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Supplementary Figures S1-S2.

Purpose: We apply positron emission tomography (PET) to elucidate changes in nanocarrier extravasation during the transition from premalignant to malignant cancer, providing insight into the use of imaging to characterize early cancerous lesions and the utility of nanoparticles in early disease.Experimental Design: Albumin and liposomes were labeled with 64Cu (half-life 12.7 hours), and longitudinal PET and CT imaging studies were conducted in a mouse model of ductal carcinoma in situ. A pharmacokinetic model was applied to estimate the tumor vascular volume and permeability.Results: From early time points characterized by disseminated hyperproliferation, the enhanced vascular permeability facilitated lesion detection. During disease progression, the vascular volume fraction increased 1.6-fold and the apparent vascular permeability to albumin and liposomes increased ∼2.5-fold to 6.6 × 10−8 and 1.3 × 10−8 cm/s, respectively, with the accumulation of albumin increasing earlier in the disease process. In the malignant tumor, both tracers reached similar mean intratumoral concentrations of ∼6% ID/cc but the distribution of liposomes was more heterogeneous, ranging from 1% to 18% ID/cc compared with 1% to 9% ID/cc for albumin. The tumor-to-muscle ratio was 17.9 ± 8.1 and 7.1 ± 0.5 for liposomes and albumin, respectively, indicating a more specific delivery of liposomes than with albumin.Conclusions: PET imaging of radiolabeled particles, validated by confocal imaging and histology, detected the transition from premalignant to malignant lesions and effectively quantified the associated changes in vascular permeability. Clin Cancer Res; 17(3); 550–9. ©2010 AACR.

PDF file - 137K, Detailed relationship between the PAP parameter derived through the image-guided model with the classical parameters of solute transport coefficient, clearance, and 30 minute albumin clearance (Cl30).

PDF file - 51K, Representative biodistribution of tissues 48 hours after nanoparticle injection (N=8). Accumulation was significantly greater for insonified tumors (p

Epac1 (exchange protein activated by cAMP) stabilizes the endothelial barrier, but detailed studies are limited by the side effects of pharmacological Epac1 modulators and transient transfections. Here, we compare the key properties of barriers between endothelial cells derived from wild-type (WT) and Epac1-knockout (KO) mice myocardium. We found that KO cell layers, unlike WT layers, had low and cAMP-insensitive trans-endothelial resistance (TER). They also had fragmented VE-cadherin staining despite having augmented cAMP levels and increased protein expression of Rap1, Rac1, RhoA, and VE-cadherin. The simultaneous direct activation of Rac1 and RhoA by CN04 compensated Epac1 loss, since TER was increased. In KO-cells, inhibition of Rac1 activity had no additional effect on TER, suggesting that other mechanisms compensate the inhibition of the Rac1 function to preserve barrier properties. In summary, Epac1 is crucial for baseline and cAMP-mediated barrier stabilization through mechanisms that are at least partially independent of Rac1.

Background Longitudinal monitoring of potential radiotherapy treatment effects can be determined by dynamic contrast-enhanced ultrasound (DCE-US). Purpose To assess functional parameters by means of DCE-US in a murine subcutaneous model of human prostate cancer, and their relationship to dose deposition and time-frame after treatment. A special focus has been placed to evaluate the vascular heterogeneity of the tumor and on the most suitable data analysis approach that reflects this heterogeneity. Material and Methods In vivo DCE-US was acquired 24 h and 48 h after radiation treatment with a single dose of 7.5 Gy and 10 Gy, respectively. Tumor vasculature was characterized pixelwise using the Brix pharmacokinetic analysis of the time-intensity curves. Results Longitudinal changes were detected ( P el, was correlated ( P ≤ 0.05) positively with microvessel density (MVD; rτ = 0.7) and negatively with necrosis (rτ = –0.6) for the treated group. Furthermore, Akep, a parameter related to transcapillary transport properties, was also correlated to MVD (rτ = 0.6, P ≤ 0.05). Conclusion DCE-US has been shown to detect vascular changes at a very early stage after radiotherapy, which is a great advantage since DCE-US is non-invasive, available at most hospitals, and is low in cost compared to other techniques used in clinical practice.

Objective An extension of single- and multi-channel blind deconvolution is presented to improve the estimation of the arterial input function (AIF) in quantitative dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). Methods The Lucy-Richardson expectation-maximization algorithm is used to obtain estimates of the AIF and the tissue residue function (TRF). In the first part of the algorithm, nonparametric estimates of the AIF and TRF are obtained. In the second part, the decaying part of the AIF is approximated by three decaying exponential functions with the same delay, giving an almost noise free semi-parametric AIF. Simultaneously, the TRF is approximated using the adiabatic approximation of the Johnson-Wilson (aaJW) pharmacokinetic model. Results In simulations and tests on real data, use of this AIF gave perfusion values close to those obtained with the corresponding previously published nonparametric AIF, and are more noise robust. Conclusion When used subsequently in voxelwise perfusion analysis, these semi-parametric AIFs should give more correct perfusion analysis maps less affected by recording noise than the corresponding nonparametric AIFs, and AIFs obtained from arteries. Significance This paper presents a method to increase the noise robustness in the estimation of the perfusion parameter values in DCE-MRI.

