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Surface properties of nanoparticles to be used for radioimmunotherapy need to be optimized to allow antibody conjugation while ensuring biocompatibility. We aimed to investigate cell adhesion and proliferation onto different coatings to be used for nanoparticles. C, CH or SiO coatings deposited onto glass coverslips by magnetron deposition as well as nitrogen functionalized materials synthetized using different reactive sputtering conditions and PPAA (plasma polymerized allylamine) coating, were compared. Amine functionalization did increase hydrophilicity in all the materials tested. Biocompatibility was assessed by measuring cell viability, morphology, attachment, spreading, and pro-inflammatory cytokine secretion. The results show that C and CN were the most biocompatible substrates while SiO and SiO N were the most toxic materials. PPAA coatings displayed unexpectedly an intermediate biocompatibility. A correlation could be observed between wettability and cell proliferation except for C coated surface, indicating that more complex processes than hydrophilicity alone are taking place that affect cell functions. © Springer Science+Business Media, LLC 2011.

We have deposited III-V alloys on 200 mm Si miscut wafers with an oxide pattern. The selective epitaxial growth (SEG) of GaAs in large windows defined by SiO2 lines on a thick strained-relaxed Ge buffer layer served as a test vehicle which allowed us to demonstrate the integration of a III-V material deposition process step in a Si manufacturing line using an industrial reactor. High quality GaAs layers with high wafer-scale thickness uniformity were achieved. In a subsequent step, SEG of InP was successfully performed on wafers with a 300 nm shallow trench isolation pattern. The seed layer morphology depended on the treatment of the Ge surface and on the growth temperature. The orientation of the trench with respect to the substrate miscut direction had an impact on the quality of the InP filling. Despite of the challenges, such an approach for the integration of III-V materials on Si substrates allowed us to obtain extended-defect-free epitaxial regions suitable for the fabrication of high-performance devices.

Introduction Tumours are not only composed of malignant cells. Indeed, blood vessels, composed of endothelial cells, are required for tumour growth. Interestingly, the induction of angiogenesis requires not only endothelial cells but also immune cells. Among them, tumor-associated macrophages (TAMs) make up to 50% of the tumour immune infiltrate. These TAMs are strong angiogenesis inducer and are in part responsible of the tumour inflammation. Two kinds of macrophages are detected in tumours: pro-inflammatory and anti-tumoral M1 macrophages and anti-inflammatory and pro-tumoral M2 macrophages. Hypoxia is another key feature of tumour microenvironment which induces angiogenesis and enhances tumour inflammation and metastasis. Two types of hypoxia occur in tumour: chronic hypoxia, which impacts cells too far from the blood vessels, and cycling hypoxia (CyH), which causes intermittent oxygenation of malignant and non-malignant cells. We have studied the impact of CyH on macrophage polarisation and activity. Secondly, the impact of CyH on the communication between macrophages and endothelial cells was investigated. Then, the impact of this dialogue on tumour growth and metastasis will be studied. Material and methods THP-1 monocytes are differentiated into M0 macrophages by PMA. M0 macrophages are then polarised into M1 macrophages with LPS and IFNg (24 hour incubation) or into M2 macrophages with IL-4 and IL-13 (48 hour incubation). After exposing these macrophages to normoxia, chronic hypoxia or CyH, the expression and secretion of pro-inflammatory factors were studied by RT-qPCR and ELISA, respectively. To investigate the communication between macrophages and endothelial cells under CyH, endothelial cells were incubated with CyH-exposed macrophage conditioned-medium and the expression of endothelial cell activation markers was analysed by RT-qPCR. Results and discussions CyH induced a pro-inflammatory phenotype in M0 macrophages and enhanced the pro-inflammatory phenotype of M1 macrophages. Indeed, an increased expression of TNFa, IL-6 and IL-1β was observed. These effects depended on NF-kB activation since IKK inhibition prevented these effects. The conditioned-media of M0, M1 and M2 macrophages exposed to CyH induced endothelial cell activation as observed by an increased IL-6, IL-8 and ICAM-1 expression. Conclusion CyH induced a pro-inflammatory phenotype in M0 and M1 macrophages via NF-kB activation. The M0, M1 and M2 macrophages exposed to cycling hypoxia induced endothelial cell activation by a secreted molecule that needs to be identified.

PURPOSE: Radioactive atoms attached to monoclonal antibodies are used in radioimmunotherapy to treat cancer while limiting radiation to healthy tissues. One limitation of this method is that only one radioactive atom is linked to each antibody and the deposited dose is often insufficient to eradicate solid and radioresistant tumors. In a previous study, simulations with the Monte Carlo N-Particle eXtended code showed that physical doses up to 50 Gy can be delivered inside tumors by replacing the single radionuclide by a radioactive nanoparticle of 5 nm diameter containing hundreds of radioactive atoms. However, tumoral and normal tissues are not equally sensitive to radiation, and previous works did not take account the biological effects such as cellular repair processes or the presence of less radiosensitive cells such as hypoxic cells. METHODS: The idea is to adapt the linear-quadratic expression to the tumor model and to determine biological effective doses (BEDs) delivered through and around a tumor. This BED is then incorporated into a Poisson formula to determine the shell control probability (SCP) which predicts the cell cluster-killing efficiency at different distances "r" from the center of the tumor. BED and SCP models are used to analyze the advantages of injecting radioactive nanoparticles instead of a single radionuclide per vector in radioimmunotherapy. RESULTS: Calculations of BED and SCP for different distances r from the center of a solid tumor, using the non-small-cell lung cancer as an example, were investigated for 90Y2O3 nanoparticles. With a total activity of about 3.5 and 20 MBq for tumor radii of 0.5 and 1.0 cm, respectively, results show that a very high BED is deposited in the well oxygenated part of the spherical carcinoma. CONCLUSIONS: For either small or large solid tumors, BED and SCP calculations highlight the important benefit in replacing the single beta-emitter 90Y attached to each antibody by a 90Y2O3 nanoparticle.

The endothelium plays a crucial role in the regulation of cardiovascular homeostasis through the release of vasoactive autacoids such as nitric oxide, prostacyclin, and a third factor or pathway termed 'endothelium-derived hyperpolarizing factor' (EDHF). Although the functional influence of NO and EDHF is sometimes reciprocal (i.e., their effects decrease or increase with the reduction in vessel diameter, respectively), recent insights led to the identification of caveolae and caveolin as common regulators of their production. In this review, we will first focus on the current understanding of the caveolin/eNOS paradigm and will then detail the most recent findings on the role of caveolae in driving EDHF-signaling pathways.

OBJECTIVE: To characterize the human ovarian xenograft revascularization process. DESIGN: Prospective experimental study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Ovarian biopsies were obtained from 12 women aged 22-35 years. INTERVENTION(S): Frozen-thawed human ovarian fragments were intraperitoneally grafted into nude mice. MAIN OUTCOME MEASURE(S): Graft perfusion was evaluated by Hoechst 33342 uptake. Murine and human vascularization was analyzed by CD31 and von Willebrand factor double immunostaining. RESULT(S): On day 3, some murine neovessels and perfused areas were located at the periphery of the fragments. Nonperfused native human vessels were present in the fragments. From day 5, perfused areas and murine endothelial areas progressively increased. Host angiogenesis initiated ovarian graft reperfusion: murine neovessels penetrated from the periphery and were colocalized with perfused areas. By day 10, the increase in perfusion and murine vascularization was significant. The center of the fragments was perfused and a significant increase was observed in human vasculature. CONCLUSION(S): Host and graft vessels contributed sequentially to graft revascularization: murine angiogenesis initiated reperfusion from day 5 and, by day 10, human angiogenesis was shown to participate in graft revascularization. Host and graft angiogenesis are potential targets to reduce the avascular period after grafting.

