Angiogenesis, the formation of new blood vessels from a pre-existing vasculature, is crucial during development and for many diseases including cancer. Despite tremendous progress in the understanding of the angiogenic process, many aspects are still not fully elucidated. Several attempts have been made to identify novel genes involved in endothelial cell biology and angiogenesis. Here we focused on Pald1, a recently identified, vascular-enriched gene encoding paladin. Our in vitro studies indicate that paladin is a lipid phosphatase catalyzing dephosphorylation of phosphatidylinositol phosphates, a process essential for endocytosis and intracellular vesicle trafficking. We confirmed paladin’s vascular expression pattern and revealed a shift from a broad endothelial cell expression during development to an arterial mural cell-restricted expression in several vascular beds in adult mice. Paladin expression in the lung, however, was not restricted to the vasculature, but was also observed in pneumocytes and myofibroblasts. Lungs of female, but not male, Pald1 null mice displayed an obstructive lung phenotype with increased alveolar air sacs that were already apparent early in the alveolarization process. Only endothelial cells, but not other main lung cell types, were affected by loss of paladin. Endothelial cell number was reduced in 4-week old mice, possibly due to increased endothelial turnover in Pald1 deficient lungs. Vascular defects were also found in the retina. Loss of paladin led to reduced retinal vascular outgrowth accompanied by a hyperdense and hypersprouting vascular front. Downstream signaling of the major angiogenic driver, vascular endothelial growth factor receptor 2 (VEGFR2) was sustained in Pald1 null mice, and VEGFR2 degradation was impaired. Furthermore, paladin inhibited endothelial cell junction stability and loss of paladin led to reduced vascular permeability. Whether the differences in VEGFR2 signaling and adherens junction stability are connected remains to be fully explored. The newly identified lipid phosphatase activity of paladin and its specific effects on VEGFR2 signaling and adherens junction stability indicate that paladin may be controlling the endocytic pathway.

Myocardial infarction (MI) is primarily caused by blockade of the coronary circulation, resulting in ischemic insult. The only available remedy is reperfusion, which induces oxidative stress and activates inflammatory responses at the site of injury. Cysteinyl leukotrienes (cysLTs) are potent pro-inflammatory mediators that exert their effects through two classical receptors: cysLT receptor 1 (CysLT1R) and cysLT receptor 2 (CysLT2R), the latter of which is prevalent in the heart and circulatory system and has been implicated in cardiovascular disease. However, although endothelial CysLT2R overexpression exacerbates MI damage and induces vascular hyperpermeability, understanding of CysLT2R activation-induced mechanisms is poor, as isolating CysLT2R-specific effects has proven difficult due to a lack of appropriate pharmacological agents. We investigate herein the role of CysLT2R activation in myocardial ischemia/reperfusion injury. We have characterized a novel CysLT2R-selective antagonist BayCysLT2 in both in vitro and in vivo systems, and establish that CysLT2R-selective antagonism attenuates exacerbated MI injury, adhesion molecule gene regulation, and myocardial neutrophil presence observed in CysLT2R overexpressing (EC) mice. We also examined effects of CysLT2R antagonism in long-term cardiac remodeling post-myocardial infarction, and found that blockade of CysLT2R post-reperfusion, regardless of whether CysLT2R is overexpressed or not, elicits a mild pathological cardiac hypertrophic response despite mitigating infarction damage to the apical ventricular wall. Finally, we created a novel mouse model (EC/KO) that expresses CysLT2R predominantly in vascular endothelium in order to identify tissue-specific mechanisms of CysLT2R activation. Surprisingly, MI injury was attenuated in EC/KO mice, indicating that both endothelial and non-endothelial CysLT2R expression subsets have roles in mediating infarction injury. Indeed, EC/KO mice demonstrated hyperpermeability in cremaster venules only when leukotrienes are applied, in contrast to EC mice. In addition, endothelial CysLT2R activation facilitates leukocyte transmigration, whereas non-endothelial CysLT2Rs regulate basal rolling leukocyte flux in microvasculature. Although much work remains to be done, the characterization of a CysLT2R-selective antagonist provides a vital tool for CysLT2R research moving forward, and our investigation of CysLT2R activation reveal the existence of a complicated and multi-faceted pathway resulting in activation of pro-inflammatory mechanisms. / Thesis (Ph.D, Physiology) -- Queen's University, 2013-09-26 10:29:03.466