Aim: Maintenance of the blood and extracellular volume requires tight control of endothelial macromolecule permeability, which is regulated by cAMP signalling. This study probes the role of the cAMP mediators rap guanine nucleotide exchange factor 3 and 4 (Epac1 and Epac2) for in vivo control of microvascular macromolecule permeability under basal conditions. Methods: Epac1 / and Epac2 / C57BL/6J mice were produced and compared with wild-type mice for transvascular flux of radio-labelled albumin in skin, adipose tissue, intestine, heart and skeletal muscle. The transvascular leakage was also studied by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) using the MRI contrast agent Gadomer-17 as probe. Results: Epac1 / mice had constitutively increased transvascular macromolecule transport, indicating Epac1-dependent restriction of baseline permeability. In addition, Epac1 / mice showed little or no enhancement of vascular permeability in response to atrial natriuretic peptide (ANP), whether probed with labelled albumin or Gadomer-17. Epac2 / and wild-type mice had similar basal and ANP-stimulated clearances. Ultrastructure analysis revealed that Epac1 / microvascular interendothelial junctions had constitutively less junctional complex. Conclusion: Epac1 exerts a tonic inhibition of in vivo basal microvascular permeability. The loss of this tonic action increases baseline permeability, presumably by reducing the interendothelial permeability resistance. Part of the action of ANP to increase permeability in wild-type microvessels may involve inhibition of the basal Epac1-dependent activity. publishedVersion

Balb/CJ mice are more sensitive to treatment with angiotensin II (ANG II) and high-salt diet compared with C57BL/6J mice. Together with higher mortality, they develop edema, signs of heart failure, and acute kidney injury. The aim of the present study was to identify differences in renal gene regulation that may affect kidney function and fluid balance, which could contribute to decompensation in Balb/CJ mice after ANG II + salt treatment. Male Balb/CJ and C57BL/6J mice were divided into the following five different treatment groups: control, ANG II, salt, ANG II + salt, and ANG II + salt + N-acetylcysteine. Gene expression microarrays were used to explore differential gene expression after treatment and between the strains. Published data from the Mouse Genome Database were used to identify the associated genomic differences. The glomerular filtration rate (GFR) was measured using inulin clearance, and fluid balance was measured using metabolic cages. Gene ontology enrichment analysis of gene expression microarrays identified glutathione transferase (antioxidant system) as highly enriched among differentially expressed genes. Balb/CJ mice had similar GFR compared with C57BL/6J mice but excreted less Na+ and water, although net fluid and electrolyte balance did not differ, suggesting that Balb/CJ mice may be inherently more prone to decompensation. Interestingly, C57BL/6J mice had higher urinary oxidative stress despite their relative protection from decompensation. In addition, treatment with the antioxidant N-acetylcysteine decreased oxidative stress in C57BL/6J mice, reduced urine excretion, and increased mortality. Balb/CJ mice are more sensitive than C57BL/6J to ANG II + salt, in part mediated by lower oxidative stress, which favors fluid and Na+ retention.

Author(s): Curry, Fitz-Roy E; Taxt, Torfinn; Rygh, Cecilie Brekke; Pavlin, Tina; Bjonrstad, Ronja; Doskeland, Stein Ove; Reed, Rolf K | Abstract: AimEpac1-/- mice, but not Epac2-/- mice have elevated baseline permeability to albumin. This study extends the investigations of how Epac-dependent pathways modulate transvascular exchange in response to the classical inflammatory agent histamine. It also evaluates the limitations of models of blood-to-tissue exchange in transgenic mice in DCE-MRI measurements.MethodsWe measured DCE-MRI signal intensity in masseter muscle of wt and Epac1-/- mice with established approaches from capillary physiology to determine how changes in blood flow and vascular permeability contribute to overall changes of microvascular flux. We used two tracers, the high molecular weight tracer (Gadomer-17, MW 17 kDa, apparent MW 30-35 kDa) is expected to be primarily limited by diffusion and therefore less dependent on changes in blood flow and the low molecular weight tracer (Dotarem (MW 0.56 kDa) whose transvascular exchange is determined by both blood flow and permeability. Paired experiments in each animal combined with analytical methods provided an internally consistent description of microvascular transport.ResultsEpac1-/- mice had elevated baseline permeability relative to wt control mice for Dotarem and Gadomer-17. In contrast to wt mice, Epac1-/- mice failed to increase transvascular permeability in response to histamine. Dotarem underestimated blood flow and vascular volume and Gadomer-17 has limited sensitivity in extravascular accumulation.ConclusionThe study suggests that the normal barrier loosening effect of histamine in venular microvessels do not function when the normal barrier tightening effect of Epac1 is already compromised. The study also demonstrated that the numerical analysis of DCE-MRI data with tracers of different molecular weight has significant limitations.

Aim Epac1−/− mice, but not Epac2−/− mice have elevated baseline permeability to albumin. This study extends the investigations of how Epac‐dependent pathways modulate transvascular exchange in response to the classical inflammatory agent histamine. It also evaluates the limitations of models of blood‐to‐tissue exchange in transgenic mice in DCE‐MRI measurements. Methods We measured DCE‐MRI signal intensity in masseter muscle of wt and Epac1−/− mice with established approaches from capillary physiology to determine how changes in blood flow and vascular permeability contribute to overall changes of microvascular flux. We used two tracers, the high molecular weight tracer (Gadomer‐17, MW 17 kDa, apparent MW 30‐35 kDa) is expected to be primarily limited by diffusion and therefore less dependent on changes in blood flow and the low molecular weight tracer (Dotarem (MW 0.56 kDa) whose transvascular exchange is determined by both blood flow and permeability. Paired experiments in each animal combined with analytical methods provided an internally consistent description of microvascular transport. Results Epac1−/− mice had elevated baseline permeability relative to wt control mice for Dotarem and Gadomer‐17. In contrast to wt mice, Epac1−/− mice failed to increase transvascular permeability in response to histamine. Dotarem underestimated blood flow and vascular volume and Gadomer‐17 has limited sensitivity in extravascular accumulation. Conclusion The study suggests that the normal barrier loosening effect of histamine in venular microvessels do not function when the normal barrier tightening effect of Epac1 is already compromised. The study also demonstrated that the numerical analysis of DCE‐MRI data with tracers of different molecular weight has significant limitations.