Purpose: Cyclic hypoxia in tumors originates from heterogeneities in RBC flux and influences not only tumor cells but also endothelial cells lining tumor blood vessels. Whether pO2 fluctuations, particularly transient reoxygenation periods, alter the well-known hypoxia-inducible factor (HIF)–dependent gene program is largely unknown. Experimental Design: We compared the transcriptomic profiles of endothelial and tumor cells exposed to cyclic hypoxia versus continuous hypoxia to uncover a possible differential effect on angiogenesis and metastases. Results: Microarray analyses identified early genes that were selectively induced by cyclic hypoxia in endothelial cells. Among them, we focused on PTGS2 because the observed increase in mRNA expression led to a significant increase in the expression and activity of cyclooxygenase-2 (COX-2; the protein product of PTGS2). HIF-1α was shown to be stabilized by cyclic hypoxia (despite reoxygenation periods) and to favor COX-2 induction as validated by the use of echinomycin and HIF-1α targeting small interfering RNA. Using a specific COX-2 inhibitor and a dedicated COX-2 targeting small interfering RNA, we documented that COX-2 accounted for the higher endothelial cell survival and angiogenic potential conferred by cyclic hypoxia. Cyclic hypoxia also led to a preferential COX-2 induction in tumor cells and, contrary to continuous hypoxia, fostered a higher metastatic take of prechallenged tumor cells. Conclusions: Our study documents that PTGS2/COX-2 is part of a cyclic hypoxia gene signature and largely accounts for the unique phenotype of endothelial and tumor cells exposed to fluctuations in pO2, thereby offering new perspectives for the clustering of tumors expressing COX-2 together with other cyclic hypoxia-responsive genes. Clin Cancer Res; 16(2); 410–9

The oxygen consumption rate of tumor cells affects tumor oxygenation and response to therapies. Highly sensitive methods for determining cellular oxygen consumption are, therefore, needed to identify treatments that can modulate this parameter. We compared the performances of three different methods for measuring cellular oxygen consumption: electron paramagnetic resonance (EPR) oximetry, the Clark electrode, and the MitoXpress fluorescent assay. To compare the assays, we used K562 cells in the presence of rotenone and hydrocortisone, compounds that are known to inhibit the mitochondrial electron transport chain to different extents. The EPR method was the only one that could identify both rotenone and hydrocortisone as inhibitors of tumor cell oxygen consumption. The Clark electrode and the fluorescence assay demonstrated a significant decrease in cellular oxygen consumption after administration of the most potent inhibitor (rotenone) but failed to show any significant effect of hydrocortisone. EPR oximetry is, therefore, the most sensitive method for identifying inhibitors of oxygen consumption on cell assays, whereas the Clark electrode offers the unique opportunity to add external compounds during experiments and still shows great sensitivity in studying enzyme and chemical reactions that consume oxygen (non-cell assays). Finally, the MitoXpress fluorescent assay has the advantage of a high-sample throughput and low bulk requirements but at the cost of a lower sensitivity.

Paclitaxel (PTX)-loaded PEGylated PLGA based nanoparticles (NP) have been previously described as more effective in vitro and in vivo than Taxol(R). The aim of this study was to test the hypothesis that our PEGylated PLGA-based nanoparticles grafted with the RGD peptide or RGD-peptidomimetic (RGDp) would target the tumor endothelium and would further enhance the anti-tumor efficacy of PTX. The ligands were grafted on the PEG chain of PCL-b-PEG included in the nanoparticles. We observed in vitro that RGD-grafted nanoparticles were more associated to Human Umbilical Vein Endothelial cells (HUVEC) by binding to alpha(v)beta(3) integrin than non-targeted nanoparticles. Doxorubicin was also used to confirm the findings observed for PTX. In vivo, we demonstrated the targeting of RGD and RGDp-grafted nanoparticles to tumor vessels as well as the effective retardation of TLT tumor growth and prolonged survival times of mice treated by paclitaxel-loaded RGD-nanoparticles when compared to non-targeted nanoparticles. Hence, the targeting of anti-cancer drug to tumor endothelium by RGD-labeled NP is a promising approach.

Tumor cells fuel their metabolism with glucose and glutamine to meet the bioenergetic and biosynthetic demands of proliferation. Hypoxia and oncogenic mutations drive glycolysis, with the pyruvate to lactate conversion being promoted by increased expression of lactate dehydrogenase A and inactivation of pyruvate dehydrogenase. The NAD(+) pool is consecutively regenerated and supports the high glycolytic flux required to produce anabolic intermediates. Glutaminolysis provides metabolic intermediates such as alpha-ketoglutarate to feed and thereby maintain the tricarboxylic acid cycle as a biosynthetic hub. Glycolysis and glutaminolysis share the capacity to generate NADPH, from the pentose phosphate pathway and through the malate conversion into pyruvate, respectively. Both pathways ultimately lead to the secretion of lactate. More than a waste product, lactate was recently identified as a major energy fuel in tumors. Lactate produced by hypoxic tumor cells may indeed diffuse and be taken up by oxygenated tumor cells. Preferential utilization of lactate for oxidative metabolism spares glucose which may in turn reach hypoxic tumor cells. Monocarboxylate transporter 1 regulates the entry of lactate into oxidative tumor cells. Its inhibition favors the switch from lactate-fuelled respiration to glycolysis and consecutively kills hypoxic tumor cells from glucose starvation. Combination with radiotherapy renders remaining cells more sensitive to irradiation, emphasizing how interference with tumor cell metabolism may complement current anticancer modalities.

Large quantities of gold nanoparticles (Au NPs) were prepared by physical vapour deposition (PVD) technique. The produced nanoparticles were transferred into water solutions [containing 1% of carboxymethylcellulose (CMC)]. To achieve this resuspension, the Au NPs were embedded in a NaCl matrix, also deposited by PVD. In order to avoid the instantaneous and irreversible aggregation of the Au NPs, a surface modification needs to be performed, for example, by coating the metallic particles with plasma polymerized polymers. In this work, we present selected conditions allowing the production of aqueous solutions of Au NPs with small diameters (ca. 10 nm). The whole process is carried out thanks to a fully automatic plasma deposition system, allowing the production of high concentration of gold nanoparticles.

Hypoxia and blood flow heterogeneities are characteristics of solid tumours and are major obstacles for therapy. Exploiting the biology of nitric oxide (NO), a small radical with multiple functions, is particularly attractive to circumvent these sources of resistance and to sensitise tumour to cytotoxic treatments such as radiotherapy and chemotherapy. Indeed, while NO mediates angiogenic effects, NO may also promote tumour perfusion, drug delivery and oxygenation. Different strategies to deliver NO to tumours and pertaining to the FECS-EJC award laureate's work are reviewed, with a focus on their therapeutic potential. The development of techniques to monitor how and to which extent NO delivery influences the phenotype of a given tumour in a given patient is also discussed.

