Vessel Leakage Research Articles

Automated detection of leakage in fluorescein angiography images with application to malarial retinopathy.

The detection and assessment of leakage in retinal fluorescein angiogram images is important for the management of a wide range of retinal diseases. We have developed a framework that can automatically detect three types of leakage (large focal, punctate focal, and vessel segment leakage) and validated it on images from patients with malarial retinopathy. This framework comprises three steps: vessel segmentation, saliency feature generation and leakage detection. We tested the effectiveness of this framework by applying it to images from 20 patients with large focal leak, 10 patients with punctate focal leak, and 5,846 vessel segments from 10 patients with vessel leakage. The sensitivity in detecting large focal, punctate focal and vessel segment leakage are 95%, 82% and 81%, respectively, when compared to manual annotation by expert human observers. Our framework has the potential to become a powerful new tool for studying malarial retinopathy, and other conditions involving retinal leakage.

Pharmacokinetic Modeling and Biodistribution Estimation Through the Molecular Communication Paradigm.

Targeted drug delivery systems (TDDSs) are therapeutic methods based on the injection and delivery of drug-loaded particles. The engineering of TDDSs must take into account both the therapeutic effects of the drug at the target delivery location and the toxicity of the drug while it accumulates in other regions of the body. These characteristics are directly related to how the drug-loaded particles distribute within the body, i.e., biodistribution, as a consequence of the processes involved in the particle propagation, i.e., pharmacokinetics. In this paper, the pharmacokinetics of TDDSs is analytically modeled through the abstraction of molecular communication, a novel paradigm in communication theory. Not only is the particle advection and diffusion, considered in our previous study, included in this model, but also are other physicochemical processes in the particle propagation, such as absorption, reaction, and adhesion. In addition, the proposed model includes the impact of cardiovascular diseases, such as arteriosclerosis and tumor-induced blood vessel leakage. Based on this model, the biodistribution at the delivery location is estimated through communication engineering metrics, such as channel delay and path loss, together with the drug accumulation in the rest of the body. The proposed pharmacokinetic model is validated against multiphysics finite-element simulations, and numerical results are provided for the biodistribution estimation in different scenarios. Finally, based on the proposed model, a procedure to optimize the drug injection rate is proposed to achieve a desired drug delivery rate. The outcome of this study is a multiscale physics-based analytical pharmacokinetic model.

Alleviating effects of α-melanocyte-stimulating hormone on retinal vessel leakage in diabetic rats

Background The primary pathogenic basis of diabetic retinopathy (DR) is microangiopathy and inflammatory procedure. Studies showed that α-melanocyte stimulating hormone (α-MSH) can play anti-oxidative stress and anti-apoptosis effects on retina via intravitreal injection and therefore mend blood-retinal barrier (BRB). However, experiment has not yet proved its mechanism. Objective This study was to investigate the protective effects of α-MSH on retinal vessel in diabetic rats. Methods Ninety clean SD rats were assigned to the normal control group, diabetes mellitus (DM) model group and α-MSH group. DM models were established by streptozocin (STZ) injection via tail vein in the rats of the DM model group and the α-MSH group, and sodium citrate buffer was injected in the same way in the normal control group. α-MSH of 3 μl (3.3 μg/μl) was intravitreally injected 1 week and 3 weeks after modeling in the rats of the α-MSH group, and normal saline solution was used in the same way in the normal control group; while no operation was performed in the DM model group. In the fifth week after modeling, Evans blue at the dose of 30 mg/ml (45 mg/kg) was injected via tail vein of rats, and retinal mount was prepared 2 hours later to examine the morphology of retinal vessels under the fluorescent microscope. The blood samples were collected from inner canthus vein for the assessment of leakage amount of Evans blue. The rat retinas were isolated after perfusion of sodium citrate buffer via the left ventricle for the observation of choroidal and retinal vessel ultrastructure by transmission electron microscopy (TEM). The relative expressions of intercellular cell adhesion molecule-1 (ICAM-1) mRNA, tumor necrosis factor-α (TNF-α), occluding mRNA and vascular endothelial growth factor (VEGF) mRNA were detected by quantitative real-time PCR. Results The body weight was lighter and the water intake was higher in the DM rats than those in the normal control rats. The blood glucose levels in the DM model group were (29.69±4.77) mmol/L at the third day and (24.64±2.72) mmol/L in the fifth week following the injection of STZ, respectively. An amount of Evans blue leaked from vessels in the rat retina of DM model group, but the retinal vessels were intact in the rats of the α-MSH group and the normal control group. The leakage amount was (10.04±8.18)μl/(g·h) in the DM model group, which was significantly more than that of (2.62±3.73)μl/(g·h) in the α-MSH group and (3.10±1.13)μl/(g·h) in the normal control group at the fifth week after modeling (P=0.035, 0.041). The ultrastructure of choriodal and retinal vessels was almost normal in the α-MSH group and the normal control group, but the swelling of choriodal vascular endothelial cells and vacuolar-like changes of the retinal pigment epithelium (RPE) were found in the DM model group. In addition, the relative expression levels of ICAM-1 mRNA and TNF-α mRNA were significantly elevated, but those of occludin mRNA were significantly declined in the DM model group compared with the α-MSH group and the normal control group (all at P<0.05). No significant difference was found in the relative expression level of VEGF mRNA among the three groups (F=0.791, P=0.466). Conclusions α-MSH ameliorates retinal vascular leakage in STZ-induced diabetic rats after intravitreal injection by improving ultrastructure of choroid and retinal vessels, down-regulating the expression of proinflammatory factors and up-regulating tight junction component in retina. Key words: α-Melanocyte-stimulating hormone; Diabetic retinopathy/therapy; Intercellular adhesion molecule-1; Tumor necrosis factor-α; Occludin; Vascular endothelial growth factor; Blood-retina barrier; Rats, Sprague Dawley

