Thomas Renné

Publications of Thomas Renné

• Regulatory mechanisms of the plasma contact system.

  Authors: Coen Maas, Thomas Renné

  Thrombosis research. 03/2012;

  The plasma contact system is a proinflammatory and procoagulant protease system. The biochemistry of the system is well established, however, its biological functions are just beginning to emerge.The plasma contact system is a proinflammatory and procoagulant protease system. The biochemistry of the system is well established, however, its biological functions are just beginning to emerge. Here, we provide a condensed overview on mechanisms involved in activation and regulation of the contact system. These recent findings will help us to better understand the role of the enigmatic system for health and disease.

• Factor XI and XII as antithrombotic targets.

  Authors: Felicitas Müller, David Gailani, Thomas Renné

  Current opinion in hematology. 09/2011; 18(5):349-55.

  Arterial and venous thrombosis are major causes of morbidity and mortality, and the incidence of thromboembolic diseases increases as a population ages. Thrombi are formed by activated platelets andArterial and venous thrombosis are major causes of morbidity and mortality, and the incidence of thromboembolic diseases increases as a population ages. Thrombi are formed by activated platelets and fibrin. The latter is a product of the plasma coagulation system. Currently available anticoagulants such as heparins, vitamin K antagonists and inhibitors of thrombin or factor Xa target enzymes of the coagulation cascade that are critical for fibrin formation. However, fibrin is also necessary for terminating blood loss at sites of vascular injury. As a result, anticoagulants currently in clinical use increase the risk of bleeding, partially offsetting the benefits of reduced thrombosis. This review focuses on new targets for anticoagulation that are associated with minimal or no therapy-associated increased bleeding. Data from experimental models using mice and clinical studies of patients with hereditary deficiencies of coagulation factors XI or XII have shown that both of these clotting factors are important for thrombosis, while having minor or no apparent roles in processes that terminate blood loss (hemostasis). Hereditary deficiency of factor XII (Hageman factor) or factor XI, plasma proteases that initiate the intrinsic pathway of coagulation, impairs thrombus formation and provides protection from vascular occlusive events, while having a minimal impact on hemostasis. As the factor XII-factor XI pathway contributes to thrombus formation to a greater extent than to normal hemostasis, pharmacological inhibition of these coagulation factors may offer the exciting possibility of anticoagulation therapies with minimal or no bleeding risk.

• The procoagulant and proinflammatory plasma contact system.

  Authors: Thomas Renné

  Seminars in immunopathology. 08/2011; 34(1):31-41.

  The contact system is a plasma protease cascade that is initiated by coagulation factor XII activation on cardiovascular cells. The system starts procoagulant and proinflammatory reactions, via theThe contact system is a plasma protease cascade that is initiated by coagulation factor XII activation on cardiovascular cells. The system starts procoagulant and proinflammatory reactions, via the intrinsic pathway of coagulation or the kallikrein-kinin system, respectively. The biochemistry of the contact system in vitro is well understood, however, its in vivo functions are just beginning to emerge. Data obtained in genetically engineered mice have revealed an essential function of the contact system for thrombus formation. Severe deficiency in contact system proteases impairs thrombus formation but does not reduce the hemostatic capacity of affected individuals. The system is activated by an inorganic polymer, polyphosphate that is released from activated platelets. Excessive inherited activation of the contact system causes a life-threatening swelling disorder, hereditary angioedema. Activation of the contact system by pathogens contributes to leakage in bacterial infections. Mast-cell-derived heparin triggers contact-system-mediated edema formation with implications for allergic disease states. Here we present an overview about the plasma contact system in occlusive and inflammatory disease and its contribution to health and pathology.

• Impaired melanoma growth in VASP deficient mice.

  Authors: Young Min Kim, Christoph Renné, Stefanie Seifert, Kai Schuh, Thomas Renné

  FEBS letters. 08/2011; 585(15):2533-6.

  Progression of tumors depends on interactions of cancer cells with the host environment. Expression of the cytoskeleton protein VASP is upregulated in various cancer entities. We analyzed the role ofProgression of tumors depends on interactions of cancer cells with the host environment. Expression of the cytoskeleton protein VASP is upregulated in various cancer entities. We analyzed the role of VASP for melanoma growth in murine allograft models. Growth of VASP expressing melanomas was retarded in VASP(-/-) versus wild-type animals. Over time tumor size was <50% in VASP(-/-) versus wild-type animals and independent of expression levels of Ena/VASP protein family members. Histological analyses showed smaller cells with impaired nutrition status and less vascularization in melanomas derived from VASP(-/-) versus counterparts from wild-type mice. Cumulatively, the data reveal a critical role of VASP in non-tumor cells in the tumor environment for melanoma growth in vivo.