Nitric oxide production in response to flow-dependent shear forces applied on the surface of endothelial cells is a fundamental mechanism of regulation of vascular tone, peripheral resistance, and tissue perfusion. This implicates the concerted action of multiple upstream “mechanosensing” molecules reversibly assembled in signalosomes recruiting endothelial nitric oxide synthase (eNOS) in specific subcellular locales, e.g., plasmalemmal caveolae. Subsequent short- and long-term increases in activity and expression of eNOS translate this mechanical stimulus into enhanced NO production and bioactivity through a complex transcriptional and posttranslational regulation of the enzyme, including by shear-stress responsive transcription factors, oxidant stress-dependent regulation of transcript stability, eNOS regulatory phosphorylations, and protein-protein interactions. Notably, eNOS expressed in cardiac myocytes is amenable to a similar regulation in response to stretching of cardiac muscle cells and in part mediates the length-dependent increase in cardiac contraction force. In addition to short-term regulation of contractile tone, eNOS mediates key aspects of cardiac and vascular remodeling, e.g., by orchestrating the mobilization, recruitment, migration, and differentiation of cardiac and vascular progenitor cells, in part by regulating the stabilization and transcriptional activity of hypoxia inducible factor in normoxia and hypoxia. The continuum of the influence of eNOS in cardiovascular biology explains its growing implication in mechanosensitive aspects of integrated physiology, such as the control of blood pressure variability or the modulation of cardiac remodeling in situations of hemodynamic overload.

The heat shock protein 90 (Hsp90) plays a crucial role in the stability of several proteins that are essential for malignant transformation. Hsp90 is therefore an interesting therapeutic target for cancer therapy. In this paper, we investigated whether an oxidative stress generated during ascorbate-driven menadione redox cycling (ascorbate/menadione), affects Hsp90 leading to the degradation of some critical proteins and cell death. Unlike 17-AAG, which inhibits Hsp90 but enhances Hsp70 levels, ascorbate/menadione-treated cells present an additional Hsp90 protein band of about 70kDa as shown by Western blot analysis, suggesting Hsp90 cleavage. This Hsp90 cleavage seems to be a selective phenomenon since it was observed in a large panel of cancer cell lines but not in non-transformed cells. Antibodies raised against either the N-terminus or the C-terminus domains of Hsp90 suggest that the site of cleavage should be located at its N-terminal part. Furthermore, antibodies raised against either the alpha- or the beta-Hsp90 isoform show that Hsp90beta is cleaved while the alpha isoform is down-regulated. We have further shown that different Hsp90 client proteins like Bcr-Abl (a chimerical protein expressed in K562 leukemia cells), RIP and Akt, were degraded when K562 cells were exposed to an oxidative stress. Both Hsp90 cleavage and Bcr-Abl degradation were observed by incubating K562 cells with another H(2)O(2)-generating system (glucose/glucose oxidase) and by incubating KU812 cells (another leukemia cell line) with ascorbate/menadione. Due to the major role of Hsp90 in stabilizing oncogenic and mutated proteins, these results may have potential clinical applications.

That Maurice Merleau-Ponty derived important inspirations from Cassirer's philosophy of symbolic forms for his theory of perception is obviously, yet is has so far hardly been studied systematically. This paper attempts to fill this gap, and develops the thesis that Merleau-Ponty's readings mistook in part the potential in Cassirer's theory of the subject and by no means exhausted it.

Heterogeneities in tumor blood flow are associated with cyclic changes in pO(2) or cyclic hypoxia. A major difference from O(2) diffusion-limited or chronic hypoxia is that the tumor vasculature itself may be directly influenced by the fluctuating hypoxic environment, and the reoxygenation phases complicate the usual hypoxia-induced phenotypic pattern. Here, we determined the cyclic hypoxia-driven pathways that modulate hypoxia inducible factor (HIF)-1alpha abundance in endothelial cells to identify possible therapeutic targets. We found that exposure of endothelial cells to cycles of hypoxia/reoxygenation led to accumulation of HIF-1alpha during the hypoxic periods and the phosphorylation of protein kinase B (Akt), extracellular regulated kinase (ERK) and endothelial nitric oxide synthase (eNOS) during the reoxygenation phases. We identified stimulation of mitochondrial respiration and activation of the phosphoinositide-3 kinase (PI3K)/Akt pathway during intervening reoxygenation periods as major triggers of the stabilization of HIF-1alpha. We also found that the NOS inhibitor nitro-l-arginine methyl ester further stimulated the cyclic hypoxia-driven HIF-1alpha accumulation and the associated gain in endothelial cell survival, thereby mirroring the effects of a PI3K/Akt inhibitor. However, combination of both drugs resulted in a net reduction in HIF-1alpha and a dramatic in decrease in endothelial cell survival. In conclusion, this study identified cyclic hypoxia, as reported in many tumor types, as a unique biological challenge for endothelial cells that promotes their survival in a HIF-1alpha-dependent manner through phenotypic alterations occurring during the reoxygenation periods. These observations also indicate the potential of combining Akt-targeting drugs with anti-angiogenic drugs, in particular those interfering with the NO pathway.

Due to its surface properties, especially the presence of amine groups, plasma polymerized allylamine (PPAA) is involved in a large range of applications. Most of them are related to biology and biochemistry. In this work, we demonstrate a quick and simple way to produce PPAA thin films by conventional DC magnetron sputtering. To the best of our knowledge, this is the first method not involving radiofrequency (RF) or microwave discharges. In this paper, we show preliminary characterizations on the produced films (bulk analysis) carried out by ion beam techniques. The Hydrogen content in the whole layers was measured by ERD. The surface composition was probed by XPS on pentafluorobenzaldehyde (PFBA) derivatized films: the maximum concentration of amine groups is 1.6%. (C) 2008 Elsevier B.V. All rights reserved.

Purpose: The biological status of nitrite recently evolved from an inactive end product of nitric oxide catabolism to the largest intravascular and tissue storage of nitric oxide (NO). Although low partial O2 pressure favors enzymatic reconversion of nitrite into NO, low pH supports a nonenzymatic pathway. Because hypoxia and acidity are characteristics of the tumor microenvironment, we examined whether nitrite injection could preferentially lead to NO production in tumors and influence response to treatments. Experimental Design: The effects of nitrite were evaluated on arteriole vasorelaxation, tumor cell respiration and tumor blood flow, oxygenation, and response to radiotherapy. Results: We first showed that a small drop in pH (−0.6 pH unit) favored the production of bioactive NO from nitrite by documenting a higher cyclic guanosine 3′,5′-monophosphate–dependent arteriole vasorelaxation. We then documented that an i.v. bolus injection of nitrite to tumor-bearing mice led to a transient increase in partial O2 pressure in tumor but not in healthy tissues. Blood flow measurements failed to reveal an effect of nitrite on tumor perfusion, but we found that O2 consumption by nitrite-exposed tumor cells was decreased at acidic pH. Finally, we showed that low dose of nitrite could sensitize tumors to radiotherapy, leading to a significant growth delay and an increase in mouse survival (versus irradiation alone). Conclusions: This study identified low pH condition (encountered in many tumors) as an exquisite environment that favors tumor-selective production of NO in response to nitrite systemic injection. This work opens new perspectives for the use of nitrite as a safe and clinically applicable radiosensitizing modality.