The Protective Role of Glutaredoxin‐1 in Endothelial Barrier Regulation and Atherosclerosis

Vascular oxidative stress is widely invoked as an important pathogenic mechanism for atherosclerosis (AS). Protein S‐glutathionylation (P‐SSG), the prevalent post‐translational oxidative modification, is emerging as a critical redox signaling mechanism in cardiovascular diseases. However, its role in AS is not clear yet. P‐SSG can be removed by glutaredoxin‐1 (Glrx), a thiol‐transferase which plays a pivotal role in redox signaling. This study is aimed to investigate the role of P‐SSG in high fat‐induced endothelial barrier dysfunction and AS through genetic manipulation of Glrx in human aortic endothelial cell (HAEC) and mice. Overexpression of Glrx attenuates high palmitate high glucose (HPHG)‐induced reversible oxidation of proteins and hyper‐permeability of HAEC monolayer to dextran. Consistently, high fat diet‐induced aortic vessel leakage indexed by dye extravasation is markedly decreased in Glrx transgenic mice when compared with WT mice. Overexpression of hGlrx‐1 in HAECs stimulates cortical actin filament formation under both basal and HPHG challenge conditions. Employing biotin‐switch assays, we showed that Glrx up‐regulation diminished HPHG‐induced oxidative modification of actin and the RhoGTPases, Rac‐1. More importantly, Glrx‐1 corrected HPHG‐induced Rac1 inactivation, a key mechanism of impaired endothelial barrier integrity. Oxidative stress‐induced endothelial barrier dysfunction is an initial step of AS, we thus assessed the high fat diet‐induced AS lesions by en face Oil red O staining in Glrx‐1 transgenic mice with ApoE deficiency background, and found that Glrx‐1 up‐regulation significantly attenuates AS lesion formation. These results suggest a causal role of P‐SSG in the pathogenesis of AS and that Glrx may have therapeutic potential for AS.

A prospective, observational study on the application of ultra-wide-field angiography in the evaluation and management of patients with anterior uveitis.

PurposeEvaluation of peripheral retinal vascular changes in anterior uveitis using ultra-wide-field fluorescein angiography.DesignA prospective, observational study of a case series of patients diagnosed with anterior uveitis.MethodsSetting: Clinical observation at an academic medical center. Patient or Study Population: A total of 65 eyes of 33 patients corresponded with the research criteria of anterior uveitis in the opinion of specialists of Peking University First Hospital. Observation Procedures: Patients were diagnosed primarily through clinical examinations and conventional fluorescein angiography. Subsequently, ultra-wide-field fluorescein angiograms were obtained for each patient. Main Outcome Measures: The main outcome was the detection of peripheral retinal changes by ultra-wide-field fluorescein angiography, and how these changes influenced the evaluation and management of the disease.ResultsPeripheral vessel leakage was detected in 27 eyes (42%) with anterior uveitis, of which 15 eyes displayed active inflammation and 12 eyes displayed inactive inflammation. Peripheral vessel leakage was found in seven of eight eyes with cystoid macular edema. Cystoid macular edema was detected in 7 of 27 eyes (26%) with peripheral vessel leakage, whereas 1 of 38 eyes (3%) did not display peripheral vessel leakage (p<0.01). 44.4% of the patients with peripheral vessel leakage had a specific etiology. The relevant treatment strategies were modified based on the results of the ultra-wide-field fluorescein angiography. 12 patients with peripheral vessel leakage and a quiescent anterior segment were added to those receiving topical glucocorticoids, while 3 patients with serious peripheral vessel leakage and an active anterior segment received a sub-Tenon injection of triamcinolone acetonide.ConclusionsUltra-wide-field fluorescein angiography was very effective in detecting peripheral retinal vascular pathology in anterior uveitis. The changes found in the periphery were important in the evaluation and management of anterior uveitis.

