretion, and results in rapid cessation of blood flow to tumors in melanoma and sarcoma xenograft model systems. To gain insight into the anti-angiogenic nature of LeTx, we utilized both developmental and pathological models of vascularization in the murine retina. By immunoblotting and immunofluorescence microscopy we observed that MAPK activation occurs in a spatially and temporally regulated manner during retinal vascular development. Intravitreal administration of LeTx caused an early delay in retinal vascular development followed by the formation of abnormal vascular tufts that co-stained with phosphorylated MAPK in the outer retinal region. Using a model of oxygen-induced retinopathy, western analysis revealed that MAPK activation preceded the onset of hypoxia and development of neovascularization in this model system. Intravitreal injection of LeTx during hypoxia resulted in a persistent lack of development and progression of all pathologic aspects of neovascularization, while having minimal effect on macrophage recruitment. VEGF levels remained at an elevated level even with the persistent lack of neovascular growth and continued hypoxic environment, implying a VEGF-independent mechanism for the effects of MKK inhibition. Additionally, immature retinal vasculature during normal development was persistently destroyed following MAPK inhibition, while mature vessels from adult animals were unaffected. Our studies not only reveal that MKK signaling plays a key role in retinal angiogenesis but also that perturbation of MKK signaling by LeTx can profoundly alter vascular morphogenesis. The persistent destruction of immature vessels during normal vascular development in the retina and in the model of oxygen-induced retinopathy suggests an extreme sensitivity of immature vessels to MKK signaling inhibition. Since MKK inhibition prevents oxygen-induced retinopathy by a mechanism independent of the VEGF-targeting strategies, this approach may be used in conjunction with existing strategies utilizing VEGF inhibitors to prevent pathologic angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2334. doi:1538-7445.AM2012-2334

Abstract 1385: Molecular changes in breast tumors following bevacizumab-based treatment: Final analysis of a randomized neoadjuvant study of bevacizumab or placebo, followed by chemotherapy with or without bevacizumab, in patients with stage II or III breast cancer

Abstract Background: Bevacizumab (bev) has been widely studied in breast cancer (BC), yet no randomized trial in BC has reported in vivo molecular effects of bev on human tumor tissue. We conducted a trial to evaluate the safety, clinical &amp; molecular effects of neoadjuvant chemotherapy plus bev for locally advanced BC. Methods: 90 pts were randomized (2:1:2:1) to 1 of 4 arms: Arm A: TAC (docetaxel, T: 75 mg/m2, doxorubicin, A: 50 mg/m2, cyclophosphamide, C: 500 mg/m2) + low dose bev (7.5 mg/kg); Arm B: TAC + low dose placebo (P); Arm C: TAC + standard dose bev (15 mg/kg); Arm D: TAC + Std-P. A run-in cycle of bev or P was followed by 6 cycles of TAC plus P or bev. Tumor biopsies pre- and 7-10 days post-run-in with bev or P were taken. Unblinding occurred post surgery (Sx): Arms A/C received maintenance bev to complete 52 wks, Arms B/D received no further P. Eligible patients were females with &amp;gt;3 cm, HER2(-) BC. Endpoints included safety and pathologic complete response (pCR) in breast &amp; lymph nodes. To assess the effects of VEGF pathway inhibition on tumor vasculature, PCR analysis using the fluidigm array platform was performed on RNA from the pre- and post-run in samples to evaluate expression of 67 genes known to play a defined role in VEGF signaling. Results: 28 pts were assigned to Arm A, 30 to Arm C, and 32 to Arms B/D. 12 pts came off Tx before surgery (Arm A:2, Arm C:6, Arms B/D:4) and 78 received all Tx, underwent Sx and are evaluable for pCR. Two pts (6%) in Arms B/D, 5 pts (18%) in Arm A and 10 pts (33%) in Arm C had wound healing complications. 17% of pts in Arm C had Gr 3 (N=4) or Gr 4 (N=1) heart failure, none in Arms A/B/D. The pCR rate in evaluable patients was 18% (14/78): 5 (19%) Arm A, 3 (13%) Arm C, 6 (21%) Arms B/D. 45 samples (20 from Arms B/D and 25 from bev arms) were included in the pair-wise analysis to identify 6 genes that were differentially expressed. Bev resulted in a decrease in expression of Dll4, Cox2, Fibronectin (FN_EIIIB), angiopoietin 2 (Angpt2) and ESM1. In addition, upregulation of the cytokine, stromal derived growth factor (SDF1) was observed. Notably, genes such as CD31 or VE-cadherin were not appreciably differentially expressed. Of the genes that were downregulated, Dll4 and Angpt2 represent genes enriched in endothelial tip cells, which guide the migration of newly formed blood vessels. The decrease in these genes likely represents the effect of bev on reducing immature, growing vasculature in the tumor. Conclusions: This trial enabled the clinical and molecular evaluation of breast cancer tumor tissue pre and post-bev. Clinically, bev was associated with more wound healing and heart failure events and similar pCR rates compared to P. Tumor expression analysis for genes in the angiogenesis pathway supports the preclinical hypothesis that bev may primarily target immature tumor vasculature. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1385. doi:1538-7445.AM2012-1385