AIMS: In endothelial cells, caveolin-1 (cav-1) is known to negatively modulate the activation of endothelial nitric oxide synthase, a key regulator of blood pressure (BP). However, the impact of genetic alteration of cav-1 on vascular nitric oxide (NO) production and BP homeostasis in vivo is unknown. METHODS AND RESULTS: We used spectral analysis of systolic blood pressure (SBP) variability in mice chronically equipped with telemetry implants to identify frequency ranges (0.05-0.4 Hz; very low frequency, VLF) specifically responding to NO, independently of changes in absolute BP or systemic neurohormone levels. VLF variability was inversely correlated to aortic vasodilator-stimulated Ser(239) phosphoprotein (VASP) phosphorylation, reflecting NO bioactivity. We show that mice deficient in cav-1 have decreased VLF variability paralleled with enhanced systemic and vascular production of NO at unchanged mean SBP levels. Conversely, VLF variability was increased upon acute injection of mice, with a peptide containing the caveolin-scaffolding domain (CSD; residues 82-101) fused to an internalization sequence of antennapedia that decreased vascular and circulating NO in vivo. CONCLUSION: These data highlight the functional importance of cav-1 for the production of bioactive NO in conduit arteries and its control of central BP variability. Given the impact of the latter on target organ damage, this raises the interest for genetic, pharmacological, or molecular interventions that modulate cav-1 expression in diseases with NO-dependent endothelial dysfunction.

Expansion of the thyroid microvasculature is the earliest event during goiter formation, always occurring before thyrocyte proliferation; however, the precise mechanisms governing this physiological angiogenesis are not well understood. Using reverse transcriptase-polymerase chain reaction and immunohistochemistry to measure gene expression and laser Doppler to measure blood flow in an animal model of goitrogenesis, we show that thyroid angiogenesis occurred into two successive phases. The first phase lasted a week and involved vascular activation; this process was thyroid-stimulating hormone (TSH)-independent and was directly triggered by expression of vascular endothelial growth factor (VEGF) by thyrocytes as soon as the intracellular iodine content decreased. This early reaction was followed by an increase in thyroid blood flow and endothelial cell proliferation, both of which were mediated by VEGF and inhibited by VEGF-blocking antibodies. The second, angiogenic, phase was TSH-dependent and was activated as TSH levels increased. This phase involved substantial up-regulation of the major proangiogenic factors VEGF-A, fibroblast growth factor-2, angiopoietin 1, and NG2 as well as their receptors Flk-1/VEGFR2, Flt-1/VEGFR1, and Tie-2. In conclusion, goiter-associated angiogenesis promotes thyroid adaptation to iodine deficiency. Specifically, as soon as the iodine supply is limited, thyrocytes produce proangiogenic signals that elicit early TSH-independent microvascular activation; if iodine deficiency persists, TSH plasma levels increase, triggering the second angiogenic phase that supports thyrocyte proliferation.

Radioimmunotherapy uses monoclonal antibodies that are still labeled with only one radioactive atom. The aim of this paper is to assess, by means of MCNPX simulations, the doses delivered around and throughout a solid tumor when the radioactive atom linked to each antibody is replaced by a 5 nm diameter nanoparticle composed of numerous radionuclides. A new model for a spherical vascularized tumor has been developed in which the antibody distributions inside the tumor can be uniform or heterogeneous. It is also possible to simulate a central necrotic core inside the tumor where the concentration of radiolabeled antibodies is assumed to be zero. Dosimetry calculations have been performed for the beta-emitting radionuclide Y290 O3. Preliminary results show that the irregularity of vasculature and the presence of a necrotic core have a noticeable influence on the deposited dose profiles. Moreover, with a total activity of 5 and 34 MBq for tumor radii of 0.5 and 1.0 cm, respectively, viable tumor cells can receive doses of up to 50 Gy, even if high nonuniformity of the total activity is observed in the tumor. These simulations still require accurate information about antibody characteristics and necrosis sizes but clearly confirm that the use of monoclonal antibodies conjugated to nanoparticles could lead to a considerable enhancement of treatment efficacy against cancer. © 2007 American Association of Physicists in Medicine.

Vascularized tumors are exposed to intermittent hypoxia, that is, hypoxia followed by periods of reoxygenation. Abnormal structure and dysfunction of tumor blood vessels are responsible for these conditions. These repeated short periods of hypoxia concern tumor cells as well as endothelial cells. However, the effects of intermittent hypoxia are poorly understood. The aim of this study was to investigate the effects of intermittent hypoxia on endothelial cells and particularly on HIF-1α, a central actor in adaptive response to hypoxia. For that, endothelial cells were exposed to four repeated cycles of 1-h hypoxia followed by 30 min of reoxygenation. We showed that repeated cycles of hypoxia/reoxygenation induced a modification in HIF-lα phosphorylation pattern: a progressive increase in HIF-1α phosphorylated form was observed during the hypoxic periods. Activation of p42/p44, Akt and PKA was observed in parallel. PKA was shown to be involved in the phosphorylation of HIF-lα under intermittent hypoxia, while p42/p44 and Akt were not. As HIF-1 activity is often associated with enhanced cell survival, a better knowledge of the effects of intermittent hypoxia on endothelial cells and the highlight of particular mechanisms induced by intermittent hypoxia are essential to understand the behavior of endothelial cells during neo-angiogenesis. © 2007 Elsevier B.V. All rights reserved.

The identification of a critical role of tumour stroma in the regulation of tumour interstitial fluid pressure and the simultaneous discovery of the impact of anti-angiogenic drugs on tumour hemodynamics have provided new potential for improving tumour delivery of anti-cancer drugs. Here, we review the most recent studies investigating how tumour-associated fibroblasts and macrophages as well as the extracellular matrix itself may be targeted to facilitate delivery of both low-molecular weight drugs and macromolecules. In addition, we summarize the current understanding of the use of vasoactive compounds, radiotherapy and vascular-disrupting agents as potential adjuvants to maximize tumour delivery of anti-cancer drugs. The impact of these strategies on the diffusive and convective modes of drug transport is discussed in the light of Fick's and Starling's laws. Finally, we discuss how transcytosis through caveolae may also be exploited to optimize the selective delivery of conventional chemotherapy to the subendothelial tumour cell compartment.

The biology of NO (nitric oxide) is poorly explained by the activity of the free radical NO (·NO) itself. Although·NO acts in an autocrine and paracrine manner, it is also in chemical equilibrium with other NO species that constitute stable stores of NO bioactivity. Among these species, S-nitrosylated hemoglobin ( S-nitrosohemoglobin; SNO-Hb) is an evolved transducer of NO bioactivity that acts in a responsive and exquisitely regulated manner to control cardiopulmonary and vascular homeostasis. In SNO-Hb, O2sensing is dynamically coupled to formation and release of vasodilating SNOs, endowing the red blood cell (RBC) with the capacity to regulate its own principal function, O2delivery, via regulation of blood flow. Analogous, physiological actions of RBC SNO-Hb also contribute to central nervous responses to blood hypoxia, the uptake of O2from the lung to blood, and baroreceptor-mediated control of the systemic flow of blood. Dysregulation of the formation, export, or actions of RBC-derived SNOs has been implicated in human diseases including sepsis, sickle cell anemia, pulmonary arterial hypertension, and diabetes mellitus. Delivery of SNOs by the RBC can be harnessed for therapeutic gain, and early results support the logic of this approach in the treatment of diseases as varied as cancer and neonatal pulmonary hypertension.