• The plasma contact system 2.0.

  Authors: Coen Maas, Chris Oschatz, Thomas Renné

  Seminars in thrombosis and hemostasis. 06/2011; 37(4):375-81.

  The contact system is a protease cascade that is initiated by factor XII activation on cardiovascular cells. The system starts procoagulant and proinflammatory reactions, via the intrinsic pathway ofThe contact system is a protease cascade that is initiated by factor XII activation on cardiovascular cells. The system starts procoagulant and proinflammatory reactions, via the intrinsic pathway of coagulation and the kallikrein-kinin system, respectively. The biochemistry of the contact system in vitro is well understood. However, activators of the system in vivo and their contributions to disease states have remained enigmatic. Recent experimental and clinical data have identified misfolded proteins, collagens, and polyphosphates as the long-sought activators of the contact system in vivo. Here we present an overview about contact system activators and their contributions to health and pathology.

• Platelet polyphosphates: the nexus of primary and secondary hemostasis.

  Authors: Felicitas Müller, Thomas Renné

  Scandinavian journal of clinical and laboratory investigation. 04/2011; 71(2):82-6.

  For decades it has been known that activated platelets promote plasma clotting and that the fibrin forming activity of activated platelets is dependent on blood coagulation factor XII. However,For decades it has been known that activated platelets promote plasma clotting and that the fibrin forming activity of activated platelets is dependent on blood coagulation factor XII. However, because factor XII deficiency is not associated with any bleeding disorder, platelet-driven factor XII activation was believed to be an in vitro artefact with no importance for coagulation in vivo. Using arterial and venous thrombosis models in factor XII deficient mice we recently demonstrated that factor XII is essential for thrombosis in vivo. However it was not known how factor XII is activated by procoagulant platelets within the growing thrombus. Here, we review our studies of the last 5 years that led to the identification of the endogenous factor XII activator. We found that platelets release polyphosphates, linear inorganic polymers of 60-100 phosphate residues that directly bound to and activated factor XII. Platelet polyphosphates potently initiate fibrin formation via the factor XII-driven intrinsic pathway. Inhibition of factor XII or polyphosphates protected mice from lethal thrombotic disease and strongly reduced clot stability in patients. Our data identify polyphosphates as the endogenous factor XII activator in vivo linking platelet activation (primary hemostasis) and fibrin production (secondary hemostasis). Targeting polyphosphate-mediated factor XII activation offers the exciting possibility for a safe anticoagulation with minimal therapy-associated bleeding.

• Mast cells increase vascular permeability by heparin-initiated bradykinin formation in vivo.

  Authors: Chris Oschatz, Coen Maas, Bernd Lecher, Thomas Jansen, Jenny Björkqvist, Thomas Tradler, Reinhard Sedlmeier, Peter Burfeind, Sven Cichon, Sven Hammerschmidt, Werner Müller-Esterl, Walter A Wuillemin, Gunnar Nilsson, Thomas Renné

  Immunity. 02/2011; 34(2):258-68.

  Activated mast cells trigger edema in allergic and inflammatory disease. We report a paracrine mechanism by which mast cell-released heparin increases vascular permeability in vivo. Heparin activatedActivated mast cells trigger edema in allergic and inflammatory disease. We report a paracrine mechanism by which mast cell-released heparin increases vascular permeability in vivo. Heparin activated the protease factor XII, which initiates bradykinin formation in plasma. Targeting factor XII or kinin B2 receptors abolished heparin-triggered leukocyte-endothelium adhesion and interfered with a mast cell-driven drop in blood pressure in rodents. Intravital laser scanning microscopy and tracer measurements showed heparin-driven fluid extravasation in mouse skin microvessels. Ablation of factor XII or kinin B2 receptors abolished heparin-induced skin edema and protected mice from allergen-activated mast cell-driven leakage. In contrast, heparin and activated mast cells induced excessive edema in mice deficient in the major inhibitor of factor XII, C1 esterase inhibitor. Allergen exposure triggered edema attacks in hereditary angioedema patients, lacking C1 esterase inhibitor. The data indicate that heparin-initiated bradykinin formation plays a fundamental role in mast cell-mediated diseases.

• Identification of SPRED2 (sprouty-related protein with EVH1 domain 2) as a negative regulator of the hypothalamic-pituitary-adrenal axis.

  Authors: Melanie Ullrich, Karin Bundschu, Peter M Benz, Marco Abesser, Ruth Freudinger, Tobias Fischer, Julia Ullrich, Thomas Renné, Ulrich Walter, Kai Schuh

  The Journal of biological chemistry. 01/2011; 286(11):9477-88.