Abstract 181: Common Angiogenic Signalling Pathways Induced by Monomeric c-Reactive Protein and FGF-2 Through Phosphatidylinositol 3-kinase

Angiogenesis is the process of new blood vessel growth from pre-existing vascular structures. The new vessels from atherosclerotic lesions may be a focus of instability, since they facilitate the infiltration of inflammatory cells and due to their tendency to leak, they may produce haemorrhagic complications. Pentameric C-reactive protein (CRP), a strong marker of inflammation, is a risk factor for cardiovascular diseases with a direct role in the development of atherosclerotic lesions. In hypoxic tissue damage, CRP dissociates irreversibly into monomeric CRP (mCRP), which was previously demonstrated to be pro-angiogenic on bovine aortic endothelial cells (BAEC). Our main study was to examine the vessel forming capability of CRP in the presence of other angiogenic factors known to be present in the micro-environment of unstable plaques with immature vasculature. Here we studied the effects of mCRP in presence or absence of FGF-2 on BAEC proliferation, migration, tube formation in Matrigel and on the vascular remodelling using spheroids, a tri-dimensional system of endothelial cell culture embedded in collagen gel. A significant synergic effect of mCRP combined with FGF-2 was observed in all angiogenesis assays used, compared to the effect of mCRP or FGF-2 alone. Using the spheroids, both mCRP and FGF-2 stimulated the length of sprouts with endothelial cells more dispersed giving an aspect of thin structures. For a better understanding of the molecular mechanisms involved, the signalling pathways were investigated by Western blotting and all the assays were performed in the presence or absence of pharmacological inhibitors of MAPK (PD98059), γ-secretase (DAPT inhibitor) and phosphatidylinositol 3-kinase (PI3K) pathways (LY294002). We showed mCRP-induced endothelial cell proliferation, migration and tube formation required activation of the PI3K pathway. MAPK activation was essential in mCRP-induced cell proliferation and differentiation (tube formation and sprouting from the core of spheroids) and γ-secretase activity was required for mCRP-induced tube formation only. For its pro-angiogenic activity, FGF-2 required all of these key pathways with the exception that γ-secretase activity was not associated with FGF-2-induced cell migration. In all assays including the over-expression of phospho-ERK, the synergistic pro-angiogenic effect of mCRP added to FGF-2 was completely inhibited by LY294002. Thus, mCRP and FGF-2 have a common signalling pathway through PI3K and an eventual deregulation of their pro-angiogenic effects due to an excessive inflammation inducing a hyper-vascularisation which could contribute to formation of unstable plaque with haemorrhagic risk, and therefore, might be prevented by targeting the key proteins of the PI3K pathway.