Objectives We investigated whether retroinfusion of liposomal endothelial nitric oxide synthase (eNOS) S1177D complementary deoxyribonucleic acid (cDNA) would affect neovascularization and function of the ischemic myocardium. Background Recently, we demonstrated the feasibility of liposomal eNOS cDNA transfection via retroinfusion in a model of acute myocardial ischemia/reperfusion. In the present study, we used this approach to target a phosphomimetic eNOS construct (eNOS S1177D) into chronic ischemic myocardium in a pig model of hibernation. Methods Pigs (n = 6/group) were subjected to percutaneous implantation of a reduction stent graft into the left anterior descending artery (LAD), inducing total occlusion within 28 days. At day 28, retroinfusion of saline solution containing liposomal green fluorescent protein or eNOS S1177D cDNA (1.5 mg/animal, 2 x 10 min) was performed. Furthermore, L-nitroarginine-methylester (L-NAME) was applied orally from day 28, where indicated. At day 28 and day 49, fluorescent microspheres were injected into the left atrium for perfusion analysis. Regional functional reserve (at atrial pacing 140/min) was assessed at day 49 by subendocardial segment shortening (SES) (sonomicrometry, percent of ramus circumflexus region). Results The eNOS S1177D overexpression increased endothelial cell proliferation as well as capillary and collateral growth at day 49. Concomitantly, eNOS S1177D overexpression enhanced regional myocardial perfusion from 62 +/- 4% (control) to 77 +/- 3% of circumflex coronary artery-perfused myocardium, unless L-NAME was co-applied (69 +/- 5%). Similarly, eNOS S1177D cDNA improved functional reserve of the LAD (33 +/- 5% vs. 7 +/- 3% of circumflex coronary artery-perfused myocardium), except for L-NAME coapplication (13 +/- 6%). Conclusions Retroinfusion of eNOS S1177D cDNA induces neovascularization via endothelial cell proliferation and collateral growth. The resulting gain of perfusion enables an improved functional reserve of the hibernating myocardium.

Tumors may evade immune responses at multiple levels, including through a defect in the lymphocyte-vessel wall interactions. The angiogenic nature of endothelial cells (EC) lining tumor blood vessels may account for such anergy. In this study, we examined whether mechanisms other than down-regulation of adhesion molecules could be involved, particularly signaling pathways dependent on the caveolae platforms. To mimic the influence of the tumor microenvironment, EC were exposed to TNF-α and the proangiogenic vascular endothelial growth factor (VEGF). We identified a dramatic inhibition of lymphocyte adhesion on activated EC following either short or long VEGF pretreatments. We further documented that VEGF did not influence the abundance of major adhesion molecules, but was associated with a defect in ICAM-1 and VCAM-1 clustering at the EC surface. We also found that overexpression of the caveolar structural protein, caveolin-1, overcame the VEGF-mediated inhibition of adhesion and restored ICAM-1 clustering. Conversely, EC transduction with a caveolin-1 small interfering RNA reduced the TNF-α-dependent increase in adhesion. Finally, we identified VEGF-induced NO production by the endothelial NO synthase as the main target of the changes in caveolin-1 abundance. We found that the NO synthase inhibitor N-nitro-l-arginine methyl ester could reverse the inhibitory effects of VEGF on lymphocyte adhesion and EC cytoskeleton rearrangement. Symmetrically, a NO donor was shown to prevent the ICAM clustering-mediated lymphocyte adhesion, thereby recapitulating the effects of VEGF. In conclusion, this study provides new insights on the mechanisms leading to the tumor EC anergy vs immune cells and opens new perspectives for the use of antiangiogenic strategies as adjuvant approaches to cancer immunotherapy.

Purpose: We hypothesized that glucocorticoids may enhance tumor radiosensitivity by increasing tumor oxygenation (pO2) through inhibition of mitochondrial respiration.Experimental Design: The effect of three glucocorticoids (hydrocortisone, dexamethasone, and prednisolone) on pO2 was studied in murine TLT liver tumors and FSaII fibrosarcomas. At the time of maximum pO2 (tmax, 30 min after administration), perfusion, oxygen consumption, and radiation sensitivity were studied. Local pO2 measurements were done using electron paramagnetic resonance. The oxygen consumption rate of tumor cells after in vivo glucocorticoid administration was measured using high-frequency electron paramagnetic resonance. Tumor perfusion and permeability measurements were assessed by dynamic contrast-enhanced magnetic resonance imaging.Results: All glucocorticoids tested caused a rapid increase in pO2. At tmax, tumor perfusion decreased, indicating that the increase in pO2 was not caused by an increase in oxygen supply. Also at tmax, global oxygen consumption decreased. When irradiation (25 Gy) was applied at tmax, the tumor radiosensitivity was enhanced (regrowth delay increased by a factor of 1.7).Conclusion: These results show the potential usefulness of the administration of glucocorticoids before irradiation.

OBJECTIVE: Myogenic tone, which has a major role in the regulation of local blood flow, refers to the ability of vascular smooth muscle to adapt its contractility to changes in transmural pressure. Although Rho-kinase is involved in myogenic tone, the pathway involved remains unclear, especially concerning translocation to the plasma membrane and activation of RhoA. As caveolae have a key role in the signal transduction of membrane-bound proteins, we tested the hypothesis that RhoA might be activated by pressure and that its activation might involve caveolin-1, which has been shown to be involved in vascular functions. METHODS: Myogenic tone was studied in isolated rat mesenteric resistance arteries (118+/-15 microm internal diameter with a pressure of 75 mmHg) submitted to pressure steps (25, 75, and 150 mmHg). Pharmacological blockade of caveolae or RhoA-Rho-kinase pathway was assessed by confocal microscopy in pressurized arteries to analyze protein co-localization and by co-immunoprecipitation in order to confirm protein interactions. Caveolin-1-deficient mice were used to confirm the role of the protein in myogenic tone. RESULTS: Pressure-induced myogenic tone was significantly reduced by RhoA inactivation with TAT-C3 (90.5% inhibition at 150 mmHg) and by the Rho-kinase inhibitor Y27632 (91.8% inhibition at 150 mmHg). In arteries pressurized at 150 mmHg, RhoA was localized to the plasma membrane (localization by confocal microscopy and increased quantity of RhoA in the membrane fraction after protein extraction). Thus, translocation of RhoA to the plasma membrane was associated with pressure-induced tone. In addition, caveolae disruption with methyl-beta-cyclodextrin reduced myogenic tone by 66% at 150 mmHg. Further, myogenic tone was significantly reduced to 24% of control in caveolin-1-deficient mice (active tone was 32.3+/-2.8 microm and 9.1+/-3.7 microm in +/+ and -/- mice, respectively, n = 5 per group), suggesting a key role of caveolin-1 in myogenic tone. Finally, RhoA and caveolin-1 co-immunoprecipitation and co-localization significantly increased when myogenic tone developed at 150 mmHg (co-localization showed 26+/-13% merging at 25 mmHg versus 97+/-21% at 150 mmHg, n = 5). Co-immunoprecipitation was prevented by TAT-C3 and by methyl beta-cyclodextrin. CONCLUSION: RhoA activation is critical for the development of myogenic tone in resistance arteries. This activation induced translocation of RhoA to the plasma membrane within caveolae, where the interaction of RhoA with caveolin-1 leads selectively to the activation of a Rho-kinase-dependent force development.

