Plasminogen activation in cancer

The subject of this thesis focusses on the role of the plasminogen activation system in angiogenesis and cancer. The plasminogen activation system regulates fibrinolysis and controls cell migration and invasion by plasmin-mediated matrix proteolysis. Plasmin is formed upon cleavage of the zymogen plasminogen by plasminogen activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). In contrast to uPA, plasminogen activation by tPA requires a cofactor, such as fibrin. Plasmin cleaves its substrates behind a basic amino-acid (lysine or arginine). Free carboxy-terminal lysines or arginines provide high affinity binding sites for plasminogen and facilitate efficient activation by plasminogen activators. Plasminogen activation can be inhibited by lysine analogues such as epsilon aminocaproic acid (?ACA) or tranexamic acid. Studies with these lysine analogues have indicated the importance of carboxy-terminal lysine residues in cancer and angiogenesis. In chapter 1 we give a literature overview of the plasminogen activation system in cancer. In addition we summarize possible ways to interfere with tumor growth by targeting this system. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a circulating carboxypeptidase B-type enzyme that specifically cleaves carboxy-terminal lysine or arginine residues from proteins. Removal of carboxy-terminal lysines and arginines from fibrin abrogates efficient plasminogen binding and consequently leads to decreased plasmin formation. Given the importance of the plasminogen activation system in angiogenesis, tumor growth and metastasis, we have studied the role of TAFI in corneal angiogenesis (chapter 2) and subcutaneous tumor growth and experimental- and spontaneous metastasis formation in TAFI-deficient mice (chapter 3). Furthermore, we have determined TAFI antigen and TAFI activity levels in patients with primary and metastasized prostate cancer (chapter 4). We further investigated the mechanism of action of endostatin (chapter 5). Endostatin is a carboxy-terminal fragment of collagen XIII, and has originally been described as one of the most potent inhibitors of angiogenesis. In animal experiments different forms of endostatin are used. Although the soluble form of endostatin inhibits tumor growth, complete tumor regression has only been observed when using insoluble endostatin. We found that insoluble endostatin, in contrast to soluble endostatin, stimulates plasminogen activation by tPA, induces plasminogen-mediated endothelial cell detachment and matrix degradation and is toxic to neuronal cells (chapter 5,6). These effects were inhibited by carboxypeptidase B treatment indicating an essential role for carboxy-terminal lysines. Insoluble proteins may occur as amorphous aggregates or as highly ordered amyloid deposits. Structure analyses revealed that insoluble endostatin forms amyloid fibers (chapter 6) which was critical for the effects on plasminogen activation and endothelial and neuronal cells (chapter 5,6,7). We further established that the presence of amyloid fibers is a common feature in other tPA-ligands, including fibrin peptides, islet amyloid polypeptide (IAPP, associated with pancreatic ß cell toxicity in type II diabetes) and amyloid ß (associated with Alzheimers disease), and responsible for their ability to stimulate tPA-mediated plasminogen activation (chapter 7). To what extent may our observations be of value for the use of endostatin and other antiangiogenic fragments? In chapter 9 we discuss the possible implications of our finding

Regulation of brain endothelial barrier function by microRNAs in health and neuroinflammation

Brain endothelial cells constitute the major cellular element of the highly specialized blood–brain barrier (BBB) and thereby contribute to CNS homeostasis by restricting entry of circulating leukocytes and blood-borne molecules into the CNS. Therefore, compromised function of brain endothelial cells has serious consequences for BBB integrity. This has been associated with early events in the pathogenesis of several disorders that affect the CNS, such as multiple sclerosis, HIV-associated neurologic disorder, and stroke. Recent studies demonstrate that brain endothelial microRNAs play critical roles in the regulation of BBB function under normal and neuroinflammatory conditions. This review will focus on emerging evidence that indicates that brain endothelial microRNAs regulate barrier function and orchestrate various phases of the neuroinflammatory response, including endothelial activation in response to cytokines as well as restoration of inflamed endothelium into a quiescent state. In particular, we discuss novel microRNA regulatory mechanisms and their contribution to cellular interactions at the neurovascular unit that influence the overall function of the BBB in health and during neuroinflammatio

MicroRNA-155 negatively affects blood-brain barrier function during neuroinflammation.

