Increased Vessel Permeability Research Articles

Constitutive excessive erythrocytosis causes inflammation and increased vascular permeability in aged mouse brain

Excessive erythrocytosis results in severely increased blood viscosity that may compromise the vascular endothelium. Using our transgenic mouse model of excessive erythrocytosis we previously showed that despite altered brain endothelial cell morphology and an activated vasculature, brain vascular integrity was largely maintained up to 4–5 months of age. We now present data showing that persistent long-term damage of the vascular wall during the later stages of adulthood (9–12 months) results in a chronic detrimental inflammatory phenotype and increased vessel permeability that likely contributes to the reduced life span of our erythropoietin overexpressing transgenic mouse. In aged transgenic animals inflammatory cells were detected in brain tissue and elevated RNA and protein levels of inflammatory markers such as IL-6 and TNFα were observed in both brain tissue and blood plasma. Additionally, increased expression of p53 and other pro-apoptotic markers, as well as decreased Bcl-xL expression in the brain vasculature, indicated ongoing cell death within the vascular compartment. Finally, abnormally elevated vascular permeability in all organs was detected in correlation with decreased expression of the tight junction protein occludin and the adherens junction protein β-catenin in brain. Thus chronic erythrocytosis results in sustained activation of inflammatory pathways, vascular dysfunction and blood–brain barrier disruption.

Modifications in Dynamic Contrast-Enhanced Magnetic Resonance Imaging Parameters After α-Particle-Emitting 227Th-trastuzumab Therapy of HER2-Expressing Ovarian Cancer Xenografts

The purpose of this study was to investigate the effect of α-particle-emitting (227)Th-trastuzumab radioimmunotherapy on tumor vasculature to increase the knowledge about the mechanisms of action of (227)Th-trastuzumab. Human HER2-expressing SKOV-3 ovarian cancer xenografts were grown bilaterally in athymic nude mice. Mice with tumor volumes 253 ± 36 mm(3) (mean ± SEM) were treated with a single injection of either (227)Th-trastuzumab at a dose of 1000 kBq/kg body weight (treated group, n=14 tumors) or 0.9% NaCl (control group, n=10 tumors). Dynamic T1-weighted contrast-enhanced magnetic resonance imaging (DCEMRI) was used to study the effect of (227)Th-trastuzumab on tumor vasculature. DCEMRI was performed before treatment and 1, 2, and 3 weeks after therapy. Tumor contrast-enhancement curves were extracted voxel by voxel and fitted to the Brix pharmacokinetic model. Pharmacokinetic parameters for the tumors that underwent radioimmunotherapy were compared with the corresponding parameters of control tumors. Significant increases of kep, the rate constant of diffusion from the extravascular extracellular space to the plasma (P<.05), and kel, the rate of clearance of contrast agent from the plasma (P<.01), were seen in the radioimmunotherapy group 2 and 3 weeks after injection, compared with the control group. The product of kep and the amplitude parameter A, associated with increased vessel permeability and perfusion, was also significantly increased in the radioimmunotherapy group 2 and 3 weeks after injection (P<.01). Pharmacokinetic modeling of MRI contrast-enhancement curves evidenced significant alterations in parameters associated with increased tumor vessel permeability and tumor perfusion after (227)Th-trastuzumab treatment of HER2-expressing ovarian cancer xenografts.

Vascular Endothelial Cadherin Expression in Lung Specimens of Patients with Sepsis-Induced Acute Respiratory Distress Syndrome and Endothelial Cell Cultures

Aims: Vascular endothelial (VE) cadherin is a cell adhesion molecule localized at endothelial cell (EC) junctions. As a major component of endothelial adherens junctions, its main function is the maintenance and regulation of EC integrity. In the acute respiratory distress syndrome (ARDS), increased vascular permeability is a major mechanism in pulmonary edema and lung dysfunction. In this study, VE-cadherin expression was investigated in ARDS lungs and control tissue as well as in an ARDS cell culture model. Methods: Lung specimens of patients with ARDS due to Gram-negative sepsis (n = 20; control lung tissue: n = 41) and cell cultures of human pulmonary microvascular ECs and human umbilical vein ECs stimulated with LPS, TNF-α and IFN-γ were stained with a VE-cadherin antibody. Staining intensity was semiquantitatively evaluated by conventional light and immunofluorescence microscopy. Results: VE-cadherin expression was statistically significantly reduced in the endothelium of all vessel types in ARDS lungs compared to control tissue. Cell cultures showing disrupted cellular borders confirmed these results. Conclusion: Reduced expression of VE-cadherin has to be considered as a major mechanism of increased vessel permeability in ARDS. The previously described vessel-type-specific expression pattern of VE-cadherin in the human lung is not influenced by ARDS.