Orchestration of every National Ignition Facility (NIF) shot cycle is managed by the Integrated Computer Control System (ICCS), which uses a scalable software architecture running code on more than...

A better understanding of the inflammatory process associated with renal ischaemia-reperfusion (IR) injury may be clinically important. In this study we examined the role of the kidney in production of inflammatory mediators by analysing renal lymph after 30 min unilateral occlusion of renal artery followed by 120 min reperfusion, as well as the effect of IR on size selectivity for proteins in both glomerular and peritubular capillaries. All measured mediators increased dramatically in renal hilar lymph, plasma and renal cortical tissue samples and returned to control levels after 120 min reperfusion. The responses were differentiated; interleukin-1β, monocyte chemoattractant protein-1 and leptin were markedly increased in plasma before reperfusion, reflecting an extrarenal response possibly induced by afferent renal nerve activity from the ischaemic kidney. Tumour necrosis factor-α was the only mediator showing elevated lymph-to-plasma ratio following 30 min reperfusion, indicating that most cytokines were released directly into the bloodstream. The IR-induced rise in cytokine levels was paralleled by a significant increase in high molecular weight plasma proteins in both lymph and urine. The latter was shown as a 14- to 166-fold increase in glomerular sieving coefficient of plasma proteins assessed by a novel proteomic approach, and indicated a temporarily reduced size selectivity of both glomerular and peritubular capillaries. Collectively, our data suggest that cytokines from the ischaemic kidney explain most of the rise in plasma concentration, and that the locally produced substances enter the systemic circulation through transport directly to plasma and not via the interstitium to lymph.

The aim of this study was to compare intratumoural heterogeneity and longitudinal changes assessed by dynamic contrast-enhanced ultrasound (DCE-US) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in prostate tumour xenografts. In vivo DCE-US and DCE-MRI were obtained 24 h pre- (day 0) and post- (day 2) radiation treatment with a single dose of 7.5 Gy. Characterization of the tumour vasculature was determined by Brix pharmacokinetic analysis of the time-intensity curves. Histogram analysis of voxels showed significant changes (p < 0.001) from day 0 to day 2 in both modalities for kep , the exchange rate constant from the extracellular extravascular space to the plasma, and kel , the elimination rate constant of the contrast. In addition, kep and kel values from DCE-US were significantly higher than those derived from DCE-MRI at day 0 (p < 0.0001) for both groups. At day 2, kel followed the same tendency for both groups, whereas kep showed this tendency only for the treated group in intermediate-enhancement regions. Regarding kep median values, longitudinal changes were not found for any modality. However, at day 2, kep linked to DCE-US was correlated to MVD in high-enhancement areas for the treated group (p = 0.05). In contrast, correlation to necrosis was detected for the control group in intermediate-enhancement areas (p < 0.1). Intratumoural heterogeneity and longitudinal changes in tumour vasculature were assessed for both modalities. Microvascular parameters derived from DCE-US seem to provide reliable biomarkers during radiotherapy as validated by histology. Furthermore, DCE-US could be a stand-alone or a complementary technique. Copyright © 2015 John Wiley & Sons, Ltd.

In dynamic contrast-enhanced magnetic resonance imaging, there has been no consensus in the choice of the pharmacokinetic model. In this paper, a new approach for assessment of the most realistic model for a given tissue is presented. Non-blind and single-channel blind deconvolution algorithms were used in quantitative magnetic resonance dynamic contrast-enhanced imaging of the mouse masseter muscle to compare the realism of two different pharmacokinetic models for the tissue residue function. The first was the adiabatic approximation tissue homogeneity model (aaJW) and the second, the two-compartment exchange model (2CXM). Normals and mice treated with the substance C48/80 were studied. C48/80 increases both blood flow and contrast leakage in muscle substantially. The obtained approximation accuracy was evaluated for both pharmacokinetic models. In addition, the arterial input functions (aifs) estimated using blind deconvolution were compared to the corresponding observed aifs. The hypothesis is that the most realistic model of the tissue residue function leads to the best fits. The non-blind deconvolution did not result in any clear answer. For blind deconvolution, the aifs of the aaJW model were very similar to the corresponding observed aifs, and clearly more so than the aifs of the 2CXM model. Also, the approximation of the observed tracer time sequences was more accurate for the aaJW than the 2CXM model. The realism of different pharmacokinetic models in describing the passage of a tracer through a microvascular bed of a single tissue could be assessed using single-channel blind deconvolution.

Objective Hyperbaric oxygen therapy (HBOT) has been used to enhance microcirculation and thereby oxygen tension in tissues. The present study aimed to investigate the effect of HBOT on radiation injury in the mandibular area of rats. Study design The left mandibles of rats were irradiated by external radiotherapy (15 Gy every other week for a total of 75 Gy). Four HBOT strategies were used: 2 prophylactic groups receiving HBOT either between each radiation treatment or immediately following terminated radiation treatment, and 2 therapeutic groups receiving HBOT after the latent period of 6 weeks after irradiation either every day (standard HBOT protocol) or 3 days a week for 6 weeks. Tissue samples of the irradiated area were taken from skin, the salivary gland, and the mandible. All tissues were stained with hematoxylin and eosin for morphologic examination. Furthermore, skin samples were stained with CD31 for blood vessel analysis. Results There was no change in blood vessel density or morphology between controls and HBOT tissues after radiation. The dentin of 2 of the 5 rats that received HBOT either normalized or was not affected by irradiation. Conclusions HBOT did not affect radiation injury of the mandibular area in rats within 12 weeks after irradiation.