Blood-brain barrier (BBB) dysfunction is a hallmark of neurological conditions such as multiple sclerosis (MS) and stroke. However, the molecular mechanisms underlying neurovascular dysfunction during BBB breakdown remain elusive. MicroRNAs (miRNAs) have recently emerged as key regulators of pathogenic responses, although their role in central nervous system (CNS) microvascular disorders is largely unknown. We have identified miR-155 as a critical miRNA in neuroinflammation at the BBB. miR-155 is expressed at the neurovascular unit of individuals with MS and of mice with experimental autoimmune encephalomyelitis (EAE). In mice, loss of miR-155 reduced CNS extravasation of systemic tracers, both in EAE and in an acute systemic inflammation model induced by lipopolysaccharide. In cultured human brain endothelium, miR-155 was strongly and rapidly upregulated by inflammatory cytokines. miR-155 up-regulation mimicked cytokine-induced alterations in junctional organization and permeability, whereas inhibition of endogenous miR-155 partially prevented a cytokine-induced increase in permeability. Furthermore, miR-155 modulated brain endothelial barrier function by targeting not only cell-cell complex molecules such as annexin-2 and claudin-1, but also focal adhesion components such as DOCK-1 and syntenin-1. We propose that brain endothelial miR-155 is a negative regulator of BBB function that may constitute a novel therapeutic target for CNS neuroinflammatory disorders

P-Glycoprotein Acts as an Immunomodulator during Neuroinflammation

Background: Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system in which autoreactive myelin-specific T cells cause extensive tissue damage, resulting in neurological deficits. In the disease process, T cells are primed in the periphery by antigen presenting dendritic cells (DCs). DCs are considered to be crucial regulators of specific immune responses and molecules or proteins that regulate DC function are therefore under extensive investigation. We here investigated the potential immunomodulatory capacity of the ATP binding cassette transporter P-glycoprotein (Pgp). P-gp generally drives cellular efflux of a variety of compounds and is thought to be involved in excretion of inflammatory agents from immune cells, like DCs. So far, the immunomodulatory role of these ABC transporters is unknown. Methods and Findings: Here we demonstrate that P-gp acts as a key modulator of adaptive immunity during an in vivo model for neuroinflammation. The function of the DC is severely impaired in P-gp knockout mice (Mdr1a/1b-/-), since both DC maturation and T cell stimulatory capacity is significantly decreased. Consequently, Mdr1a/1b-/- mice develop decreased clinical signs of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Reduced clinical signs coincided with impaired T cell responses and T cell-specific brain inflammation. We here describe the underlying molecular mechanism and demonstrate that P-gp is crucial for the secretion of pro-inflammatory cytokines such as TNF-alpha and IFN-gamma. Importantly, the defect in DC function can be restored by exogenous addition of these cytokines. Conclusions: Our data demonstrate that P-gp downmodulates DC function through the regulation of pro-inflammatory cytokine secretion, resulting in an impaired immune response. Taken together, our work highlights a new physiological role for P-gp as an immunomodulatory molecule and reveals a possible new target for immunotherap

MicroRNAs regulate human brain endothelial cell-barrier function in inflammation: implications for multiple sclerosis.

Blood-brain barrier (BBB) dysfunction is a major hallmark of many neurological diseases, including multiple sclerosis (MS). Using a genomics approach, we defined a microRNA signature that is diminished at the BBB of MS patients. In particular, miR-125a-5p is a key regulator of brain endothelial tightness and immune cell efflux. Our findings suggest that repair of a disturbed BBB through microRNAs may represent a novel avenue for effective treatment of MS

Sphingosine 1-phosphate receptor 5 mediates the immune quiescence of the human brain endothelial barrier

BACKGROUND: The sphingosine 1-phosphate (S1P) receptor modulator FTY720P (Gilenya®) potently reduces relapse rate and lesion activity in the neuroinflammatory disorder multiple sclerosis. Although most of its efficacy has been shown to be related to immunosuppression through the induction of lymphopenia, it has been suggested that a number of its beneficial effects are related to altered endothelial and blood–brain barrier (BBB) functionality. However, to date it remains unknown whether brain endothelial S1P receptors are involved in the maintenance of the function of the BBB thereby mediating immune quiescence of the brain. Here we demonstrate that the brain endothelial receptor S1P(5) largely contributes to the maintenance of brain endothelial barrier function. METHODS: We analyzed the expression of S1P(5) in human post-mortem tissues using immunohistochemistry. The function of S1P(5) at the BBB was assessed in cultured human brain endothelial cells (ECs) using agonists and lentivirus-mediated knockdown of S1P(5). Subsequent analyses of different aspects of the brain EC barrier included the formation of a tight barrier, the expression of BBB proteins and markers of inflammation and monocyte transmigration. RESULTS: We show that activation of S1P(5) on cultured human brain ECs by a selective agonist elicits enhanced barrier integrity and reduced transendothelial migration of monocytes in vitro. These results were corroborated by genetically silencing S1P(5) in brain ECs. Interestingly, functional studies with these cells revealed that S1P(5) strongly contributes to brain EC barrier function and underlies the expression of specific BBB endothelial characteristics such as tight junctions and permeability. In addition, S1P(5) maintains the immunoquiescent state of brain ECs with low expression levels of leukocyte adhesion molecules and inflammatory chemokines and cytokines through lowering the activation of the transcription factor NFκB. CONCLUSION: Our findings demonstrate that S1P(5) in brain ECs contributes to optimal barrier formation and maintenance of immune quiescence of the barrier endothelium