Nuclear magnetic resonance spectroscopy of fluorine-19 ((19)F NMR) has proven useful for evaluating kinetics of fluorinated chemotherapy drugs in tumors in vivo. This work investigated how three perfusion-enhancing vascular modifiers (BQ123, thalidomide, and Botulinum neurotoxin type A [BoNT-A]) would affect the chemotherapeutic efficacy of gemcitabine, a fluorinated drug widely used in human cancer treatment. Murine tumor growth experiments demonstrated that only BoNT-A showed a strong trend to enhance tumor growth inhibition by gemcitabine (1.7 days growth delay, P = 0.052, Student t-test). In accord with these results, (19)F NMR experiments showed that only BoNT-A increased significantly the uptake of gemcitabine in tumors (50% increase, P = 0.0008, Student t-test). Further experiments on gemcitabine kinetics (NMR vs time) and distribution ((19)F MRI) confirmed the uptake-enhancing properties of BoNT-A. The results of this study demonstrate that (19)F NMR can monitor modulation of the pharmacokinetics of fluorinated chemotherapy drugs in tumors. The results also show that (19)F NMR data can give a strong indication of the effectiveness of perfusion-enhancing vascular modifiers for improving gemcitabine chemotherapy in murine tumors. (19)F NMR is a promising tool for preclinical evaluation of such vascular modifiers and may ultimately be used in the clinic to monitor how these modifiers affect chemotherapy.

Recently, a new generation of highly promising inhibitors bearing β-keto-enol functionality has emerged. Reported herein is the first synthesis and use of novel designed drugs based on the β-keto-enol group embedded with heterocyclic moieties such as pyrazole, pyridine, and furan, prepared in a one-step procedure by mixed Claisen condensation. All the newly synthesized compounds were characterized by FT-IR, ¹H-NMR, (13)C-NMR, ESI/LC-MS, elemental analysis, and evaluated for their in vitro antiproliferative activity against breast cancer (MDA-MB241) human cell lines and fungal strains (Fusarium oxysporum f.sp albedinis FAO). Three of the synthesized compounds showed potent activity against fungal strains with IC50 values in the range of 0.055-0.092 µM. The results revealed that these compounds showed better IC50 values while compared with positive controls.

PEGylated chitosan-based nanoparticles offer attractive platforms for siRNA cocktail delivery into tumors. Still, therapeutic efficacy requires us to select a rational combination of siRNAs and an efficient tumor delivery after systemic administration. Here, we showed that non-covalent PEGylation of chitosan-based nanoparticles loaded with siRNA targeting two key transporters of energy fuels for cancer cells, namely the lactate transporter MCT1 and the glutamine transporter ASCT2, could lead to significant antitumor effects. As a ligand, we tested variations of the prototypical RGD peptidomimetic (RGDp). A higher siRNA delivery was obtained with naphthyridine-containing RGDp randomly conjugated on the PEG chain by clip photochemistry and the use of a lipophilic linker than when using traditional chain-end grafting and RGDp with a hydrophilic linker. The antiproliferative effects resulting from ASCT2 and MCT1 silencing were validated separately in vitro in conditions mimicking specific metabolic profiles of cancer cells and in vivo upon concomitant delivery. The combination of those siRNA and the selected components of targeted RGDp nanoparticles led to a dramatic tumor growth inhibition upon peri-tumoral but also systemic administration in mice. Altogether these data emphasize the convenience of using non-covalent PEGylated chitosan particles to produce sheddable stealth protection compatible with an efficient siRNA delivery in tumors.

Measuring intracellular metabolism has increasingly led to important insights in biomedical research. (13)C tracer analysis, although less information-rich than quantitative (13)C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting (13)C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments. ispartof: Current Opinion in Biotechnology vol:34 pages:189-201 ispartof: location:England status: published

Transgenic and gene knockout rodent models are primordial to study pathophysiological processes in cardiovascular research. Over time, cardiac MRI has become a gold standard for in vivo evaluation of such models. Technical advances have led to the development of magnets with increasingly high field strength, allowing specific investigation of cardiac anatomy, global and regional function, viability, perfusion or vascular parameters. The aim of this report is to provide a review of the various sequences and techniques available to image mice on 7-11.7 T magnets and relevant to the clinical setting in humans. Specific technical aspects due to the rise of the magnetic field are also discussed.

We investigated the impact of oxygenation status (measured by EPR oximetry) on the uptake of (18)F-FDG (measured by PET) in two different tumor models during a carbogen breathing challenge. We observed a significant drop in (18)F-FDG uptake under carbogen breathing that suggests a rapid metabolic adaptation to the oxygen environment.

Radioimmunotherapy has shown that the use of monoclonal antibodies combined with a radioisotope like 131I or 90Y still remains ineffective for solid and radioresistant tumour treatment. Previous simulations have revealed that an increase in the number of 90Y labelled to each antibody or nanoobject could be a solution to improve treatment output. It now seems important to assess the treatment output and toxicity when radionuclides such as 90Y, 177Lu, 131I, 124I, and 188Re are used. Tumour control probability (TCP) and normal tissue complication probability (NTCP) curves versus the number of radionuclides per nanoobject were computed with MCNPX to evaluate treatment efficacy for solid tumours and to predict the incidence of surrounding side effects. Analyses were carried out for two solid tumour sizes of 0.5 and 1.0 cm radius and for nanoobject (i.e., a radiolabelled antibody) distributed uniformly or nonuniformly throughout a solid tumour (e.g., Non-small-cell-lung cancer (NSCLC)). 90Y and 188Re are the best candidates for solid tumour treatment when only one radionuclide is coupled to one carrier. Furthermore, regardless of the radionuclide properties, high values of TCP can be reached without toxicity if the number of radionuclides per nanoobject increases.

Hypoxia is a common feature in tumors associated with an increased resistance of tumor cells to therapies. In addition to O2 diffusion–limited hypoxia, another form of tumor hypoxia characterized by fluctuating changes in pO2 within the disorganized tumor vascular network is described. Here, we postulated that this form of intermittent hypoxia promotes endothelial cell survival, thereby extending the concept of hypoxia-driven resistance to the tumor vasculature. We found that endothelial cell exposure to cycles of hypoxia reoxygenation not only rendered them resistant to proapoptotic stresses, including serum deprivation and radiotherapy, but also increased their capacity to migrate and organize in tubes. By contrast, prolonged hypoxia failed to exert protective effects and even seemed deleterious when combined with radiotherapy. The use of hypoxia-inducible factor-1α (HIF-1α)–targeting small interfering RNA led us to document that the accumulation of HIF-1α during intermittent hypoxia accounted for the higher resistance of endothelial cells. We also used an in vivo approach to enforce intermittent hypoxia in tumor-bearing mice and found that it was associated with less radiation-induced apoptosis within both the vascular and the tumor cell compartments (versus normoxia or prolonged hypoxia). Radioresistance was further ascertained by an increased rate of tumor regrowth in irradiated mice preexposed to intermittent hypoxia and confirmed in vitro using distinctly radiosensitive tumor cell lines. In conclusion, we have documented that intermittent hypoxia may condition endothelial cells and tumor cells in such a way that they are more resistant to apoptosis and more prone to participate in tumor progression. Our observations also underscore the potential of drugs targeting HIF-1α to resensitize the tumor vasculature to anticancer treatments. (Cancer Res 2006; 66(24): 11736-44)