Background Epithelial to mesenchymal transition (EMT) is considered to be important for cancer invasion and metastasis. Tumour hypoxia, in addition to Transforming Growth Factor-β (TGF-β) and Notch, amongst others, have been suggested to be involved in EMT. We therefore investigated if hypoxia, TGF-β1 and the Notch ligand Jagged-1 alone induced morphological changes with corresponding EMT signatures in different epithelial breast cancer cell lines in vitro. Furthermore, we also studied whether or not TGF-β1, or Jagged-1 in combination with hypoxia added any effect on EMT. Methods The cells were exposed to normoxia or hypoxia alone or in combination with TGF-β1 or Jagged-1. Morphological responses to treatment were investigated by light microscopy, and changes in markers for EMT and hypoxia were evaluated by western blot analysis and immunofluorescence studies. Results One of the four cell lines (MCF7) became elongated and highly multipolar, indicative of EMT, following hypoxia, TGF-β1 and Jagged-1 treatment per se with the most distinct morphological shift seen with Jagged-1 treatment in combination with hypoxia. Also, when regarding hypoxia, MCF7 cells showed the greatest change in EMT-markers of the four cell lines tested, but these changes were not consistent with a typical EMT pattern. The morphology of BT474 cells was not altered following Jagged-1 treatment, however, Jagged-1 increased E-cadherin levels. Morphology was changed following TGF-β1 treatment of BT474 cells, but it did not affect E-cadherin levels. Neither Jagged-1 nor TGF-β1 altered the levels of Vimentin in the BT474 cell line. The E-cadherin responses to hypoxia varied with end-point in both MCF7 and BT474 cells, and in most cases were not consistent with EMT. Conclusion Our results using four different breast cancer cell lines in vitro do not provide evidence that EMT is induced by hypoxia alone or in combination with TGF-β1 or the Notch ligand Jagged-1. The inconsistency in morphological appearance and EMT-markers, as well as the time dependent variation in E-cadherin responses could not support EMT. Importantly, there was not one single common response pattern to the stimuli used, suggesting that cell lines with different hormone statuses display individual traits that respond differently to the stimuli applied. Thus, based on the present results, common statements that single factors by themselves can induce EMT seem questionable.

Background: Imatinib causes increased turnover of stromal collagen, reduces collagen fibril diameter, enhances extracellular fluid turnover and lowers interstitial fluid pressure (IFP) in the human colonic carcinoma KAT-4/HT-29 (KAT-4) xenograft model. Methods: We compared the effects of imatinib on oxygen levels, vascular morphology and IFP in three experimental tumor models differing in their content of a collagenous extracellular matrix. Results: Neither the KAT4 and CT-26 colonic carcinoma models, nor B16BB melanoma expressed PDGF beta-receptors in the malignant cells. KAT-4 tumors exhibited a well-developed ECM in contrast to the other two model systems. The collagen content was substantially higher in KAT-4 than in CT-26, while collagen was not detectable in B16BB tumors. The pO(2) was on average 5.4, 13.9 and 19.3 mmHg in KAT-4, CT-26 and B16BB tumors, respectively. Treatment with imatinib resulted in similar pO(2)-levels in all three tumor models but only in KAT-4 tumors did the increase reach statistical significance. It is likely that after imatinib treatment the increase in pO(2) in KAT-4 tumors is caused by increased blood flow due to reduced vascular resistance. This notion is supported by the significant reduction observed in IFP in KAT-4 tumors after imatinib treatment. Vessel area varied between 4.5 and 7% in the three tumor models and was not affected by imatinib treatment. Imatinib had no effect on the fraction of proliferating cells, whereas the fraction of apoptotic cells increased to a similar degree in all three tumor models. Conclusion: Our data suggest that the effects of imatinib on pO(2)-levels depend on a well-developed ECM and provide further support to the suggestion that imatinib acts by causing interstitial stroma cells to produce a less dense ECM, which would in turn allow for an increased blood flow. The potential of imatinib treatment to render solid tumors more accessible to conventional treatments would therefore depend on the degree of tumor desmoplasia.

Background: Tumor hypoxia is relevant for tumor growth, metabolism, resistance to chemotherapy and metastasis. We have previously shown that hyperoxia, using hyperbaric oxygen treatment (HBOT), attenuates tumor growth and shifts the phenotype from mesenchymal to epithelial (MET) in the DMBA-induced mammary tumor model. This study describes the effect of HBOT on tumor growth, angiogenesis, chemotherapy efficacy and metastasis in a triple negative MDA-MB-231 breast cancer model, and evaluates tumor growth using a triple positive BT-474 breast cancer model. Materials and methods: 5 x 105 cancer cells were injected s.c. in the groin area of NOD/SCID female mice. The BT-474 group was supplied with Progesterone and Estradiol pellets 2-days prior to tumor cell injection. Mice were divided into controls (1 bar, pO2 = 0.2 bar) or HBOT (2.5 bar, pO2 = 2.5 bar, 90 min, every third day until termination of the experiments). Treatment effects were determined by assessment of tumor growth, proliferation (Ki67-staining), angiogenesis (CD31-staining), metastasis (immunostaining), EMT markers (western blot), stromal components collagen type I, Itgb1 and FSP1 (immunostaining) and chemotherapeutic efficacy (5FU). Results: HBOT significantly suppressed tumor growth in both the triple positive and negative tumors, and both MDA-MB-231 and BT-474 showed a decrease in proliferation after HBOT. No differences were found in angiogenesis or 5FU efficacy between HBOT and controls. Nevertheless, HBOT significantly reduced both numbers and total area of the metastastatic lesions, as well as reduced expression of N-cadherin, Axl and collagen type I measured in the MDA-MB-231 model. No change in stromal Itgb1 and FSP1 was found in either tumor model. Conclusion: Despite the fact that behavior and prognosis of the triple positive and negative subtypes of cancer are different, the HBOT had a similar suppressive effect on tumor growth, indicating that they share a common oxygen dependent anti-tumor mechanism. Furthermore, HBOT significantly reduced the number and area of metastatic lesions in the triple negative model as well as a significant reduction in the EMT markers N-cadherin, Axl and density of collagen type I. publishedVersion