Abstract 4955: Pegylated recombinant human hyaluronidase PH20 (PEGPH20) increases tumor perfusion in mouse xenografts and phase 1 cancer patients.

Abstract Pegylated recombinant human hyaluronidase PH20 (PEGPH20) is an investigational therapeutic agent under clinical development for use alone or in combination with other cancer therapies for the treatment of patients with non-hematologic malignancies that may accumulate hyaluronan (HA), a glycosaminoglycan that is a significant component of the extracellular matrix of many solid tumors. HA accumulation has been correlated with local invasion, the presence of distal metastasis, higher tumor grade, and poorer overall survival. Preclinical studies have demonstrated that sustained HA removal, accomplished with PEGPH20, decreases tumor interstitial pressure, increases vascular area, inhibits tumor growth and enhances chemotherapeutic activity in HA-rich xenografts and genetically engineered mouse tumor models. As dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is a clinically translatable MRI perfusion protocol, we used DCE-MRI imaging to evaluate the effects of PEGPH20 treatment on tumor vascular permeability, vascular surface area and/or blood flow. In mice, prostate cancer PC3 cells were implanted adjacent to the right tibial periosteum. When tumors reached ∼500 mg, animals were staged into vehicle or PEGPH20 (4.5 mg/kg) treatment groups. Utilizing gadopentetate dimeglumine (Gd-DTPA) as a contrast agent, pre-treatment baseline DCE scans of the tumors were acquired. Animals were subsequently IV dosed with test article and post-treatment scans were acquired at 24 h. Initial area under the curve (IAUC) and Ktrans were calculated voxel-by-voxel using a 2-compartment PK model. Although vehicle alone did not increase or decrease Ktrans or IAUC, PEGPH20 treatment increased Ktrans (1/sec) and IAUC (mmol.s) by 74% and 78.7%, respectively. Concurrent to preclinical studies, DCE-MRI imaging was conducted pre- and post- PEGPH20 in 9 patients in a Phase 1 dose-escalation, safety, tolerability, PK/PD study of PEGPH20 treatment in patients with advanced solid tumors. Patients were treated with 1.6, 3 or 5 μg/kg PEGPH20 1-2/weekly. Baseline, day 1, day 2-5 and end-of-cycle (EOC) scans were acquired (w/ Gd-DTPA) in 3 patients. Images were fit on a pixel-by-pixel basis to a 2-compartment, 3-parameter, PK model and Ktrans and extracellular volume (Ve) calculated. Results suggest an early and rapid increase in tumor perfusion in target lesions, compared to baseline (mean ΔKtrans = 428%; N=3; Day 2-5 scans), as well as an expansion of the extracellular space with a reduction of these parameters towards baseline by EOC (mean ΔKtrans = 28% above baseline). Taken together, these studies suggest that PEGPH20 increases vessel permeability, blood flow and/or vessel surface area in HA-rich solid tumors. Additional preclinical and clinical studies, complete with DCE-MRI imaging, are ongoing to further evaluate PEGPH20 in the treatment HA-rich malignancies. Citation Format: Curtis B. Thompson, Patrick McConville, Ron Korn, Jenifer Baranski, Meridith Baugher, Deanne Lister, Erin Trachet, Jacob Y. Hesterman, Jack Hoppin, Joy Zhu, Daniel C. Maneval. Pegylated recombinant human hyaluronidase PH20 (PEGPH20) increases tumor perfusion in mouse xenografts and phase 1 cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4955. doi:10.1158/1538-7445.AM2013-4955

Effect of Low-Intensity Focused Ultrasound on the Middle Ear in a Mouse Model of Acute Otitis Media