Quantitative Analysis of Peripheral Vasculitis, Ischemia, and Vascular Leakage in Uveitis Using Ultra-Widefield Fluorescein Angiography

To investigate the relationships between peripheral vasculitis, ischemia, and vascular leakage in uveitis using ultra-widefield fluorescein angiography (FA). Cross-sectional, consecutive case series. Consecutive ultra-widefield FA images were collected from 82 uveitis patients (82 eyes) in a single center. The extent of peripheral vasculitis, capillary nonperfusion, and vessel leakage were quantified. Parameters included: (1) foveal avascular zone area and macular leakage, (2) peripheral diffuse capillary leakage and ischemia, (3) peripheral vasculitis, and (4) leakage from neovascularization. Central macular thickness measurements were derived with optical coherence tomography. Main outcome measures were correlations between central and peripheral fluorangiographic changes as well as associations between visual function, ultra-widefield FA-derived metrics, and central macular thickness. Although central leakage was associated with peripheral leakage (r= 0.553, P= .001), there was no association between foveal avascular zone size and peripheral ischemia (r= 0.114, P= .324), regardless of the underlying uveitic diagnosis. Peripheral ischemia was, however, correlated to neovascularization-related leakage (r= 0.462, P= .001) and focal vasculitis (r= 0.441, P= .001). Stepwise multiple regression analysis revealed that a poor visual acuity was independently associated with foveal avascular zone size and central macular thickness (R(2)-adjusted= 0.45, P= .001). We present a large cohort of patients with uveitis imaged with ultra-widefield FA and further describe novel methods for quantification of peripheral vascular pathology, in an attempt to identify visually significant parameters. Although we observed that relationships exist between peripheral vessel leakage, vasculitis, and ischemia, it was only macular ischemia and increased macular thickness that were independently associated with a reduced visual acuity.

Focal transient CNS vessel leak provides a tissue niche for sequential immune cell accumulation during the asymptomatic phase of EAE induction

Peripheral immune cells are critical to the pathogenesis of neurodegenerative diseases including multiple sclerosis (MS) (Hendriks et al., 2005; Kasper and Shoemaker, 2010). However, the precise sequence of tissue events during the early asymptomatic induction phase of experimental autoimmune encephalomyelitis (EAE) pathogenesis remains poorly defined. Due to the spatial–temporal constrains of traditional methods used to study this disease, most studies had been performed in the spine during peak clinical disease; thus the debate continues as to whether tissue changes such as vessel disruption represent a cause or a byproduct of EAE pathophysiology in the cortex. Here, we provide dynamic, high-resolution information on the evolving structural and cellular processes within the gray matter of the mouse cortex during the first 12 asymptomatic days of EAE induction. We observed that transient focal vessel disruptions precede microglia activation, followed by infiltration of and directed interaction between circulating dendritic cells and T cells. Histamine antagonist minimizes but not completely ameliorates blood vessel leaks. Histamine H1 receptor blockade prevents early microglia function, resulting in subsequent reduction in immune cell accumulation, disease incidence and clinical severity.

New concepts in the appraisal and management of birdshot retinochoroiditis, a global perspective.