Emerging preclinical studies support the concept of a transient “normalization” of tumor vasculature during the early stage of antiangiogenic treatment, with possible beneficial effects on associated radiotherapy or chemotherapy. One key issue in this area of research is to determine whether this feature is common to all antiangiogenic drugs and whether the phenomenon occurs in all types of tumors. In the present study, we characterized the evolution of the tumor oxygenation (in transplantable liver tumor and FSAII tumor models) after administration of SU5416, an antagonist of the vascular endothelial growth factor receptor. SU5416 induced an early increase in tumor oxygenation [measured by electronic paramagnetic resonance (EPR)], which did not correlate with remodeling of the tumor vasculature (assessed by CD31 labeling using immunohistochemistry) or with tumor perfusion (measured by dynamic contrast enhanced-magnetic resonance imaging). Inhibition of mitochondrial respiration (measured by EPR) was responsible for this early reoxygenation. Consistent with these unique findings in the tumor microenvironment, we found that SU5416 potentiated tumor response to radiotherapy but not to chemotherapy. In addition to the fact that the characterization of the tumor oxygenation is essential to enable correct application of combined therapies, our results show that the long-term inhibition of oxygen consumption is a potential novel target in this class of compounds. (Cancer Res 2006; 66(19): 9698-704)

OBJECTIVES: Amlodipine is a calcium channel blocker (CCB) known to stimulate nitric oxide production from endothelial cells. Whether this ancillary property can be related to the capacity of amlodipine to concentrate and alter the structure of cholesterol-containing membrane bilayers is a matter of investigation. Here, we reasoned that since the endothelial nitric oxide synthase is, in part, expressed in cholesterol-rich plasmalemmal microdomains (e.g., caveolae and rafts), amlodipine could interfere with this specific locale of the enzyme and thereby modulate NO production in endothelial cells. METHODS AND RESULTS: Using a method combining lubrol-based extraction and subcellular fractionation on sucrose gradient, we found that amlodipine, but not verapamil or nifedipine, induced the segregation of endothelial NO synthase (eNOS) from caveolin-enriched low-density membranes (8+/-2% vs. 42+/-3% in untreated condition; P

Maturation of tumor vasculature involves the recruitment of pericytes that protect the endothelial tubes from a variety of stresses, including antiangiogenic drugs. Mural cells also provide mature tumor blood vessels with the ability to either relax or contract in response to substances present in the tumor microenvironment. The observed cyclic alterations in tumor blood flow and the associated deficit in chemotherapeutic drug delivery could in part arise from this vasomodulatory influence. To test this hypothesis, we focused on endothelin-1 (ET-1), which, besides its autocrine effects on tumor cell growth, is a powerful vasoconstrictor. We first document that an ETA receptor antagonist induced relaxation of microdissected tumor arterioles and selectively and quantitatively increased tumor blood flow in experimental tumor models. We then combined dye staining of functional vessels, fluorescent microsphere-based mapping, and magnetic resonance imaging to identify heterogeneities in tumor blood flow and to examine the reversibility of such phenomena. Data from all these techniques concurred to show that administration of an ETA receptor antagonist could reduce the extent of underperfused tumor areas, proving the key role of vessel tone variations in tumor blood flow heterogeneity. We also provide evidence that ETA antagonist administration could, despite an increase in tumor interstitial fluid pressure, improve access of cyclophosphamide to the tumor compartment and significantly influence tumor growth. In conclusion, tumor endogenous ET-1 production participates largely in the temporal and spatial variations in tumor blood flow. ETA antagonist administration may wipe out such heterogeneities, thus representing an adjuvant strategy that could improve the delivery of conventional chemotherapy to tumors. [Mol Cancer Ther 2006;5(6):1620–7]

Virtually all cell types within the myocardium express caveolae, where cell-specific isoforms of caveolin both maintain the structural organisation of these cholesterol-rich of the plasmalemma and serve as scaffolds for the dynamic constitution of "signalosomes", or hubs concentrating numerous transmembrane signaling proteins and their effectors. Analysis of the phenotype of mice with genetic deletion or overexpression of specific caveolin isoforms has provided key evidence for the importance of caveolins and caveolae in several aspects of the cardiovascular biology, including vascular contractility, lipid metabolism, angiogenesis, or the control of cardiac hypertrophy. Among specific protein-protein interactions involving caveolins in cardiac tissue, these genetic models unequivocally confirmed the functional importance of the dynamic association of the endothelial isoform of nitric oxide synthase (eNOS) for its post-translational regulation in endothelial cells and cardiac myocytes, which bears on the enzyme's capacity to modulate nitric oxide (NO)-dependent endothelial function, angiogenesis, and excitation-contraction coupling. We will review the current understanding of this regulation of eNOS (and potentially other NOS isoforms) through protein-protein interactions involving several G-protein-coupled receptors and other allosteric modulators in the context of emerging paradigms on the regulation of cardiac function by NO.

PURPOSE: Structural and functional abnormalities in the tumor vascular network are considered factors of resistance of solid tumors to cytotoxic treatments. To increase the efficacy of anticancer treatments, efforts must be made to find new strategies for transiently opening the tumor vascular bed to alleviate tumor hypoxia (source of resistance to radiotherapy) and improve the delivery of chemotherapeutic agents. We hypothesized that Botulinum neurotoxin type A (BoNT-A) could interfere with neurotransmitter release at the perivascular sympathetic varicosities, leading to inhibition of the neurogenic contractions of tumor vessels and therefore improving tumor perfusion and oxygenation. EXPERIMENTAL DESIGN: To test this hypothesis, BoNT-A was injected locally into mouse tumors (fibrosarcoma FSaII, hepatocarcinoma transplantable liver tumor), and electron paramagnetic resonance oximetry was used to monitor pO(2) in vivo repeatedly for 4 days. Additionally, contrast-enhanced magnetic resonance imaging was used to measure tumor perfusion in vivo. Finally, isolated arteries were mounted in wire myograph to monitor specifically the neurogenic tone developed by arterioles that were co-opted by the surrounding growing tumor cells. RESULTS: Using these tumor models, we showed that local administration of BoNT-A (two sites; dose, 29 units/kg) substantially increases tumor oxygenation and perfusion, leading to a substantial improvement in the tumor response to radiotherapy (20 Gy of 250-kV radiation) and chemotherapy (cyclophosphamide, 50 mg/kg). This observed therapeutic gain results from an opening of the tumor vascular bed by BoNT-A because we showed that BoNT-A could inhibit neurogenic tone in the tumor vasculature. CONCLUSIONS: The opening of the vascular bed induced by BoNT-A offers a way to significantly increase the response of tumors to radiotherapy and chemotherapy.