We hypothesized that low-intensity focused ultrasound (LIFU) increases vessel permeability and antibacterial drug activity in the mouse middle ear. We determined appropriate settings by applying LIFU to mouse ears with the external auditory canal filled with normal saline and performed histologic and immunohistologic examination. Acute otitis media was induced in mice with nontypable Haemophilus influenzae, and they were given ampicillin (50, 10, or 2 mg/kg) intraperitoneally once daily for 3 days with or without LIFU (1.0 W/cm2, 20% duty cycle, 30 s). In the LIFU(+) groups receiving the 2- and 10-mg/kg doses, viable bacteria counts, number of inflammatory cells and IL-1β and TNF-α levels in middle ear effusion were significantly lower than in the LIFU(−) groups on the same doses. Severity of AOM also tended to be reduced more in the LIFU(+) groups than in the LIFU(−) groups. LIFU application with antibiotics may be effective for middle ear infection.

The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury.

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after stroke.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-012-1076-3) contains supplementary material, which is available to authorized users.

In Vivo Imaging of Neurovascular Remodeling After Stroke

Stroke is one of the leading causes of adult disability throughout the world and, even though neural mechanisms of loss of function have been extensively studied, many aspects of poststroke cerebral responses remain poorly understood. Of particular interest is the mounting evidence of the capacity of the adult brain to reorganize after injury, which is believed to contribute to limitation of the extent of neural dysfunction and to restoration of affected neural functions. Processes such as neuronal plasticity, glial proliferation, and neovascularization may be essential for preservation or recovery of function after stroke and may conceivably go hand-in-hand at nearby and remote sites of active tissue reorganization.1,2 Specifically, the acute initiation of neurovascular remodeling, stimulating the proliferation and growth of existing arteries/arterioles (ie, arteriogenesis) or new capillaries (ie, angiogenesis), may be critical to facilitate the survival and restructuring of neural tissue, which ultimately may contribute to functional recovery at later stages.3 A variety of vascular imaging strategies is available for in vivo detection, characterization, and quantification of these processes, which can significantly aid in elucidation of the role of neurovascular remodeling after stroke, as discussed in this review. Because most of the described imaging modalities are present in experimental and clinical settings, this raises significant opportunities for translational studies. Growth and remodeling of existing and nascent vascular networks in health and disease involve a number of critical steps that have been comprehensively described by Risau4 and Carmeliet.5 Arteriogenesis involves adaptive growth and proliferation of preexisting (collateral) arteries and arterioles in response to increase in intravascular shear forces. The increased shear stress leads to upregulation of cell adhesion molecules, followed by accumulation of monocytes and other leukocytes that release cytokines and growth factors around the proliferating and maturating arteries.6 Although arteriogenic growth of collateral …

Brown spider (Loxosceles intermedia) venom triggers endothelial cells death by anoikis

Brown spider (Loxosceles sp.) venom affects the endothelium of vessels and triggers disruptive activity in the subendothelial matrix. The vascular disorders observed after venom exposure include leukocyte and platelet activation, disseminated intravascular coagulation, an increase in vessel permeability and hemorrhage into the dermis. In this study, we report additional evidence regarding the mechanism of endothelial cell cytotoxicity induced by Loxosceles intermedia venom. Exposure to venom led to endothelial cell detachment in a time-dependent manner. Loss of cell anchorage and cell–cell adhesion following venom exposure was accompanied by changes in the distribution of the α5β1 integrin and VE-cadherin. An ultrastructural analysis of cells treated with venom revealed morphological alterations characteristic of apoptosis. Moreover, after venom exposure, the ratio between Bax and Bcl-2 proteins was disturbed in favor of Bax. In addition, late apoptosis was only observed in cells detached by the action of venom. Accordingly, there was no increase in apoptosis when cells were exposed to L. intermedia venom in suspension, suggesting that the loss of cell anchorage provides the signal to initiate apoptosis. Thus, L. intermedia venom likely triggers endothelial cell death indirectly through an apoptotic mechanism known as anoikis.