Background: The increased vascular permeability seen after burn contribute to morbidity and mortality as it interferes with organ function and the healing process. Large efforts have been made to explore underlying pathophysiological mechanisms that generate increased vascular permeability after burns. Many different substances have been proposed as mediators of which histamine, serotonin and oxygen radicals are claimed most important. However, no specific blocker has convincingly been shown to be clinically effective. Early work has claimed increased histamine plasma-concentrations in humans after burn and data from animal models pointed at histamine as an important mediator. Modern human clinical studies investigating the role of histamine as a mediator of the generalized post burn increase in vascular permeability are lacking. less thanbrgreater than less thanbrgreater thanMethod: We examined histamine turnover by measuring the urinary excretion of histamine and methyl histamine for 48 h after burns in 8 patients (mean total burn surface area 24%). less thanbrgreater than less thanbrgreater thanResults: Over time, in this time frame and compared to healthy controls we found a small increase in the excretion of histamine, but no increase of its metabolite methylhistamine. less thanbrgreater than less thanbrgreater thanConclusion: Our findings do not support that histamine is an important mediator of the increased systemic vascular permeability seen after burn. / <p>Funding Agencies|Research and Development Unit, Jamtland County Council, Sweden||</p>

No description available.

La perméabilité vasculaire est une caractéristique cruciale de l’angiogenèse. Les acteurs principaux sont les cellules endothéliales qui la régulent en réponse à divers facteurs perméabilisant, tels que le « Vascular Endothelial Growth Factor » (VEGF). Dans le contexte pathologique du cancer, les cellules tumorales produisent de grandes quantités de VEGF qui stimulent la perméabilité, ce qui leur permet d’infiltrer le réseau vasculaire. Il est connu que la tyrosine kinase Src contrôle cette modulation de la perméabilité. Puisque notre laboratoire a préalablement démontré que la phosphatase de type récepteur (PTP) DEP-1 est impliquée dans l’activation de Src en réponse au VEGF, nous avons émis l'hypothèse que DEP-1 pourrait aussi jouer un rôle dans la perméabilité des cellules endothéliales. Grâce à des expériences de transfections d’ARN interférant, nous démontrons que DEP-1 est important pour la régulation de la phosphorylation de la VE-Cadhérine, un médiateur critique de la perméabilité. L’impact de DEP-1 sur la dissociation de jonctions intercellulaires est également démontré par microscopie à immunofluorescence de cellules endothéliales. DEP-1 est également nécessaire à l’augmentation de la perméabilité induite par VEGF in vitro. Deux résidus tyrosine retrouvés dans la queue carboxy-terminale de DEP-1 sont essentiels à l’activation de Src en réponse au VEGF. Suite à la transfection d’un plasmide encodant DEP-1 muté pour ces deux résidus, nous démontrons aussi leur implication dans la régulation de la perméabilité in vitro par DEP-1. Ces travaux permettent ainsi d’approfondir nos connaissances sur un nouveau régulateur potentiel de la perméabilité vasculaire. / Endothelial cell permeability is a crucial step of angiogenesis. The main actors behind permeability are endothelial cells who accomplish this in response to permeabilizing factors, most notably Vascular Endothelial Growth Factor (VEGF). In a pathological context, migrating tumor cells produce great quantities of VEGF that stimulate an increase of vascular permeability, which allows them to intravasate into the vasculature. Src has been shown to mediate this process. Our laboratory has previously shown that the protein tyrosine phosphatase DEP-1 is involved in the regulation of VEGF-dependant activation of Src. These data thus suggested that DEP-1 might play a role in endothelial cell permeability. Here, we show through siRNA experiments that DEP-1 is important for the regulatory phosphorylation of VE-Cadherin which is critical for the induction of permeability. The impact of DEP-1 on intercellular junction dissociation is also demonstrated through immunofluorescence microscopy of endothelial cells. We further show that DEP-1 is absolutely required for the VEGF-dependent increase of permeability as illustrated by in vitro permeability assay on siRNA-transfected endothelial cells. Finally, we show that tyrosine residues in DEP-1’s carboxy-terminal tail, which are crucial for mediating Src activity in response to VEGF, are implicated in VEGF-dependant increase in permeability by transfecting plasmids coding for DEP-1 mutants of these tyrosine residues. These findings shed light on a novel potential key regulator of in vivo permeability.