Acute hypoxia (transient cycles of hypoxia-reoxygenation) is known to occur in solid tumors and is generally believed to be caused by tumor blood flow instabilities. It was recently demonstrated that T2*-weighted (T2*w) gradient echo (GRE) MRI is a powerful non-invasive method for investigating periodic changes in tumor pO2 and blood flow associated with acute hypoxia. Here, the possible correlation between tumor vessel immaturity, vessel functionality and T2*w GRE signal fluctuations was investigated. Intramuscularly implanted FSa II fibrosarcoma-bearing mice were imaged at 4.7 T. Maps of spontaneous fluctuations of MR signal intensity in tumor tissue during air breathing were obtained using a T2*w GRE sequence. This same sequence was also employed during air-5% CO2 breathing (hypercapnia) and carbogen breathing (hypercapnic hyperoxia) to obtain parametric maps representing vessel maturation and vessel function, respectively. Vascular density, vessel maturation and vessel perfusion were also assessed histologically by using CD31 labeling, alpha-smooth muscle actin immunoreactivity and Hoechst 33242 labeling, respectively. About 50% of the tumor fluctuations occurred in functional tumor regions (responsive to carbogen) and 80% occurred in tumor regions with immature vessels (lack of response to hypercapnia). The proportion of hypercapnia-responsive voxels were found to be twice as great in fluctuating than in non-fluctuating tumor areas (P: 0.22 vs 0.13). Similarly, the proportion of functional voxels was somewhat greater in fluctuating tumor areas (P: 0.54 vs 0.43). The mean values of MR signal changes during hypercapnia (VD) and during carbogen breathing (VF) (significant voxels only) were also larger in fluctuating than in non-fluctuating tumor areas (P < 0.05). This study demonstrated that adequate vessel functionality and advanced vessel maturation could explain at least in part the occurrence of spontaneous T2*w GRE signal fluctuations. Functionality and maturation are not required for signal fluctuations, however, because a large fraction of fluctuations could still occur in non-perfused and/or immature vessels.

We hypothesized that nonsteroidal antiinflammatory drugs (NSAIDs) might enhance tumor radiosensitivity by increasing tumor oxygenation (pO2), via either a decrease in the recruitment of macrophages or from inhibition of mitochondrial respiration. The effect of four NSAIDs (diclofenac, indomethacin, piroxicam, and NS-398) on pO2 was studied in murine TLT liver tumors and FSaII fibrosarcomas. At the time of maximum pO2 (tmax, 30 minutes after administration), perfusion, oxygen consumption, and radiation sensitivity were studied. Local pO2 measurements were done using electron paramagnetic resonance. Tumor perfusion and permeability measurements were assessed by dynamic contrast-enhanced magnetic resonance imaging. The oxygen consumption rate of tumor cells after in vivo NSAID administration was measured using high-frequency electron paramagnetic resonance. Tumor-infiltrating macrophage localization was done with immunohistochemistry using CD11b antibody. All the NSAIDs tested caused a rapid increase in pO2. At tmax, tumor perfusion decreased, indicating that the increase in pO2 was not caused by an increase in oxygen supply. Also at tmax, global oxygen consumption decreased but the amount of tumor-infiltrating macrophages remained unchanged. Our study strongly indicates that the oxygen effect caused by NSAIDs is primarily mediated by an effect on mitochondrial respiration. When irradiation (18 Gy) was applied at tmax, the tumor radiosensitivity was enhanced (regrowth delay increased by a factor of 1.7). These results show the potential utility of an acute administration of NSAIDs for radiosensitizing tumors, and shed new light on the mechanisms of NSAID radiosensitization. These results also provide a new rationale for the treatment schedule when combining NSAIDs and radiotherapy.

Caveolae are plasmalemmal invaginations formed by the sequestration of cholesterol and glycosphingolipids with self-associating molecules named caveolins, resulting in a platform for the assembly of signaling complexes at the surface of the cell. The enrichment of the endothelial nitric oxide synthase in caveolae and its direct interaction with caveolin both account for the exquisite regulation of nitric oxide production in cardiovascular tissues. Dissection of the angiogenic signaling cascade downstream vascular endothelial growth factor recently led to recognition that although the former enables the compartmentation of endothelial nitric oxide synthase and optimizes the process leading to its activation, the latter maintains the enzyme in its inactivated state in the absence of stimulation. Alteration in caveolin abundance or subcellular location may lead endothelial cells or cardiac myocytes to favor one mode of regulation over the other and thereby alter the subtle equilibrium governing nitric oxide production in these cells.

Activation of an innate immune response is among the first lines of defense after tissue injury. Restoring blood flow to the site of injured tissue is often a necessary prerequisite for mounting an initial immune response to pathogens and for subsequent initiation of a successful repair of wounded tissue. The multiple links among pathogen recognition and suppression, increased angiogenesis, and tissue repair are the topics of this review, which examines of the roles of antimicrobial peptides, mammalian toll-like receptors (TLRs), inflammatory cytokines, and putative “danger” signals, among other signaling pathways, in triggering, sustaining, and then terminating an angiogenic response.

Nitric oxide (NO) is a powerful angiogenic mediator acting downstream of vascular endothelial growth factor (VEGF). Both the endothelial NO synthase (eNOS) and the VEGFR-2 receptor colocalize in caveolae. Because the structural protein of these signaling platforms, caveolin, also represses eNOS activity, changes in its abundance are likely to influence the angiogenic process in various ways. In this study, we used mice deficient for the caveolin-1 gene (Cav −/− ) to examine the impact of caveolae suppression in a model of adaptive angiogenesis obtained after femoral artery resection. Evaluation of the ischemic tissue perfusion and histochemical analyses revealed that contrary to Cav +/+ mice, Cav −/− mice failed to recover a functional vasculature and actually lost part of the ligated limbs, thereby recapitulating the effects of the NOS inhibitor l -NAME administered to operated Cav +/+ mice. We also isolated endothelial cells (ECs) from Cav −/− aorta and showed that on VEGF stimulation, NO production and endothelial tube formation were dramatically abrogated when compared with Cav +/+ ECs. The Ser1177 eNOS phosphorylation and Thr495 dephosphorylation but also the ERK phosphorylation were similarly altered in VEGF-treated Cav −/− ECs. Interestingly, caveolin transfection in Cav −/− ECs redirected the VEGFR-2 in caveolar membranes and restored the VEGF-induced ERK and eNOS activation. However, when high levels of recombinant caveolin were reached, VEGF exposure failed to activate ERK and eNOS. These results emphasize the critical role of caveolae in ensuring the coupling between VEGFR-2 stimulation and downstream mediators of angiogenesis. This study also provides new insights to understand the paradoxical roles of caveolin (eg, repressing basal enzyme activity but facilitating activation on agonist stimulation) in cardiovascular pathophysiology.

Although derived from the host tissue, the tumor vasculature is under the influence of the tumor microenvironment and needs to adapt to the resistance to blood flow inherent to the dynamics of tumor growth. Such vascular remodeling can offer selective targets to pharmacologically modulate tumor perfusion and thereby improve the efficacy of conventional anticancer treatments. Radiotherapy and chemotherapy can, indeed, take advantage of a better tumor oxygenation and drug delivery, respectively, both partly dependent on the tumor blood supply. Here, we showed that isolated tumor arterioles mounted in a pressure myograph have the ability, contrary to size-matched healthy arterioles, to contract in response to a transluminal pressure increase. This myogenic tone was exquisitely dependent on the endothelin-1 pathway because it was completely abolished by the selective endothelin receptor A (ETA) antagonist BQ123. This selectivity was additionally supported by the large increase in endothelin-1 abundance in tumors and the higher density of the ETA receptors in tumor vessels. We also documented by using laser Doppler microprobes and imaging that administration of the ETA antagonist led to a significant increase in tumor blood flow, whereas the perfusion in control healthy tissue was not altered. Finally, we provided evidence that acute administration of the ETA antagonist could significantly stimulate tumor oxygenation, as determined by electron paramagnetic resonance oximetry, and increase the efficacy of low-dose, clinically relevant fractionated radiotherapy. Thus, blocking the tumor-selective increase in the vascular endothelin-1/ETA pathway led us to unravel an important reserve of vasorelaxation that can be exploited to selectively increase tumor response to radiotherapy.