A vaccine targeting angiomotin induces an antibody response which alters tumor vessel permeability and hampers the growth of established tumors

Angiomotin (Amot) is one of several identified angiostatin receptors expressed by the endothelia of angiogenic tissues. We have shown that a DNA vaccine targeting Amot overcome immune tolerance and induce an antibody response that hampers the progression of incipient tumors. Following our observation of increased Amot expression on tumor endothelia concomitant with the progression from pre-neoplastic lesions to full-fledged carcinoma, we evaluated the effect of anti-Amot vaccination on clinically evident tumors. Electroporation of plasmid coding for the human Amot (pAmot) significantly delayed the progression both of autochthonous tumors in cancer prone BALB-neuT and PyMT genetically engineered mice and transplantable TUBO tumor in wild-type BALB/c mice. The intensity of the inhibition directly correlated with the titer of anti-Amot antibodies induced by the vaccine. Tumor inhibition was associated with an increase of vessels diameter with the formation of lacunar spaces, increase in vessel permeability, massive tumor perivascular necrosis and an effective epitope spreading that induces an immune response against other tumor associated antigens. Greater tumor vessel permeability also markedly enhances the antitumor effect of doxorubicin. These data provide a rationale for the development of novel anticancer treatments based on anti-Amot vaccination in conjunction with chemotherapy regimens.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-012-9263-3) contains supplementary material, which is available to authorized users.

Imaging in arthritis: quantifying effects of therapeutic intervention using MRI and molecular imaging.

Modern imaging techniques are becoming increasingly important in assessing the course of arthritis and in permitting measurement of response to treatment as part of the follow-up of patients. They include ultrasonography (US), MRI, PET/CT, and biofluorescence. In patients with rheumatoid arthritis, clinical evaluation is significantly less sensitive than either US or MRI in detecting synovitis. As a result, imaging is a useful alternative to achieving proper assessment of disease activity. The different areas in which the new imaging techniques could help practicing rheumatologists and internal physicians include the following: early and differential diagnosis of arthritis, evaluation of disease activity, prognosis, assessment of treatment efficacy, assessment of remission, and evaluation of subclinical disease. MRI is probably the best imaging method to study disease activity in RA, because it can study all the joints with similar efficacy, has been sufficiently standardised, and yields data on inflammation that can be quantified. Different methods, developed to score synovitis activity, are increasingly used in clinical trials. The main application of PET/CT in rheumatology is the diagnosis and follow-up of large vessel vasculitis. More recently, also RA disease activity has been evaluated, allowing a panoramic view of the patient. Molecular imaging studies molecular and cellular processes in intact living organisms in a non-invasive fashion. In fluorescence, dyes, that emit light upon excitation by a light source and are read by a camera, can be used to show inflamed areas where neoangiogenesis, vasodilatation, and increased vessel permeability are present. These dyes can be coupled with different compounds including antibodies and drugs.

Imaging in arthritis: quantifying effects of therapeutic intervention using MRI and molecular imaging.

Modern imaging techniques are becoming increasingly important in assessing the course of arthritis and in permitting measurement of response to treatment as part of the follow-up of patients. They include ultrasonography (US), MRI, PET/CT, and biofluorescence. In patients with rheumatoid arthritis, clinical evaluation is significantly less sensitive than either US or MRI in detecting synovitis. As a result, imaging is a useful alternative to achieving proper assessment of disease activity. The different areas in which the new imaging techniques could help practicing rheumatologists and internal physicians include the following: early and differential diagnosis of arthritis, evaluation of disease activity, prognosis, assessment of treatment efficacy, assessment of remission, and evaluation of subclinical disease. MRI is probably the best imaging method to study disease activity in RA, because it can study all the joints with similar efficacy, has been sufficiently standardised, and yields data on inflammation that can be quantified. Different methods, developed to score synovitis activity, are increasingly used in clinical trials. The main application of PET/CT in rheumatology is the diagnosis and follow-up of large vessel vasculitis. More recently, also RA disease activity has been evaluated, allowing a panoramic view of the patient. Molecular imaging studies molecular and cellular processes in intact living organisms in a non-invasive fashion. In fluorescence, dyes, that emit light upon excitation by a light source and are read by a camera, can be used to show inflamed areas where neoangiogenesis, vasodilatation, and increased vessel permeability are present. These dyes can be coupled with different compounds including antibodies and drugs.

Imaging in arthritis: quantifying effects of therapeutic intervention using MRI and molecular imaging.