Background The progression of damage in the renal cortex has not been investigated in the nonclipped kidney of the two-kidney, one-clip model of renal hypertension. In other hypertensive models, damage has been found to progress from the juxtamedullary cortex (JMC) and outward, which has been attributed to early vascular effects. Method The present study investigated the relation between perivascular deposition of collagen and structural damage after 16 and 24 weeks of hypertension in the nonclipped kidney in rats. Results Periarterial collagen density in the kidney was significantly increased already 16 weeks after clipping, at that time tubulointerstitial damage was not evident. After 24 weeks of clipping, periarterial collagen was further increased, and tubulointerstitial damage had developed in the JMC, whereas the outer cortex was protected. Interstitial collagen was not significantly increased in any cortex part during the course of the experiment. Collagen type I a1 mRNA was increased in the JMC after 24 weeks, and α smooth muscle actin histochemistry and collagen type I a2 in-situ hybridization identified myofibroblasts around the arteries after 16 and 24 weeks as the major source of this increase. Conclusion Fibrosis in the nonclipped kidney of renal hypertensive rats starts around the juxtamedullary resistance vessels and then progresses in the JMC, whereas the outer cortex is protected. This suggests that pressure-induced injury to the vasculature attracts or activates fibroblasts in the perivascular area, which may allow damage to progress by impairing vessel function.

Non-technical summary When vascular volume is expanded, atrial natriuretic peptide (ANP) released from the heart acts to restore plasma volume by increasing renal water excretion, causing vasodilatation and increasing vascular permeability to water and macromolecules. Previous experiments using mice with selective deletion of ANP receptors in vascular endothelial cells emphasized the importance of vascular permeability regulation by ANP in plasma volume restoration because this genetic manipulation of ANP action on vascular permeability limited the restoration of vascular volume after an acute increase in plasma volume. Here we demonstrate retention of intravenously infused fluid in wild-type mice in which the response to endogenous ANP was attenuated by the pharmacological agent rolipram that stabilized the endothelial barrier by tightening adhesion between adjacent endothelial cells. The strategy may provide novel approaches to the clinical problem of maintenance of vascular volume after acute intravenous fluid infusion.

A new prototype of a Long-Strip Multi-Gap Resistive Plate Chamber (LMRPC) for the STAR Muon Telescope Detector (MTD) at RHIC has been developed. This prototype has an active area of 52 x 90 cm(2) and consists of six 250 mu m wide gaps. Each detector has 12 strips, read-out at both ends, which are each 3.8 cm wide and 90 cm long with 0.6 cm intervals. In cosmic-ray tests, the efficiency was larger than 95% and the time resolution was similar to 75 ps for the 94% Freon, 5% iso-butane, and 1% SF6 gas mixture. There was good uniformity in the performance across the different strips. The module was also tested in a proton beam at IHEP in Beijing. The efficiency was close to 100% and the best timing resolution achieved was 55 ps for the 90% Freon, 5% iso-butane, and 5% SF6 gas mixture. Trigger scans along and across the strip direction were also performed. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.

Inhibition of phosphodiesterase 4 (PDE4) to increase endothelial cAMP and stabilize the endothelial barrier attenuates acute inflammatory increases in vascular permeability. We extended this approach to attenuate physiological increases in vascular permeability in response to atrial natriuretic peptide (ANP), which acts with the kidney to regulate plasma volume. We measured blood-to-tissue albumin clearance and changes in plasma volume in isoflurane-anaesthetized mice (C57BL/6J) pre-treated with rolipram (8 mg kg−1i.p., 30 min). Rolipram significantly reduced albumin permeability, measured using a dual-label fluorescence method, in skin and skeletal muscle compared with ANP alone (500 ng kg−1 min−1). Skin and muscle tissue accounted for 70% of the reduction in whole body albumin clearance taking into account albumin clearance in gastrointestinal (GI) tissue, heart and kidney. The action of ANP and rolipram to modify albumin clearances in duodenum and jejunum could be accounted for by local increases in vascular perfusion to increase surface area for exchange. ANP increased haematocrit from 40.6% to 46.8%, corresponding to an average loss of 22% plasma fluid volume (227 μl), and this was almost completely reversed with rolipram. Renal water excretion accounted for less than 30% of plasma fluid loss indicating that reduced albumin permeability and reduced filtration into vasodilated GI tissue were the predominant actions of PDE4 inhibition. Similar fluid retention was measured in mice with endothelial-restricted deletion of the guanylyl cyclase-A receptor for ANP. Stabilizing the endothelial barrier to offset ANP-induced increases in vascular permeability may be part of a strategy to maintain plasma volume.