Birdshot retinochoroiditis (BRC) is a relatively recently described entity. BRC uveitis is predominant in the posterior segment with dual, independent retinal, and choroidal inflammation. The disease has no known extra-ocular inflammation sites and yet features the strongest known HLA association: HLA-A29 is present in close to 100% of cases. Aim in this mini-review was to readjust the appraisal of BRC in the light of a global approach including the full array of investigational procedures. Historical background and the genesis of the disease name were searched. Global disease description including both the retina and the choroid was given. Retinal involvement was clearly characterized, pointing toward the profuse leakage of retinal vessels of all sizes in early disease and widespread atrophy in under treated patients. The importance of exploration of choroidal disease, unavailable until the early 1990s before the advent of indocyanine green angiography (ICGA) was stressed, allowing early diagnosis of disease. Despite its proven importance to explore the choroid, ICGA is still sparsely used. Existing diagnostic criteria were found to be clearly inappropriate not allowing early diagnosis and are in need to be revised, taking into account both retinal and choroidal aspects of the disease, in order to make early diagnosis possible and hence allow proper management .

The effect of AMA0428, a novel and potent ROCK inhibitor, in a model of neovascular age-related macular degeneration.

Rho kinase (ROCK) is associated with VEGF-driven angiogenesis, as well as with inflammation and fibrosis. Therefore, the effect of AMA0428, a novel ROCK inhibitor, was studied in these processes, which highly contribute to the pathogenesis of neovascular AMD. The effect of AMA0428 (0.5-5.0 μM) on human umbilical vein endothelial cells (HUVECs), human brain microvascular endothelial cells (HBMECs), and human brain microvascular pericytes (HBVPs) was determined using cell viability (WST-1), apoptosis (caspase 3/7), and migration (scratch and under-agarose) assays. The in vivo response was investigated using a laser-induced choroidal neovascularization (CNV) mouse model, in which intravitreal injections of AMA0428, murine anti-VEGF-R2 mAb (DC101), or placebo was given. Outcome was assessed by analysis of inflammation (CD45), angiogenesis (FITC-dextran), vessel leakage (Texas Red-conjugated Dextran and FITC-labeled lectin) and fibrosis (Sirius Red/Collagen I). The AMA0428 dose-dependently reduced proliferation and VEGF-induced migration of HUVEC and HBMEC (P < 0.05). No significant effect was seen on HBVP proliferation; however, migration and pericyte recruitment were enhanced (P < 0.05) by AMA0428 administration. There was no apoptosis induction. The AMA0428 significantly reduced CNV and vessel leakage 2 weeks after laser treatment, comparable to DC101. In addition, AMA0428 inhibited inflammation on day 5 by 42% (P < 0.05) and collagen deposition on day 30 by 43% (P < 0. 05), whereas DC101 had no effect on inflammation nor on fibrosis. The results suggest that targeting ROCK with AMA0428 not only reduces neoangiogenesis, but also blocks inflammation and fibrosis (contrary to VEGF suppression). These results point to a potential therapeutic benefit of ROCK inhibition in neovascular AMD.

Ebola virus: melatonin as a readily available treatment option.

There is currently an urgent need for a viable, cheap, and readily available treatment for the Ebola virus outbreak in West Africa. Here, it is proposed that melatonin may have significant utility in helping the management of this outbreak. Optimizing natural killer (NK) cell responses seems crucial to surviving Ebola virus infection. Melatonin increases NK cell cytotoxicity significantly, suggesting efficacy in managing the Ebola virus. Under conditions of challenge, melatonin increases heme oxygenase-1 (HO-1), which inhibits Ebola virus replication. Melatonin also has protective effects in cases of septic shock, which, although bacterial, has similar end-point presentations involving blood vessel leakage. Melatonin's effects on haemorrhage are mediated primarily by a decrease in pro-inflammatory cytokines. By optimizing the appropriate immune response, melatonin is likely to afford protection to those at high risk of Ebola viral infection, as well as having direct impacts on the course of infection per se. Although no direct data pertain to the utility of melatonin in the management of the Ebola virus, convergent bodies of data suggest its utility, which is reviewed in this article.

Numerical Study on the Heat Transfer in the Leakage of Pressure Vessels Considering the Joule-Thomson Cooling Effect