Modern imaging techniques are becoming increasingly important in assessing the course of arthritis and in permitting measurement of response to treatment as part of the follow-up of patients. They include ultrasonography (US), MRI, PET/CT, and biofluorescence. In patients with rheumatoid arthritis, clinical evaluation is significantly less sensitive than either US or MRI in detecting synovitis. As a result, imaging is a useful alternative to achieving proper assessment of disease activity. The different areas in which the new imaging techniques could help practicing rheumatologists and internal physicians include the following: early and differential diagnosis of arthritis, evaluation of disease activity, prognosis, assessment of treatment efficacy, assessment of remission, and evaluation of subclinical disease. MRI is probably the best imaging method to study disease activity in RA, because it can study all the joints with similar efficacy, has been sufficiently standardised, and yields data on inflammation that can be quantified. Different methods, developed to score synovitis activity, are increasingly used in clinical trials. The main application of PET/CT in rheumatology is the diagnosis and follow-up of large vessel vasculitis. More recently, also RA disease activity has been evaluated, allowing a panoramic view of the patient. Molecular imaging studies molecular and cellular processes in intact living organisms in a non-invasive fashion. In fluorescence, dyes, that emit light upon excitation by a light source and are read by a camera, can be used to show inflamed areas where neoangiogenesis, vasodilatation, and increased vessel permeability are present. These dyes can be coupled with different compounds including antibodies and drugs.

Leukocyte-endothelial cell interaction is necessary for photodynamic therapy induced vascular permeabilization

Photodynamic therapy (PDT) affects vascular barrier function and thus increases vessel permeability. This phenomenon may be exploited to facilitate targeted drug delivery and may lead to a new clinical application of photodynamic therapy. Here, we investigate the role of leukocyte recruitment for PDT-induced vascular permeabilization. Fluorescein isothiocyanate dextran (FITC-D, 2,000 kDa) was injected intravenously 120 minutes after focal PDT on striated muscle in nude mice bearing dorsal skinfold chambers (Visudyne® 800 µg/kg, fluence rate 300 mW/cm2 , light dose of 200 J/cm2). Leukocyte interaction with endothelial cells was inhibited by antibodies functionally blocking adhesion molecules ("MABS-PDT" group, n = 5); control animals had PDT but no antibody injection (group "PDT", n = 7). By intravital microscopy, we monitored leukocyte rolling and sticking in real-time before, 90 and 180 minutes after PDT. The extravasation of FITC-D from striated muscle vessels into the interstitial space was determined in vivo during 45 minutes to assess treatment-induced alterations of vascular permeability. PDT significantly increased the recruitment of leukocytes and enhanced the leakage of FITC-D. Neutralization of adhesion molecules before PDT suppressed the rolling of leukocytes along the venular endothelium and significantly reduced the extravasation of FITC-D as compared to control animals (156 ± 27 vs. 11 ± 2 (mean ± SEM, number of WBC/30 seconds mm vessel circumference; P < 0.05) at 90 minutes after PDT and 194 ± 21 vs. 14 ± 4 at 180 minutes after PDT). In contrast, leukocyte sticking was not downregulated by the antibody treatment. Leukocyte recruitment plays an essential role in the permeability-enhancing effect of PDT.

Leukocyte rolling and adhesion via ICAM-1 signals to endothelial permeability. Focus on “Leukocyte rolling and adhesion both contribute to regulation of microvascular permeability to albumin via ligation of ICAM-1”