This review will summarize current knowledge on the role of the extracellular matrix (ECM) in general and on the interstitial fluid pressure (P(if)) in particular with regard to their importance in transcapillary exchange. The fluid volume in the interstitial space is normally regulated within narrow limits by automatic re-adjustment of the interstitial hydrostatic and colloid osmotic pressures in response to perturbations in capillary filtration and by the lymphatics. Contrary to this commonly accepted view, P(if) can become an active force and create a fluid flux across the capillaries in several inflammatory reactions and trauma situations rather than limit the changes occurring. The molecular mechanisms involved in the lowering of P(if) include the release of cellular tension exerted on the collagen and microfibril networks in the connective tissue via the collagen-binding beta(1)-integrins, thereby allowing the glycosaminoglycan ground substance, which is normally underhydrated, to expand and take up fluid. Several growth factors and cytokines, including the platelet-derived growth factor BB, are able to reverse a lowering of P(if) and restore the normal compaction of the ECM. The magnitude of the lowering of P(if) varies with the inflammatory response. In several inflammatory reactions, a lowering of P(if) to -5 to -10 mmHg is seen, which will increase capillary filtration by 10-20 times since the normal capillary filtration pressure is usually 0.5-1 mmHg (skin and skeletal muscle). Unless this lowering of P(if) is taken into account, the enhanced solute flux resulting from an inflammatory response will be ascribed to an increased capillary permeability.

Atrial natriuretic peptide (ANP) via its guanylyl cyclase-A (GC-A) receptor participates in regulation of arterial blood pressure and vascular volume. Previous studies demonstrated that concerted renal diuretic/natriuretic and endothelial permeability effects of ANP cooperate in intravascular volume regulation. We show that the microvascular endothelial contribution to the hypovolaemic action of ANP can be measured by the magnitude of the ANP-induced increase in blood-to-tissue albumin transport, measured as plasma albumin clearance corrected for intravascular volume change, relative to the corresponding increase in ANP-induced renal water excretion. We used a two-tracer method with isotopically labelled albumin to measure clearances in skin and skeletal muscle of: (i) C57BL6 mice; (ii) mice with endothelium-restricted deletion of GC-A (floxed GC-A × tie2-Cre: endothelial cell (EC) GC-A knockout (KO)); and (iii) control littermates (floxed GC-A mice with normal GC-A expression levels). Comparison of albumin clearances in hypervolaemic EC GC-A KO mice with normovolaemic littermates demonstrated that skeletal muscle albumin clearance with ANP treatment accounts for at most 30% of whole body clearance required for ANP to regulate plasma volume. Skin microcirculation responded to ANP similarly. Measurements of permeability to a high molecular mass contrast agent (35 kD Gadomer) by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) enabled repeated measures in individual animals and confirmed small increases in muscle and skin microvascular permeability after ANP. These quantitative methods will enable further evaluation of the contribution of ANP-dependent microvascular beds (such as gastro-intestinal tract) to plasma volume regulation.

Heparan sulfate (HS) chains bind and modulate the signaling efficiency of many ligands, including members of the fibroblast growth factor (FGF) and platelet-derived growth factor families. We previously reported the structure of HS synthesized by embryonic fibroblasts from mice with a gene trap mutation of Ext1 that encodes a glycosyltransferase involved in HS chain elongation. The gene trap mutation results in low expression of Ext1, and, as a consequence, HS chain length is substantially reduced. In the present study, Ext1 mutant and wild-type mouse embryonic fibroblasts were analyzed for the functional consequences of the Ext1 mutation for growth factor signaling and interaction with the extracellular matrix. Here, we show that the phosphorylation of ERK1/2 in response to FGF2 stimulation was markedly decreased in the Ext1 mutant fibroblasts, whereas neither PDGF-BB nor FGF10 signaling was significantly affected. Furthermore, Ext1 mutants displayed reduced ability to attach to collagen I and to contract collagen lattices, even though no differences in the expression of collagen-binding integrins were observed. Reintroduction of Ext1in the Ext1 mutant fibroblasts rescued HS chain length, FGF2 signaling, and the ability of the fibroblasts to contract collagen. These data suggest that the length of the HS chains is a critical determinant of HS-protein interactions and emphasize the essential role of EXT1 in providing specific binding sites for growth factors and extracellular matrix proteins.

We study the beam-energy and system-size dependence of phi meson production (using the hadronic decay mode phi -> K(+) K(-)) by comparing the new results from Cu + Cu collisions and previously reported Au + Au collisions at root s(NN) = 62.4 and 200 GeV measured in the STAR experiment at RHIC. Data presented in this Letter are from mid-rapidity (vertical bar y vertical bar ) and net strangeness = 2 (Xi). The enhancement for phi mesons is observed to be higher at root s(NN) = 200 GeV compared to 62.4 GeV. These observations for the produced phi(s (s) over bar) mesons clearly suggest that, at these collision energies, the source of enhancement of strange hadrons is related to the formation of a dense partonic medium in high energy nucleus-nucleus collisions and cannot be alone due to canonical suppression of their production in smaller systems. (C) 2009 Elsevier B.V. All rights reserved.

The administration of insulin is recommended to patients with severe sepsis and hyperglycemia. Previously, we demonstrated that insulin may have direct anti-inflammatory properties and counteracted fluid losses from the circulation by normalizing the interstitial fluid pressure (PIF). PIFis one of the Starling forces determining fluid flux over the capillary wall, and a lowered PIFis one of the driving forces in early edema formation in inflammatory reactions. Here we demonstrate that insulin restores a lipopolysaccharide (LPS)-lowered PIFvia a mechanism involving integrin αvβ3. In C57 black mice ( n = 6), LPS lowered PIFfrom −0.2 ± 0.2 to −1.6 ± 0.3 ( P < 0.05) and after insulin averaged −0.8 ± 0.2 mmHg ( P = 0.098 compared with after LPS). Corresponding values in wild-type BALB/c mice ( n = 5) were −0.8 ± 0.1, −2.1 ± 0.3 ( P < 0.05), and −0.8 ± 0.3 mmHg ( P < 0.05 compared with LPS) after insulin administration. In BALB/c integrin β3-deficient (β3−/−) mice ( n = 6), LPS lowered PIFfrom −0.1 ± 0.2 to −1.5 ± 0.3 mmHg ( P < 0.05). Insulin did not, however, restore PIFin these mice (averaged −1.7 ± 0.3 mmHg after insulin administration). Cell-mediated collagen gel contraction can serve as an in vitro model for in vivo measurements of PIF. Insulin induced αvβ3-integrin-dependent collagen gel contraction mediated by C2C12 cells. Our findings suggest a beneficiary effect of insulin for patients with sepsis with regard to the fluid balance, and this effect may in part be due to a normalization of PIFby a mechanism involving the integrin αvβ3.