The cooling effect caused by the Joule-Thomson (JT) effect in the leakage of pressure vessels has gained more and more attention and worries from natural gas transportation industries as it may influence the validity of the conventional leak-before-break approach. Currently, leak-before-break (LBB) design codes for pressure vessels do not take into account of the subsequent low temperature depression and the drop in fracture toughness in the aftermath of a leak. This study is to ensure the integrity of the compressed natural gas (CNG) containment in marine transportation vessels being proposed to transport the gas in highly pressurized (up to 250bar) cylinders. A numerical method was proposed to simulate the heat transfer between the high pressure argon gas leaking through a narrow crack and the surrounding metal which is caused by the Joule-Thomson cooling effect of the leaking argon. A one-way coupling model was developed to calculate the gas pressure drop at different crack depth, followed by the temperature drop. A heat transfer simulation then calculated the temperature in the metal in the vicinity of the crack. The Redlich-Kwong (RK) equation with a relatively high accuracy was adopted to evaluate the temperature change of the leaking argon. The modified Petukhov correlation by Adams et al. was employed to evaluate the heat transfer coefficient of leaking argon. A MATLAB code was developed to iteratively calculate the properties of leaking gas in a crack which was fitted as formula as boundary conditions in heat transfer simulation of COMSOL program. Under the maximum internal pressure of 91bar, the simulated temperature drop of the test plate in the vicinity of the crack caused by the Joule-Thomson cooling effect is 16.2°C. An experiment rig designed and tested under the same conditions showed a good agreement between the simulation and experiment at the obtained lowest temperature in the test plate.

Gene Therapy with Endogenous Inhibitors of Angiogenesis for Neovascular Age-Related Macular Degeneration: Beyond Anti-VEGF Therapy.

Age-related macular degeneration (AMD) is the leading cause of substantial and irreversible vision loss amongst elderly populations in industrialized countries. The advanced neovascular (or “wet”) form of the disease is responsible for severe and aggressive loss of central vision. Current treatments aim to seal off leaky blood vessels via laser therapy or to suppress vessel leakage and neovascular growth through intraocular injections of antibodies that target vascular endothelial growth factor (VEGF). However, the long-term success of anti-VEGF therapy can be hampered by limitations such as low or variable efficacy, high frequency of administration (usually monthly), potentially serious side effects, and, most importantly, loss of efficacy with prolonged treatment. Gene transfer of endogenous antiangiogenic proteins is an alternative approach that has the potential to provide long-term suppression of neovascularization and/or excessive vascular leakage in the eye. Preclinical studies of gene transfer in a large animal model have provided impressive preliminary results with a number of transgenes. In addition, a clinical trial in patients suffering from advanced neovascular AMD has provided proof-of-concept for successful gene transfer. In this mini review, we summarize current theories pertaining to the application of gene therapy for neovascular AMD and the potential benefits when used in conjunction with endogenous antiangiogenic proteins.

THE GIFTS OF TIME

9 5 R T H E G I F T S O F T I M E D A V I D M A S O N To stand in the kitchen high up in the trees watching a sapling sway, the canopy of leaves and needles stirred by an undersea, and stare, a mug of co√ee in the hand, is all of time. No necessary task impels a rush to dress and find the keys. Decades have served for that. It’s time to breathe. Time also for a long gray ship to turn and for a young man standing on the bridge to wonder if that distant speck is bird or continent. The young man, older now, can hear the heartbeat of an ailing girl. He moves the stethoscope, tells her to breathe, and knows the murmur is her leaking blood, and he is only one, and in the time it takes to breathe he too is gone forever. He too is like the stir of swaying trees, the muddy cli√s eroded by the surf. Stand here and listen to the trees and know their generation too will fall away. The cli√s will fall away. The voices die. ≤ There was another ship, another time, but going nowhere. It steamed both day and night. It made quite a business of making clouds. The sky poured from its stack, its boilers the same, and the ship’s hull tugged at cables and lines 9 6 Y lashed to a gravel bulkhead by the road. It tugged like a leashed dog with boundless hope but never left the shore, that cloudy ship with laborers who strove inside the hull. It rained inside. The men were always wet, the women too, working wet, and wet when they quit work and stepped out to the clouds exhaled from cigarettes they cupped in hands, talking of food they would like to eat again and letters they would like to read, dry-eyed. They too felt time rising from the gray stack. Time is the kitchen high up in the trees and time is the cloudy ship, time is the shore. The people hadn’t known the time before. Only when it slowed and swayed and clouded out, only when the co√ee in the hand went cool could anyone be sure they’d touched the hours or the year of gull cries from an open throat. A current stirs the trees like tidal grass. Stand in the kitchen looking out to sea through stands of waving limbs and feel the wind, the leaking vessels of the blood go down. ≤ No one can make up time. The sea would laugh, the crowded rocks whisper among themselves. The co√ee has gone cold. The names are gone. They are another generation gone. The room is time, the room is out of time. The fissured road will fall into the waves. The ruined millionaire will watch his house tip like a sandbox toy and slide away. A colony of ants will have its say remembered by the beetle rolling dung. An old man dances, knowing he is young. A woman dances in the breaking day. ...