THE VASCULAR ENDOTHELIUM plays a vital role in the inflammatory response by induction and surface expression of adhesion molecules and chemokines. At sites of acute inflammation, blood flow is increased and postcapillary venules exhibit increased permeability (leakiness) and support the influx of blood leukocytes, primarily neutrophils. The relationship between neutrophil influx and increased vessel permeability has been a topic of investigation for decades. Many investigations have reported a direct neutrophil-dependent increase in vessel permeability during inflammation using in vivo and in vitro models (reviewed in Ref. 4). One aspect that has received increased attention is whether endothelial cell adhesion molecules contribute to the regulation of vessel permeability, in addition to their well-characterized role in neutrophil rolling and adhesion. Intercellular adhesion molecule-1 (ICAM-1, also known as CD54) is an immunoglobulin superfamily member that is abundantly expressed on the endothelial cell surface and is enriched at endothelial cell borders in vivo and in vitro (2, 12, 14, 17). ICAM-1 surface expression is upregulated in endothelial cells by proinflammatory cytokines like TNF-, IL-1, IFN-, or bacterial endotoxins, and ICAM-1 serves as a receptor for leukocyte 2-integrins (LFA-1 and Mac-1) (reviewed in Ref. 15). In an experiment of nature, patients with leukocyte adhesion deficiency-1 (LAD-1) have a severe primary immune deficiency in which blood neutrophils fail to localize to sites of inflammation or injury. This defect occurs because patients’ neutrophils either lack 2-integrins or contain mutations in these molecules. Studies using neutrophils from LAD-1 patients or 2-integrin-deficient mice clearly demonstrate that these cells have normal selectin-dependent rolling on activated endothelium but fail to stably arrest and transmigrate. Additional studies have demonstrated that endothelial-expressed ICAM-1 contributes to both kinetics of leukocyte rolling and arrest in vivo as determined by intravital microscopy of the cremaster muscle microcirculation in ICAM-1 / mice (16). In this issue of American Journal of Physiology-Cell Physiology, Sumagin and colleagues (18) use elegant in vivo studies to demonstrate that microvascular permeability in the cremaster muscle model is regulated by leukocyte engagement of ICAM-1. The authors monitored permeability (Ps) in cremaster microcirculation by efflux of luminal fluorescent-tagged albumin by fluorescence confocal intravital microscopy. Prior studies by Sarelius and colleagues (19) in the same model demonstrated that ICAM-1 engagement induced a localized increase in vessel permeability in unstimulated arterioles and in TNF-stimulated venules. In agreement with this finding ,4ho f TNF- stimulation of arterioles and venules in ICAM-1 knockout (KO) animals did not result in increased vessel permeability as compared with wild-type (WT) mice under identical conditions. Similar results were seen with 2-integrin KO mice. In the current study, the authors provide further evidence linking ICAM-1 expression to regulation of endothelial permeability. Comparisons between unstimulated venules and TNF--treated venules reveal that neutrophil rolling regulates venule permeability in unstimulated venules while leukocyte adhesion regulates venule permeability in TNF--stimulated venules. The 2-integrin-blocking antibodies, which inhibit neutrophil arrest on endothelium, reduced the vessel permeability, and this was further reduced by treatment with a combination of 2-integrin and P-selectin blocking antibodies that inhibits both rolling and arrest. Interestingly, the reduction in venule permeability observed by blockade of P-selectin and 2-integrin function were suggested to be due to insufficient ICAM-1 engagement due to the absence of rolling leukocytes. To examine the role that neutrophils play, circulating neutrophils were depleted by antineutrophil GR-1 antibody treatment. Notably, depletion of neutrophils caused a marked decrease in

Cilengitide inhibits metastatic bone colonization in a nude rat model

Integrins αvβ3 and αvβ5 are considered to play an important role in the pathogenesis of breast cancer bone metastases. This study investigates the effects of the αvβ3/αvβ5 integrin-specific inhibitor cilengitide during early metastatic bone colonization. The impact of cilengitide on the migration, invasion and proliferation of MDA-MB-231 human breast carcinoma cells as well as on bone resorption by osteoclasts was investigated in vitro. For in vivo experiments, nude rats were treated with cilengitide for 30 days starting one day after site-specific tumor cell inoculation in the hind leg, and the course of metastatic changes in bone was followed using flat-panel volumetric computed tomography (VCT) and magnetic resonance imaging (MRI). Vascular changes in bone metastases were investigated using dynamic contrast-enhanced (DCE-) MRI-derived parameters amplitude A and exchange rate coefficient kep. In vitro, cilengitide treatment resulted in a decrease in proliferation, migration and invasion of MDA-MB-231 cells, as well as of osteoclast activity. In vivo, the development of bone metastasis in the hind leg of rats was not prevented by adjuvant cilengitide treatment, but cilengitide reduced the volumes of osteolytic lesions and respective soft tissue tumors of developing bone metastases as assessed with VCT and MRI, respectively. DCE-MRI revealed significant changes in the A and kep parameters including decreased relative blood volume and increased vessel permeability after cilengitide treatment indicating vessel remodeling. In conclusion, during early pathogenic processes of bone colonization, cilengitide treatment exerted effects on tumor cells, osteoclasts and vasculature reducing the skeletal lesion size of experimental skeletal metastases.