ABSTRACT Vascular permeability and angiogenesis are crucial events in the rodent and human uterus in early pregnancy and are regulated by vascular endothelial growth factor (VEGF) and prostaglandins liberated from arachidonic acid by cyclooxygenase 2 (COX2). These events coincide with the typical morphological features of the receptive uterus and are regulated by synchronized release of ovarian hormones (oestrogen and progesterone). However, administration of follicle stimulating hormone (FSH) and human chorionic gonadotropin (hCG), commonly used in assisted reproduction, affect the synchrony of the hormonal milieu, particularly by increasing oestrogen levels. This causes detrimental changes to the uterine morphology and affects vascular permeability at the site of implantation. In the present study, the expression of COX2 and VEGF was compared between control and hyperstimulated rat uteri during the peri-implantation period using immunohistochemistry and Western blot analysis. While in control pregnant rats COX2 and VEGF immunolocalization occurred in the luminal epithelial cells and stroma on consecutive days, strong immunolocalization of COX2 and VEGF occurred in the luminal epithelial cells but was inhibited in the stroma of the hyperstimulated rats. This appears to have resulted in the suppression of stromal decidualization and vascular permeability. Western blot analysis did not show any results. This may be due to low concentrations of the protein in the sample. Since vascular permeability and angiogenesis are critical to the process of implantation and are influenced by VEGF and COX2, disturbance of the pattern of these two proteins by hyperstimulation may contribute to the low implantation rate in IVF programes. immunohistochemistry and Western blot analysis. While in control pregnant rats COX2 and VEGF immunolocalization occurred in the luminal epithelial cells and stroma on consecutive days, strong immunolocalization of COX2 and VEGF occurred in the luminal epithelial cells but was inhibited in the stroma of the hyperstimulated rats. This appears to have resulted in the suppression of stromal decidualization and vascular permeability. Western blot analysis did not show any results. This may be due to low concentrations of the protein in the sample. Since vascular permeability and angiogenesis are critical to the process of implantation and are influenced by VEGF and COX2, disturbance of the pattern of these two proteins by hyperstimulation may contribute to the low implantation rate in IVF programes.

MY-24 is an aristeromycin derivative previously shown to protect AG129 type I and II interferon receptor knockout mice from lethal challenge with Tacaribe virus (TCRV). TCRV is nonpathogenic to humans, but is closely related to the highly pathogenic New World arenaviruses that cause often-fatal viral hemorrhagic fever syndromes. Remarkably, MY-24 prevented mortality without reducing TCRV burden in the circulation or tissues. To investigate the mechanism by which MY-24 protects AG129 mice against TCRV infection, we first characterized the natural history of disease in the model with an emphasis on cytokine responses and vascular integrity to establish the best times to evaluate the effects of MY-24 treatment on host responses believed to contribute to pathogenesis and fatal outcome. We found that viral replication in the blood and in various tissues precedes a hyperproduction of proinflammatory mediators that may lead to the destabilization of the endothelial barrier and increased vascular leakage believed to contribute to terminal shock associated with severe cases of hemorrhagic fever. We also found slightly reduced virus titers in certain tissues from MY-24-treated mice, suggesting that there may be a weak antiviral effect; however, TCRV was not cleared from lung, spleen, brain or kidney in recovering animals out to 40 days post-infection, indicative of the establishment of chronic infection in mice that are able to survive the initial challenge. Neutralizing antibodies do not appear to play a major role in the antiviral effect of MY-24, whereas reductions in several key proinflammatory cytokines in mice treated with MY-24 may serve to reduce vascular leakage caused by TCRV infection.

Le mélanome cutané est l'un des cancers les plus agressifs et mortel capables de dissémination métastatique à distance. L’intravasation cellules tumorales dans le vaisseau sanguin dans les tumeurs primaires et l'extravasation sont des étapes importantes dans la formation de métastases. Ces étapes impliquent la perturbation de la barrière endothéliale par des cellules tumorales afin de faciliter leur migration transendothéliale et la colonisation métastatique. Cependant, le processus par lequel les cellules tumorales modulent l'intégrité des jonctions vasculaires est encore mal compris. Afin de déterminer les facteurs de perméabilité sécrétés par les cellules métastatiques, nous avons identifié la protéine matricielle SPARC comme un facteur critique contribuant à la perméabilité vasculaire et l'extravasation des cellules tumorales. Nous montrons que SPARC sécrété par les cellules de mélanome induit une perméabilité vasculaire via l'ouverture des jonctions intercellulaires des monocouches endothéliales et entraîne la migration transendothéliale de cellules de mélanome. In vivo, l’extinction de SPARC mène à une diminution drastique dans la colonisation à court et long terme des poumons et de la perméabilité des capillaires pulmonaires. A l’inverse, sa surexpression augmente les capacités d’extravasation et les métastases. / Cutaneous melanoma is one of the most aggressive cancers capable of distant and lethal metastatic spread. Tumor cell intravasation into blood vessel at primary tumor sites and subsequent extravasation are critical steps in the formation of metastases. These steps entail disruption of the endothelial barrier by tumor cells to facilitate their transendothelial passage and metastatic seeding. However, the way by which tumor cells modulate vascular junction integrity is still poorly understood. In an attempt to determine permeability factors secreted by metastatic cells, we identified the matricellular protein SPARC as a critical signaling factor that contributes to elevated vascular permeability and tumor cell extravasation. We show that SPARC released by melanoma cells enhances vascular leakiness by inducing opening of intercellular junctions of endothelial monolayers and drives melanoma cell transendothelial migration. In vivo vascular permeability and metastatic assays demonstrate that SPARC deficiency abrogates tumor-induced permeability of lung capillaries and prevents extravasation from blood vessels and metastasis, whereas overexpression of SPARC increases the lung metastatic potential of melanoma cells. Mechanistically, SPARC-induced endothelial gap formation and transmigration is dependent on vascular cell adhesion molecule (VCAM1) and p38 MAPK signaling pathway in endothelial cells. Importantly, blocking VCAM1 impedes melanoma cell extravasation. The clinical relevance of our findings is highlighted by the high levels of SPARC detected in tumor cells from human pulmonary melanoma lesions.