Exendin-4 promotes endothelial barrier enhancement via PKA- and Epac1-dependent Rac1 activation.

Among emerging antidiabetic agents, glucagon-like peptide-1 (GLP-1)-based therapies carry special cardiovascular implications, exerting both direct and indirect effects. The control of vascular permeability is of pivotal importance in vascular pathologies. The objective of the present study was to determine the effect of GLP-1 on endothelial barrier function and assess the underlying mechanism(s). Here we show for the first time that the stable GLP-1 analog exendin-4 attenuated the leakage of subcutaneous blood vessels in mice indexed by dye extravasation caused by injections of thrombin. Moreover, in cultured endothelial cells, exendin-4 significantly prevented the thrombin-induced FITC-dextran permeability of endothelial monolayers via GLP-1 receptor. Immunofluorescence microscopy reveals that exendin-4 abrogates detrimental effects of thrombin on VE-cadherin and the F-actin cytoskeleton, with decreased stress fiber and gap formation. Importantly, exendin-4 reduced thrombin-induced tyrosine phosphorylation of VE-cadherin at Y731 and Y658. Moreover, small GTPase Rac1 was significantly activated as a result of exendin-4 treatment. The efficacy of exendin-4 to counteract the barrier-compromising effect of thrombin was blunted when Rac1 was inactivated by Rac1 inhibitor NSC-23766. Inhibition of PKA activity or small-interfering RNA for exchange protein directly activated by cAMP 1 (Epac1) decreased exendin-4-induced Rac1 activation and barrier enhancement, indicating the participation of both PKA and Epac1 in the barrier-stabilizing effect of exendin-4 elicited on thrombin-impaired barrier function. Thus, our findings have uncovered an unpredicted role for exendin-4 in the coordination of vascular permeability and clarified the molecular underpinnings that contribute to barrier restoration initiated by exendin-4.

Attenuated transforming growth factor beta signaling promotes metastasis in a model of HER2 mammary carcinogenesis.

IntroductionTransforming growth factor beta (TGFβ) plays a major role in the regulation of tumor initiation, progression, and metastasis. It is depended on the type II TGFβ receptor (TβRII) for signaling. Previously, we have shown that deletion of TβRII in mammary epithelial of MMTV-PyMT mice results in shortened tumor latency and increased lung metastases. However, active TGFβ signaling increased the number of circulating tumor cells and metastases in MMTV-Neu mice. In the current study, we describe a newly discovered connection between attenuated TGFβ signaling and human epidermal growth factor receptor 2 (HER2) signaling in mammary tumor progression.MethodsAll studies were performed on MMTV-Neu mice with and without dominant-negative TβRII (DNIIR) in mammary epithelium. Mammary tumors were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence staining. The levels of secreted proteins were measured by enzyme-linked immunosorbent assay. Whole-lung mount staining was used to quantitate lung metastasis. The Cancer Genome Atlas (TCGA) datasets were used to determine the relevance of our findings to human breast cancer.ResultsAttenuated TGFβ signaling led to a delay tumor onset, but increased the number of metastases in MMTVNeu/DNIIR mice. The DNIIR tumors were characterized by increased vasculogenesis, vessel leakage, and increased expression of vascular endothelial growth factor (VEGF). During DNIIR tumor progression, both the levels of CXCL1/5 and the number of CD11b+Gr1+ cells and T cells decreased. Analysis of TCGA datasets demonstrated a significant negative correlation between TGFBR2 and VEGF genes expression. Higher VEGFA expression correlated with shorter distant metastasis-free survival only in HER2+ patients with no differences in HER2-, estrogen receptor +/- or progesterone receptor +/- breast cancer patients.ConclusionOur studies provide insights into a novel mechanism by which epithelial TGFβ signaling modulates the tumor microenvironment, and by which it is involved in lung metastasis in HER2+ breast cancer patients. The effects of pharmacological targeting of the TGFβ pathway in vivo during tumor progression remain controversial. The targeting of TGFβ signaling should be a viable option, but because VEGF has a protumorigenic effect on HER2+ tumors, the targeting of this protein could be considered when it is associated with attenuated TGFβ signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0425-7) contains supplementary material, which is available to authorized users.