Abstract SY16-01: The impact of oxygen availability on angiogenesis: HIF/Notch pathway interactions

Abstract The therapeutic manipulation of angiogenesis holds great promise for treating diverse pathological conditions including cancer, macular degeneration, atherosclerosis, and peripheral arterial disease (PAD). However, the ability to reliably inhibit (or promote) angiogenesis in clinical settings requires a thorough appreciation of the complex molecular events that coordinate vessel sprouting and remodeling. It is well established that these processes are strongly influenced by localized O2 deprivation (hypoxia), which can arise as a consequence of ischemic insult or rapid cell division during solid tumor growth. Elucidating the mechanisms by which hypoxic stress modulates the activity of vascular endothelial cells (ECs) is therefore critical to our understanding of disease-associated angiogenesis. Hypoxia-inducible factor-2α (HIF-2α) is highly expressed in embryonic vascular endothelial cells (ECs) and activates the expression of target genes whose products modulate vascular function and angiogenesis. We have described previously a genetic model designed to test the physiologic consequences of deleting HIF-2α in murine endothelial cells. Surprisingly, mice with HIF-2α-deficient ECs developed normally but displayed a variety of phenotypes, including increased vessel permeability, aberrant endothelial cell ultrastructure, and pulmonary hypertension. Moreover, these animals exhibited defective tumor angiogenesis associated with increased hypoxic stress and tumor cell apoptosis. Immortalized HIF-2α-deficient ECs displayed decreased adhesion to extracellular matrix proteins and expressed reduced levels of transcripts encoding fibronectin, integrins, endothelin B receptor, angiopoietin 2, and delta-like ligand 4 (DLL4). Surprisingly, EC-specific HIF-2α deletion produced increased numbers of newly formed vessels in ischemic muscles and autochthonous tumors; however, these vessels fail to remodel effectively, thereby reducing overall vascular area, blood flow, and tissue oxygenation. Purified HIF-2α-deficient ECs display increased migration, invasion, and tube formation in vitro, as well as reduced hypoxic expression of important angiogenic molecules including angiopoietin 2 (Ang2) and adrenomedullin (ADM-1). Interestingly, similar effects on tumor angiogenesis were observed upon inhibition of the delta-like ligand 4 (Dll4)/Notch pathway, and we further demonstrate that HIF-2α specifically regulates hypoxic expression of Dll4, as well as downstream effectors of the Dll4/Notch pathway both in vivo and in vitro. Collectively, these data identify novel functions for HIF-2α, both in restricting angiogenic sprouting and promoting vascular remodeling, and identify specific molecular effectors underlying these effects, including Dll4, Ang2, and ADM-1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY16-01. doi:10.1158/1538-7445.AM2011-SY16-01

Experimental mycotoxic nephropathy in pigs provoked by a mouldy diet containing ochratoxin A and fumonisin B1

Mycotoxic nephropathy was induced in eighteen young pigs by mouldy diets containing 0.5 ppm ochratoxin A (OTA) and/or 10 ppm fumonisin B1 (FB1) for three months. While the most obvious damages provoked by OTA were seen in the kidneys as expressed by the strong degenerative changes in proximal tubules and fibrosis in kidneys, FB1 was found to induce an increase in permeability of vessels mainly in lung, brain, cerebellum or kidneys and slight to moderate degenerative changes in kidneys. Pathomorphological damages in pigs exposed to both mycotoxins simultaneously present a combination of the main lesions provoked by each mycotoxin alone being stronger in their expression. Biochemical investigations as expressed by the increase of serum creatinine, urea and enzyme activity of ASAT/ALAT and by the decrease of serum cholesterol, total protein, albumin and glucose were strongest in pigs exposed to both mycotoxins simultaneously as can be anticipated form the strongest lesions in the kidneys. Both mycotoxins and their combination were found to disturb powerfully humoral immune response in all experimental pigs as expressed by the strong decrease in antibody titer against Morbus Aujesky at days 21 and 35 after vaccination. Having in mind that the feed levels of the both mycotoxins as well as the exposure time and the pathological findings corresponded to those in some spontaneous cases of porcine nephropathy in Bulgaria and South Africa, it can be concluded that the same mycotoxins are involved in the observed field cases of that nephropathy.