Simultaneous optical imaging and MRI of a dorsal skin-fold window chamber mouse model is investigated as a novel methodology to study the tumor microenvironment. Simultaneous imaging with two modalities allows for cross-validation of results, integration of the capabilities of the two modalities in one study and mitigation of invasive factors, such as surgery and anesthesia, in an in-vivo experiment. To make this investigation possible, three optical imaging systems were developed that operated inside the MRI scanner. One of the developed systems was applied to estimate vascular kinetic parameters of tumors in a dorsal skin-fold window chamber mouse model with simultaneous optical and MRI imaging. The target of imaging was a molecular agent that was dual labeled with both optical and MRI contrast agents. The labeling of the molecular agent, characteristics of the developed optical systems, the methodologies of measuring vascular kinetic parameters using optical imaging and MRI data, and the obtained results are described and illustrated.

La perméabilité vasculaire est une caractéristique cruciale de l’angiogenèse. Les acteurs principaux sont les cellules endothéliales qui la régulent en réponse à divers facteurs perméabilisant, tels que le « Vascular Endothelial Growth Factor » (VEGF). Dans le contexte pathologique du cancer, les cellules tumorales produisent de grandes quantités de VEGF qui stimulent la perméabilité, ce qui leur permet d’infiltrer le réseau vasculaire. Il est connu que la tyrosine kinase Src contrôle cette modulation de la perméabilité. Puisque notre laboratoire a préalablement démontré que la phosphatase de type récepteur (PTP) DEP-1 est impliquée dans l’activation de Src en réponse au VEGF, nous avons émis l'hypothèse que DEP-1 pourrait aussi jouer un rôle dans la perméabilité des cellules endothéliales. Grâce à des expériences de transfections d’ARN interférant, nous démontrons que DEP-1 est important pour la régulation de la phosphorylation de la VE-Cadhérine, un médiateur critique de la perméabilité. L’impact de DEP-1 sur la dissociation de jonctions intercellulaires est également démontré par microscopie à immunofluorescence de cellules endothéliales. DEP-1 est également nécessaire à l’augmentation de la perméabilité induite par VEGF in vitro. Deux résidus tyrosine retrouvés dans la queue carboxy-terminale de DEP-1 sont essentiels à l’activation de Src en réponse au VEGF. Suite à la transfection d’un plasmide encodant DEP-1 muté pour ces deux résidus, nous démontrons aussi leur implication dans la régulation de la perméabilité in vitro par DEP-1. Ces travaux permettent ainsi d’approfondir nos connaissances sur un nouveau régulateur potentiel de la perméabilité vasculaire. / Endothelial cell permeability is a crucial step of angiogenesis. The main actors behind permeability are endothelial cells who accomplish this in response to permeabilizing factors, most notably Vascular Endothelial Growth Factor (VEGF). In a pathological context, migrating tumor cells produce great quantities of VEGF that stimulate an increase of vascular permeability, which allows them to intravasate into the vasculature. Src has been shown to mediate this process. Our laboratory has previously shown that the protein tyrosine phosphatase DEP-1 is involved in the regulation of VEGF-dependant activation of Src. These data thus suggested that DEP-1 might play a role in endothelial cell permeability. Here, we show through siRNA experiments that DEP-1 is important for the regulatory phosphorylation of VE-Cadherin which is critical for the induction of permeability. The impact of DEP-1 on intercellular junction dissociation is also demonstrated through immunofluorescence microscopy of endothelial cells. We further show that DEP-1 is absolutely required for the VEGF-dependent increase of permeability as illustrated by in vitro permeability assay on siRNA-transfected endothelial cells. Finally, we show that tyrosine residues in DEP-1’s carboxy-terminal tail, which are crucial for mediating Src activity in response to VEGF, are implicated in VEGF-dependant increase in permeability by transfecting plasmids coding for DEP-1 mutants of these tyrosine residues. These findings shed light on a novel potential key regulator of in vivo permeability.