Systemic treatment with glutathione PEGylated liposomal methylprednisolone (2B3201) improves therapeutic efficacy in a model of ocular inflam mation. Invest Ophthalmol Vis Sci

PURPOSE. Ocular inflammation is associated with the loss of visual acuity and subsequent blindness. Since their development, glucocorticoids have been the mainstay of therapy for ocular inflammatory diseases. However, the clinical benefit is limited by side effects due to the chronic use and generally high dosage that is required for effective treatment. We have developed the G-Technology to provide a means for sustained drug delivery, increased drug half-life, and reduced bodily drug exposure. Glutathione PEGylated liposomal methylprednisolone (2B3-201) has been developed as treatment for neuroinflammatory conditions and was evaluated in ocular inflammation. METHODS. The efficacy of 2B3-201 was investigated in rats with experimental autoimmune uveitis (EAU). Rats received 10 mg/kg of 2B3-201 intravenously at disease onset and at peak of the disease. The same dose of free methylprednisolone served as control treatment. Clinical signs of ocular inflammation were assessed by slit-lamp and immunohistochemistry. CONCLUSIONS. In this study, we show that systemic treatment with 2B3-201, a glutathione PEGylated liposomal methylprednisolone formulation, resulted in a superior efficacy in rats with EAU. Altogether, our findings hold promise for the development of a safe and more convenient systemic treatment for uveitis

SOX-18 controls endothelial-specific claudin-5 gene expression and barrier function

Members of the claudin family constitute tight junction strands and are major determinants in specificity and selectivity of paracellular barriers. Transcriptional control of claudin gene expression is essential to establish individual claudin expression patterns and barrier properties. Using full genome expression profiling, we now identify sex-determining region Y-box (SOX)-18, a member of the SOX family of high-mobility group box transcription factors, as one of the most differentially induced genes during establishment of the endothelial barrier. We show that overexpression of SOX-18 and a dominant-negative mutant thereof, as well as SOX-18 silencing, greatly affect levels of claudin-5 (CLDN5). The relevance of an evolutionary conserved SOX-binding site in the CLDN5 promoter is shown using sequential promoter deletions, as well as point mutations. Furthermore, SOX-18 silencing abrogates endothelial barrier function, as measured by electric cell-substrate impedance sensing. Thus an obligatory role for SOX-18 in the regulation of CLDN5 gene expression in an endothelial-specific and cell density-dependent manner is established, as well as a crucial, nonredundant role for specifically SOX-18 in the formation of the endothelial barrie

Diapedesis of monocytes is associated with MMP-mediated occludin disappearance in brain endothelial cells

The blood-brain barrier (BBB), a selective barrier formed by endothelial cells and dependent on the presence of tight junctions, is compromised during neuroinflammation. A detailed study of tight junction dynamics during transendothelial migration of leukocytes has been lacking. Therefore, we retrovirally expressed green fluorescent protein (GFP) fused to the N-terminus of the tight junction protein occludin in the rat brain endothelial cell line GP8/3.9. Confocal microscopy analyses revealed that GFP-occludin colocalized with the intracellular tight junction protein, ZO-1, localized at intercellular connections, and was absent at cell borders lacking apposing cells. Using live cell imaging we found that monocytes scroll over the brain endothelial cell surface toward cell-cell contacts, induce gap formation, which is associated with local disappearance of GFP-occludin, and subsequently traverse the endothelium paracellularly. Immunoblot analyses indicated that loss of occludin was due to protein degradation. The broad spectrum matrix metalloproteinase (MMP) inhibitor BB-3103 significantly inhibited endothelial gap formation, occludin loss, and the ability of monocytes to pass the endothelium. Our results provide a novel insight into the mechanism by which leukocytes traverse the BBB and illustrate that therapeutics aimed at the stabilization of the tight junction may be beneficial to resist a neuroinflammatory attack