Congenital prekallikrein deficiency

The congenital deficiency of prekallikrein (PK) is a rare condition in which there is a peculiar discrepancy between a severe in vitro defect and absence of bleeding. The gene controlling PK synthesis is located on chromosome 4 and consists of 14 exons and 15 introns. Only approximately 80 cases of PK deficiency have been described in the literature. Owing to the lack of bleeding, most cases go undetected or, if detected, go unreported. Occasional bleeding or thrombosis have been reported in a few patients but this was only due to the presence of associated risk factors. It is certain that the defect does not protect from thrombosis. Diagnosis is based on the presence of a great prolongation of partial thromboplastin time and normal prothrombin time and thrombin time. The long partial thromboplastin time is fully corrected by the addition of normal plasma or normal serum and presents the unusual feature of shortening on long incubation times. Platelet and vascular tests are normal. Immunological studies allow differentiation into two types, namely cases of true deficiency, which are approximately 70% of the total, and cases with abnormal forms. PK is a glycoprotein synthesized in the liver as a single-chain peptide of 88000 Da. It mostly circulates (∼75%) as a complex with high-molecular-weight kininogen. It is cleaved by FXIIa into a heavy chain and a light chain (catalytic domain), held together by disulfide bonds. Molecular biology techniques have so far only been applied to eleven families, and these studies do not yet allow definite phenotype/genotype conclusions. The exons involved are 5, 8, 11, 14 and 15. The noncoagulative effects of PK, mainly based on the effect of kallikrein, have been studied less, since they appear to be the result of the involvement of other components of the contact phase. Kallikrein can mainly affect the formation of bradykinin from high-molecular-weight kininogen and the activation of pro-urokinase to urokinase. Bradykinin causes inflammation, vasodilatation and an increase in vessel permeability. The activation of pro-urokinase results in enhanced fibrinolysis. However, fibrinolysis has been reported to be normal or defective in these patients.

Intravitreal Bevacizumab (Avastin&amp;#174;) for Diabetic Retinopathy at 24-months: The 2008 Juan Verdaguer-Planas Lecture

Diabetic retinopathy (DR) remains the major threat to sight in the working age population. Diabetic macular edema (DME) is a manifestation of DR that produces loss of central vision. Proliferative diabetic retinopathy (PDR) is a major cause of visual loss in diabetic patients. In PDR, the growth of new vessels is thought to occur as a result of vascular endothelial growth factor (VEGF) release into the vitreous cavity as a response to ischemia. Furthermore, VEGF increases vessel permeability leading to deposition of proteins in the interstitium that facilitate the process of angiogenesis and macular edema. This review demonstrates multiple benefits of intravitreal bevacizumab (IVB) on DR including DME and PDR at 24 months of follow up. The results indicate that IVB injections may have a beneficial effect on macular thickness and visual acuity (VA) in diffuse diabetic macular edema. Therefore, in the future this new therapy could replace or complement focal/grid laser photocoagulation in DME. In PDR, this new option could be an adjuvant agent to pan-retina photocoagulation so that more selective therapy may be applied. In addition, we report a series of patients in which tractional retinal detachment developed or progressed after adjuvant preoperative IVB in severe PDR.

Viral G protein-coupled receptor up-regulates Angiopoietin-like 4 promoting angiogenesis and vascular permeability in Kaposi's sarcoma

Kaposi's sarcoma (KS) is an enigmatic vascular tumor thought to be a consequence of dysregulated expression of the human herpesvirus-8 (HHV-8 or KSHV)-encoded G protein-coupled receptor (vGPCR). Indeed, transgenic animals expressing vGPCR manifest vascular tumors histologically identical to human KS, with expression of the viral receptor limited to a few cells, suggestive of a paracrine mechanism for vGPCR tumorigenesis. Both human and vGPCR experimental KS lesions are characterized by prominent angiogenesis and vascular permeability attributed to the release of angiogenic molecules, most notably vascular endothelial growth factor. However, the relative contribution of these paracrine mediators to the angiogenic and exudative phenotype of KS lesions remains unclear. Here we show that vGPCR up-regulation of Angiopoietin-like 4 (ANGPTL4) plays a prominent role in promoting the angiogenesis and vessel permeability observed in KS. Indeed, ANGPTL4 expression is a hallmark of vGPCR experimental and human KS lesions. Inhibition of ANGPTL4 effectively blocks vGPCR promotion of the angiogenic switch and vascular leakage in vitro and tumorigenesis in vivo. These observations suggest that ANGPTL4 is a previously unrecognized target for the treatment of patients with KS. As angiogenesis and increased vessel permeability are common themes in all solid tumors, these findings may have a broad impact on our understanding and treatment of cancer.