There is an increasing focus on the tumor microenvironment in carcinogenesis. Integrins are important receptors and adhesion molecules in this environment and have been shown to be involved in cell adhesion, proliferation, differentiation and migration. The present study aimed to evaluate the effect of stromal integrin β3-deficiency on tumor growth, angiogenesis, interstitial fluid pressure (PIF), fibrosis and metastasis in a murine breast cancer (4T1) and a prostate tumor (RM11) model. We showed that stromal integrin β3-deficiency led to an elevation in PIF that correlated to a shift towards thicker collagen fibrils in the 4T1 mammary tumor. In the RM11 prostate carcinoma model there was no effect of integrin β3-deficiency on PIF and collagen fibril thickness. These findings support the notion that changes in the collagen scaffold influence PIF, and also indicate that there must be important crosstalk between the stroma and tumor cells, in a tumor cell line specific manner. Furthermore, stromal integrin β3-deficiency had no effect on tumor growth or angiogenesis in both tumor models and no effect on lung metastasis in the 4T1 mammary tumor model. In conclusion, the stromal β3 integrin influence PIF, possibly via its effect on the structure of the collagen network, in a tumor cell line dependent manner.

Research on the biology of the tumor stroma has the potential to lead to development of more effective treatment regimes enhancing the efficacy of drug-based treatment of solid malignancies. Tumor stroma is characterized by distorted blood vessels and activated connective tissue cells producing a collagen-rich matrix, which is accompanied by elevated interstitial fluid pressure (IFP), indicating a transport barrier between tumor tissue and blood. Here, we show that the collagen-binding proteoglycan fibromodulin controls stroma structure and fluid balance in experimental carcinoma. Gene ablation or inhibition of expression by anti-inflammatory agents showed that fibromodulin promoted the formation of a dense stroma and an elevated IFP. Fibromodulin-deficiency did not affect vasculature but increased the extracellular fluid volume and lowered IFP. Our data suggest that fibromodulin controls stroma matrix structure that in turn modulates fluid convection inside and out of the stroma. This finding is particularly important in relation to the demonstration that targeted modulations of the fluid balance in carcinoma can increase the response to cancer therapeutic agents.

This study describes the biological effects of hyperoxic treatment on BT4C rat glioma xenografts in vivo with special reference to tumor growth, angiogenesis, apoptosis, general morphology and gene expression parameters. One group of tumor bearing animals was exposed to normobaric hyperoxia (1 bar, pO(2) = 1.0) and another group was exposed to hyperbaric hyperoxia (2 bar, pO(2) = 2.0), whereas animals housed under normal atmosphere (1 bar, pO(2) = 0.2) served as controls. All treatments were performed at day 1, 4 and 7 for 90 min. Treatment effects were determined by assessment of tumor growth, vascular morphology (immunostaining for von Willebrand factor), apoptosis by TUNEL staining and cell proliferation by Ki67 staining. Moreover, gene expression profiles were obtained and verified by real time quantitative PCR. Hyperoxic treatment caused a approximately 60% reduction in tumor growth compared to the control group after 9 days (p < 0.01). Light microscopy showed that the tumors exposed to hyperoxia contained large "empty spaces" within the tumor mass. Moreover, hyperoxia induced a significant increase in the fraction of apoptotic cells ( approximately 21%), with no significant change in cell proliferation. After 2 bar treatment, the mean vascular density was reduced in the central parts of the tumors compared to the control and 1 bar group. The vessel diameters were significantly reduced (11-24%) in both parts of the tumor tissue. Evidence of induced cell death and reduced angiogenesis was reflected by gene expression analyses.Increased pO(2)-levels in experimental gliomas, using normobaric and moderate hyperbaric oxygen therapy, caused a significant reduction in tumor growth. This process is characterized by enhanced cell death, reduced vascular density and changes in gene expression corresponding to these effects.

Neurogenic inflammation is known to induce lowering of interstitial fluid pressure (Pif) in mouse skin. This study examined the possible role of mast cell activation secondary to neuropeptide release in lowering of Pifby using KitW/ KitW-vmice, which are devoid of mast cells, including connective tissue mast cells (CTMCs). Pifwas measured in paw skin of anesthetized (fentanyl-fluanison and midazolam, 1:1) mice with glass capillaries connected to a servo-controlled counterpressure system. In contrast to wild-type mice, intravenous administration of mast cell-activating compound 48/80 induced no lowering of Pifin KitW/ KitW-vmice. Intravenous challenge with substance P (SP), calcitonin gene-related peptide (CGRP), or capsaicin induced a significant ( P < 0.05) lowering of Pifin wild-type mice to −2.16 ± 0.28, −1.96 ± 0.11, and −2.22 ± 0.19 mmHg, respectively, compared with vehicle (−0.49 ± 0.11 mmHg). In KitW/ KitW-vmice the Pifresponse to SP was completely abolished (−0.53 ± 0.32 mmHg) while the response to CGRP and capsaicin was attenuated (−1.33 ± 0.13 and −1.42 ± 0.13 mmHg, respectively) although significantly ( P < 0.05) lowered compared with vehicle. Immunohistochemical analysis revealed no difference in distribution or density of SP- and CGRP-immunoreactive fibers in paws of KitW/ KitW-vcompared with wild-type mice. We conclude that lowering of Pifnormally depends on mast cells. However, the sensory nerves can also elicit a lowering of Pifthat is independent of mast cells.