Delivery of Molecular and Nanoscale Medicine to Tumors: Transport Barriers and Strategies

Tumors are similar to organs, with unique physiology giving rise to an unusual set of transport barriers to drug delivery. Cancer therapy is limited by nonuniform drug delivery via blood vessels, inhomogeneous drug transport into tumor interstitium from the vascular compartment, and hindered transport through tumor interstitium to the target cells. Four major abnormal physical and physiological properties contribute to these transport barriers. Accumulated solid stress compresses blood vessels to diminish the drug supply to many tumor regions. Immature vasculature with high viscous and geometric resistances and reduced pressure gradients leads to sluggish and heterogeneous blood flow in tumors to further limit drug supply. Nonfunctional lymphatics coupled with highly permeable blood vessels result in elevated hydrostatic pressure in tumors to abrogate convective drug transport from blood vessels into and throughout most of the tumor tissue. Finally, a dense structure of interstitial matrix and cells serves as a tortuous, viscous, and steric barrier to diffusion of therapeutic agents. In this review, we discuss the origins and implications of these barriers. We then highlight strategies for overcoming these barriers by modulating either drug properties or the tumor microenvironment itself to enhance the delivery and effectiveness of drugs in tumors.

Abstract 1306: Tumor vasculature and angiogenic profile of pediatric pilocytic astrocytoma

Abstract Introduction: Pilocytic astrocytomas (WHO, grade I astrocytomas) are the most frequent brain tumors in children. Despite a 5-year survival of 90%, outcome is not always favourable, mainly because of surgical morbidity and tumor progression. To reduce this morbidity as well as mortality, new therapeutic modalities are needed. As target in cancer treatment, angiogenesis is extensively investigated, and formed an effective and well tolerated treatment in recurrent adult glioblastomas (WHO, grade IV astrocytomas). However, little is known about angiogenesis in low grade pediatric astrocytomas. So this study aimed to characterize tumor vasculature and the angiogenic profile of 59 pediatric pilocytic astrocytomas to obtain insights into potential angiogenic related therapeutic targets in these tumors. Materials and methods: Microvessel density (MVD), vessel maturity in terms of basement membrane and pericytes coverage, and turnover of both endothelial and tumor cells, and vascular endothelial growth factor (VEGF) expression were evaluated in tumor tissue, immunohistochemically stained with respectively CD34, collagen IV, Smooth Muscle Actin (SMA), Ki67/CD34, caspase-3/CD34, and VEGF(-A-D). Collagen IV and SMA slides were quantified using morphometry software. As indicator for vessel stabilization, the angiopoietin (ANGPT)-1/ANGPT-2 balance was calculated using Real Time RT-PCR. These data were compared with previously described 62 adult glioblastomas,(Sie et al, J Neurosurg 2009) often seen as a model for studying angiogenesis. Results: Pilocytic astrocytoma had fewer but wider vessels than glioblastoma. Slightly thicker pericyte coverage and a higher ANGPT-1/ANGPT-2 balance were seen in pilocytic astrocytoma (respectively p = 0.016, and p = 0.001). Turnover of endothelial and tumor cells were lower in pilocytic astrocytoma (p &amp;lt; 0.001). Within pilocytic astrocytoma, a positive correlation was found between pericytes and basement membrane coverage (r: 0.377, p = 0.005). Higher ANGPT-1/ANGPT-2 balance was correlated with less apoptotic endothelial cells (r: −0.480, p = 0.007). Comparing both tumors, no significant difference was found in basement membrane coverage, the fraction of vessels covered with pericytes, and VEGF-A expression. Proliferating tumor cells were positively correlated with proliferating endothelial cells (r: 0.636, p &amp;lt; 0.001) and VEGF-B expression (r: 0.290, p = 0.029). Conclusion: Despite the fact that pilocytic astrocytomas showed slightly less immature and instable vasculature compared with glioblastoma, a large overlap in the angiogenic profile was seen between both tumors. These findings suggest promising possibilities for targeting angiogenesis with anti-VEGF as therapeutic strategy in pilocytic astrocytoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1306.