Engineered Antibody Therapies Coming of Age for Aging Brains

Engineered Antibody Therapies Coming of Age for Aging Brains

Abstract 2071A: Anti-VEGF treatment in orthotopic breast cancer xenografts: Dynamic contrast-enhanced micro-CT correlates with 3D multispectral fluorescence histology

Abstract Aim: Here, we evaluate the ability of dynamic contrast-enhanced micro-computed tomography (DCE micro-CT) in combination with 3D fluorescence ultramicroscopy (UM) to assess the early physiological and morphological treatment response of orthotopic breast tumors to anti-angiogenic drug therapy. Methods: Non-invasive DCE micro-CT was performed on breast cancer xenografts (KPL-4) that were treated twice with an antibody against the vascular endothelial growth factor (VEGF) or a control antibody. Tumor physiology was assessed in different tumor regions of interests measuring the relative blood volume (rBV) and the permeability-surface area product (PS). Parametric maps were calculated on a voxel-by-voxel basis to evaluate intra-tumor vascular heterogeneity. Mean physiological measurements were compared with morphological measures of the tumor vascular architecture (microvessel density (MVD) and relative vessel volume (rVV)) as obtained by 3-dimensional fluorescence UM. Additionally, vessel leakage was assessed by UM quantifying the penetration strength of a co-injected fluorescence-labeled therapeutic antibody into the tumor tissue. Results: Examination of the parametric maps revealed significantly different spatial patterns of intra-tumoral blood supply (rBV) between anti-VEGF-treated tumors and control. While a characteristic rim vascularization (high rBV values in the tumor periphery) was observed in the control tumors, treated tumors showed a widely homogeneous blood supply. Compared with high-resolution UM, these physiological rBV maps showed excellent agreement with the morphological appearance of the 3D tumor vascular architecture. Assessment of the mean physiology showed a significant decrease of rBV (p&amp;lt;0.01) and PS (p&amp;lt;0.05) in the tumor periphery after anti-angiogenic treatment. The reduction of rBV correlated well with a significant reduction of rVV (p&amp;lt;0.01) in the corresponding tumor region. The decrease in PS was found consistent with a significantly reduced antibody uptake (p&amp;lt;0.01) in the tumor tissue after treatment. No treatment effect was observed by tumor volume. Conclusions: DCE micro-CT used along with UM provides comprehensive and complementary information of the physiological and morphological anti-angiogenic treatment response in breast cancer xenografts. This technology may help to improve current standard methods in the assessment of anti-angiogenic drug efficacy in preclinical drug development. Citation Format: Thomas Pöschinger, Anja Renner, Fabian Eisa, Michael Dobosz, Robert Brauweiler, Willi A. Kalender, Werner Scheuer. Anti-VEGF treatment in orthotopic breast cancer xenografts: Dynamic contrast-enhanced micro-CT correlates with 3D multispectral fluorescence histology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2071A. doi:10.1158/1538-7445.AM2014-2071A

P16.33 * AN IN SILICO ESTIMATION OF THE PHARMACOKINETIC PROFILE AND THE DISPOSITION OF GD-DTPA IN BRAIN TUMOR LESIONS OF DIFFERENT VASCULATURE THROUGH PBPK MODELS

Brain tumor lesions (BTL), i.e. high grade gliomas, are known to have a prominence of vasculature, which is promoted through hypoxia mechanisms and differential expression of vascular endothelial growth factor (VEGF). Imaging techniques such as dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) with intra-venous (i.v.) administration of a Gd-based contrast agent (GBCA) are successfully used for the diagnosis and characterization of the BTLs. Tracer kinetics plays an important role in DCE-MRI by assessing the vessel leakage through estimation of the transfer and disposition of GBCAs in a lesion. Physiologically-based pharmacokinetic modeling (PBPK) represents a well-documented approach to estimate in silico the disposition of pharmacologic agents in the body. In this work, we sought to i)present a whole-body PBPK approach in order to estimate the PK profile of Gd-DTPA (Gadopentetic acid, Magnevist®) and ii)evaluate the impact of vascular fraction of tracer's extravascular-extracellular disposition in a simulated BTL. PK profile was assessed through the application of Simcyp® simulator platform and the generation of whole-body PBPK model. BTL was introduced as an additional compartment (∼5% of total brain weight) with tissue characteristics matching those of a brain tumor. In silico clinical trials (ISCTs) were designed by integrating literature data for a virtual population of patients with cancer for Simcyp® and Gd-DTPA properties. The ISCTs were generated for a representative individual and concentrations were estimated for 15 minutes following i.v. bolus injection of Gd-DTPA (0.1 mmol/kg). Given the simulated BTL's size, all parameters were kept constant except for capillary fraction in order to evaluate the impact of vasculature. RESULTS: from the whole-body PBPK simulations estimate the maximum plasma concentration of Gd-DTPA to be 3.0 mM whereas the intracranial blood concentration to be 1.7 mM. Regarding the simulated BTL, concentrations varied between 1.5-1.7 mM for the extravascular-extracellular space following modulation of tissue percentage in capillaries. Finally, zero concentrations are predicted for brain parenchyma and intracellular tissues due to the presence of blood-brain barrier and the absence of cellular intake, transfer or passive mediated, for Gd-DTPA, respectively. The presented in silico approach was capable of assessing the PK profiles of Gd-DTPA and especially its disposition in the extravascular-extracellular space of a putative BTL. The need for exploitation of the imaging techniques with studies correlating image analysis with clinical outcome and in silico tools is of great interest. The incorporation of PBPK models along with in silico tools for tumor growth and/or clinical data could reveal novel approaches for improving DCE-MRI techniques applied in diagnosis and treatment protocols (i.e. novel anti-VEGF treatments).