Aim: To investigate the ability of the microdialysis technique to measure capillary selectivity of different sized plasma proteins induced by local administration of platelet activating factor (PAF). Methods: We used hollow plasmapheresis fibres with 3 cm membrane (cut off 3000 kDa) placed on the back of anaesthetized rats. Results: Platelet activating factor (50 μg mL - 1 ) administered locally via the fibre, increased extravasation of radiolabelled 1 2 5 I-HSA from plasma to the microdialysis fibre by approximately 900% compared both to baseline and the control fibre within 70 min (n = 6, P < 0.05). The extravasation in the control fibre did not change over time. HPLC measurement of plasma proteins in the microdialysis perfusate also demonstrated decreased capillary selectivity for proteins in the diameter range of 73 A, 56 A and 39 A after local administration of PAF (n = 6, P < 0.05). PAF also significantly lowered interstitial fluid (P i f ) pressure after subcutaneous administration (50 μg, mL - 1 ). Mean arterial pressure (MAP) after intravenous injection of PAF (0.4 μg kg - 1 ) fell instantly by about 50 mmHg, and stabilized at 50 mmHg after 15 min (n = 6). MAP was unaltered when PAF was given through the microdialysis fibre (n = 4). Both total tissue water (TTW) and extravasation of albumin, measured as the plasma-to-tissue clearance (E-alb) showed a significant increase after PAF (n = 7, P < 0.05). Conclusions: The present study demonstrates that PAF induces plasma protein extravasation and decrease capillary selectivity of different sized plasma proteins. It also increases transcapillary fluid flux, and lowers P i f , indicating a role for PAF in the interstitium for generation of transcapillary transport of water and large molecules followed by formation of oedema.

Interstitial fluid pressure (P(if)) is important for maintaining constant interstitial fluid volume. In several acute inflammatory reactions, a dramatic lowering of P(if) has been observed, increasing transcapillary filtration pressure and favoring initial and rapid edema formation. This lowering of P(if) seems to involve dynamic beta(1)-integrin-mediated interactions between connective tissue cells and extracellular matrix (ECM) fibers. beta(1)-Integrins are adhesion receptors responsible for the attachment of connective tissue cells to the ECM providing a force-transmitting physical link between the ECM and cytoskeleton. Disruption of actin filaments leads to lowering of P(if) and edema formation, suggesting a role for actin filaments. The aim of this study was to further investigate the role of the cytoskeleton in the control of P(if) by studying the effect of microtubuli fixation using paclitaxel and docetaxel. P(if) was measured with the micropuncture technique. Albumin extravasation (E(alb)) was measured using (125)I-labeled albumin. Paclitaxel and docetaxel were tested locally on foot skin in female Wistar rats. Paclitaxel (6 mg/ml) reduced P(if) from -1.5 +/- 1.0 mmHg in controls to -4.9 +/- 2.6 mmHg after 30 min (P0.05) in a dose-dependent manner (P0.05). Docetaxel caused a similar lowering of P(if). Both paclitaxel and docetaxel increased E(alb) compared with Cremophor EL and saline control (P0.05). Pretreatment with phalloidin before paclitaxel, causing fixation of actin filaments, abolished the lowering of P(if) caused by paclitaxel. This study confirms several previous studies demonstrating that connective tissue cells influence P(if) and edema formation.

We tested the hypothesis that hyperbaric oxygen (HBO) alone and with chemotherapy (5-FU) attenuates tumor growth of DMBA-induced tumors in rats. Six series were performed: (1) Controls (air and vehicle 0.9% NaCl i.p.), (2) 5-FU (0.2 mg/kg i.p.), (3) HBO (2 bar for 90 min and vehicle), (4) HBO and 5-FU, (5) HBO (11 days) and air (next 12 days), (6) HBO (23 days). All treatments were applied on days 1, 4, 7, 10 (Series 1-4), as well as on days 14, 17 and 23 (Series 5-6). Tumor diameter increased by 76.7 and 41.2% in untreated controls and in the 5-FU group, respectively, after 10 days. Tumor size fell by 17-24.2% in the HBO groups and by 35.5% when combined with 5-FU (P < 0.05 compared to HBO). HBO treatment reduced the total number of blood vessels in the tumors. After completion of HBO treatment tumor size increased, but statistically insignificant, during the next 12 days.

In the present study we investigated four variables using factorial design to decide if any of these could explain the variations in the control measurements of interstitial fluid pressure (Pif) in rat trachea that were experienced. This approach requires only a fraction of the animals normally needed when studying each factor separately. Pif in tracheal tissue was measured with the servocontrolled counterpressure system using sharpened micropipettes. The measurements were performed over a period of 60 min and are presented as mean for every 15 min period. The factors investigated in the study were: three strains of female rats (Strain) two brands of diets (Food); two breeder companies (Source); and finally two batches of the same set of animals to repeat the experiment twice (Week), using a total of 48 animals. There was a highly significant effect within Strain the first week (p=0.007), but this response was not observed the second week. The interaction between Strain×Week was significant (p=0.007) while the main effects Strain or Week alone were not significant. The response pattern for Strain and Food was inconsistent for the two experimental weeks studied. These experiments made it possible for us to simultaneously test several factors and exclude these factors as the reason for the observed changes in our experiments since the experiments did not allow the conclusion that one or several of these factors could explain the variation in Pif.