Tissue injury by microbeam irradiation depends on the stage of vascular maturation

PurposeThe aim of this study is to explore the effects of Microbeam Radiation (MR) on vascular biology using the chick chorio‐allantoic membrane (CAM).MethodsCAMs were irradiated with multiple planar synchrotron X‐ray beams at doses between 100 and 300 Gy and were evaluated morphologically and in vivo at days 8 and 12.ResultsIn vivo monitoring and morphological investigations of day 8 CAM immature vasculature 6 hr after MR revealed a near total destruction of the capillary plexus within the beams’ width. Surprisingly, arteries and veins were not affected. Conversely, at day 12, only well defined lesions in the microvasculature (mature ones, covered by pericytes) were observed.After 300 Gy MR, TEM revealed enlargement of the interendothelial cell junctions which could explain the oedema, the microvessels along the beam path showed signs of disruption and apoptosis. The remaining vasculature recovered rapidly and CAM regained its normal thickness. Between 1 hr and 6 hr no additional morphological changes were observed.Conclusions The effects of MR are most likely mediated by capillary damage. The vascular toxicity of MR depends on the stage of capillary maturation: the immature vessels are much more vulnerable than the mature ones. The physiological effects of MR appear within a short time, the most important structural alterations being present between 15 and 60 minutes after irradiation.

E10A, an adenovirus carrying human endostatin gene, in combination with docetaxel treatment inhibits prostate cancer growth and metastases.

E10A, a replication-defective adenovirus carrying human endostatin gene, has finished Phase I clinical trials for solid cancers. We assessed whether the combination of E10A with docetaxel would enhance antiangiogenic activities and inhibit prostate cancer growth and metastases. Combination use of conditioned medium from prostate cancer cells infected by E10A and docetaxel exerted synergistic inhibition of HUVECs proliferation, migration and tube formation, compared with either agent alone. In prostate cancer s.c. xenograft models, combined therapy resulted in significant tumour growth inhibition and survival improvement. The antitumoural effect was tightly correlated with a remarkable decrease in tumour cell proliferation, microvessel, especially immature vasculature and significant increase in apoptosis induction. Systemic administration of E10A and docetaxel also effectively inhibited orthotopic growth and metastases of prostate cancer and achieved better in vivo antiangiogenic effects than either agent alone. Our data indicate that E10A in combination with docetaxel exert enhanced antiangiogenic activities and inhibit prostate cancer growth and metastases. Therefore, this approach may be an effective treatment for advanced prostate cancer and deserves more extensive investigation.

Update on risk factors and future perspectives for preterm infants

Abstract Purpose To give an update on risk factors for retinopathy of prematurity with special focus on postnatal growth and growth factors Methods The relationship between birth weight, serum levels of IGF‐I as well as postnatal longitudinal growth and ROP will be presented. Preventive measures will be discussed. Results Birth weight data on 451 infants demonstrated initially a significant difference in BW between different ROP stages but when taking gestational age and sex into account the significance was eliminated. Recently, a new diagnostic tool based on weekly neonatal measurements of body weight and serum insulin‐like growth factor 1 (IGF‐I) levels, was shown to be predictive for ROP development. The algorithm “Weight IGF‐I Neonatal ROP” (WINROP™) predicted early (mean 10 weeks) all infants who later developed proliferative ROP requiring treatment. The WINROP algorithm was then taken one step further using only serial weight measurements (n=700), excluding blood sampling for measuring IGF‐I. With this approach WINROP predicted all infants who later developed proliferative ROP requiring treatment (100% sensitivity) and correctly identified 75% of those who did not develop proliferative ROP, and thus would not need any ophthalmologic screening. We have also shown a close relationship between postnatal growth, severe ROP and poor brain development. Conclusion For decades, neonatal intensive care has focused on survival of the most immature babies. Time has come to find methods to ameliorate the nutrition for the children born very preterm. It is known that IGF‐I is essential for growth and development of the immature vasculature of the eye. Intervention with substitution of IGF‐I to the very preterm babies to raise IGF‐I up to normal intrauterine levels might be beneficial. Commercial interest