The effect of a rock inhibitor ama0428 in a model of wet age‐related macular degeneration

AbstractPurpose Rho kinase (ROCK) is associated with VEGF‐driven angiogenesis and is involved in inflammation and fibrosis. Therefore, the effect of a locally acting ROCK inhibitor, AMA0428, was studied in wet age‐related macular degeneration (AMD).Methods The effect of AMA0428 on human brain microvascular endothelial cells (HBMEC), human brain vascular pericytes (HBVP) and human tenon fibroblasts (HTF) was determined by measuring cell viability (WST‐1), apoptosis (caspase 3/7) and 2 migration assays (scratch and under‐agarose) The in vivo response was investigated using a laser‐induced choroidal neovascularization (CNV) mouse model. Intravitreal injections were given on day 0, 4, 10 and 20 with AMA0428, murine anti‐VEGFR Ab (DC101) or placebo. Outcome was assessed by analysis of inflammation (CD45), angiogenesis (FITC‐dextran), vessel leakage (Texas Red‐conjugated Dextran and FITC‐labeled lectin) and fibrosis (Collagen I).Results AMA0428 dose‐dependently reduced proliferation and VEGF‐induced migration of HBMEC and HTF. No significant effect was seen on HBVP proliferation; however, migration and pericyte recruitment were increased. There was no apoptosis induction. AMA0428 significantly reduced CNV and vessel leakage 2 weeks after lasering, comparable to DC101. In addition, AMA0428 inhibited inflammation on day 5 by 20% and collagen deposition on day 30 by 39% while DC101 had no effect on inflammation nor fibrosis.Conclusion Our data suggest that targeting ROCK with AMA0428 not only reduces neoangiogenesis, but also blocks inflammation and fibrosis (contrary to anti‐VEGF). These results point to a potential therapeutic benefit of ROCK inhibition in wet AMD.

A new kind of labyrinth-like capillary is responsible for leakage from human choroidal neovascular endothelium, as investigated by high-resolution electron microscopy.

This study reports the clinicopathologic findings of leaky sites in pathological vessels after submacular removal of choroidal neovascular membranes (CNV). As the site that causes fluid exudation from neovascular vessels is unknown, specific attention was focused on the formation of fenestrations, cellular junctions, and morphologic alteration which can cause endothelial leakage. Choroidal neovascular membranes of 15 patients who underwent submacular surgery for CNV were investigated. Five patients received bevacizumab treatment before surgery, and another five received photodynamic therapy before surgery. The remaining five did not receive any other treatment before surgery. All membranes were embedded for transmission electron microscopy. CNVs were analyzed for pathological cell-to-cell connections, fenestrations, or other pathological conditions which can cause leakage of plasma. The morphology of the newly formed blood channels was very variable, and in principle was not different in treated and untreated patients. The sources of leakage in neovascular choroidal vessels were caused by insufficient endothelial cell connections and by capillaries with microvillar projections into the vessel lumen which blocked cellular perfusion but still allowed the flow of plasma. Fenestrations were only infrequently observed. A newly discovered type of pathological capillary, called a labyrinth capillary, is very likely responsible for the permanent leakage of fluid. Due to the small vessel lumen, thrombocytes cannot enter these capillaries to close the leakages. Fenestrations did not appear to play a significant role in vascular leakiness.