  Sprouty-related proteins with EVH1 (enabled/vasodilator-stimulated phosphoprotein homology 1) domain (SPREDs) are inhibitors of MAPK signaling. To elucidate SPRED2 in vivo function, we characterizedSprouty-related proteins with EVH1 (enabled/vasodilator-stimulated phosphoprotein homology 1) domain (SPREDs) are inhibitors of MAPK signaling. To elucidate SPRED2 in vivo function, we characterized body homeostasis in SPRED2(-/-) mice. They showed a doubled daily water uptake, induced by elevated serum osmolality, originating from increased blood salt load. Accordingly, serum aldosterone was doubled, accompanied by augmented adrenal aldosterone synthase (AS) expression. Surprisingly, serum vasopressin (AVP) was unaltered, and, as evidenced by halved angiotensin II (Ang II) levels, the renin angiotensin system (RAS) was down-regulated. Adrenocorticotropic hormone (ACTH) was significantly elevated in SPRED2(-/-) mice, together with its secretagogue corticotropin-releasing hormone (CRH) and its downstream target corticosterone. ERK phosphorylation in brains was augmented, and hypothalamic CRH mRNA levels were elevated, both contributing to the increased CRH release. Our data were supported by CRH promoter reporter assays in hypothalamic mHypoE-44 cells, revealing a SPRED-dependent inhibition of Ets (ERK/E-twenty-six)-dependent transcription. Furthermore, SPRED suppressed CRH production in these cells. In conclusion, our study suggests that SPRED2 deficiency leads to an increased MAPK signaling, which results in an augmented CRH promoter activity. The subsequent CRH overproduction causes an up-regulation of downstream hypothalamic-pituitary-adrenal (HPA) hormone secretion. This constitutes a possible trigger for the observed compulsive grooming in SPRED2(-/-) mice and may, together with hyperplasia of aldosterone-producing cells, contribute to the hyperaldosteronism and homeostatic imbalances.

• A role for factor XIIa-mediated factor XI activation in thrombus formation in vivo.

  Authors: Qiufang Cheng, Erik I Tucker, Meghann S Pine, India Sisler, Anton Matafonov, Mao-Fu Sun, Tara C White-Adams, Stephanie A Smith, Stephen R Hanson, Owen J T McCarty, Thomas Renné, András Gruber, David Gailani

  Blood. 11/2010; 116(19):3981-9.

  Mice lacking factor XII (fXII) or factor XI (fXI) are resistant to experimentally-induced thrombosis, suggesting fXIIa activation of fXI contributes to thrombus formation in vivo. It is not clearMice lacking factor XII (fXII) or factor XI (fXI) are resistant to experimentally-induced thrombosis, suggesting fXIIa activation of fXI contributes to thrombus formation in vivo. It is not clear whether this reaction has relevance for thrombosis in pri mates. In 2 carotid artery injury models (FeCl(3) and Rose Bengal/laser), fXII-deficient mice are more resistant to thrombosis than fXI- or factor IX (fIX)-deficient mice, raising the possibility that fXII and fXI function in distinct pathways. Antibody 14E11 binds fXI from a variety of mammals and interferes with fXI activation by fXIIa in vitro. In mice, 14E11 prevented arterial occlusion induced by FeCl(3) to a similar degree to total fXI deficiency. 14E11 also had a modest beneficial effect in a tissue factor-induced pulmonary embolism model, indicating fXI and fXII contribute to thrombus formation even when factor VIIa/tissue factor initiates thrombosis. In baboons, 14E11 reduced platelet-rich thrombus growth in collagen-coated grafts inserted into an arteriovenous shunt. These data support the hypothesis that fXIIa-mediated fXI activation contributes to thrombus formation in rodents and primates. Since fXII deficiency does not impair hemostasis, targeted inhibition of fXI activation by fXIIa may be a useful antithrombotic strategy associated with a low risk of bleeding complications.

• Inhibition of bradykinin receptor B1 protects mice from focal brain injury by reducing blood-brain barrier leakage and inflammation.

  Authors: Furat Raslan, Tobias Schwarz, Sven G Meuth, Madeleine Austinat, Michael Bader, Thomas Renné, Klaus Roosen, Guido Stoll, Anna-Leena Sirén, Christoph Kleinschnitz

  Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 03/2010; 30(8):1477-86.

  Kinins are proinflammatory and vasoactive peptides that are released during tissue damage and may contribute to neuronal degeneration, inflammation, and edema formation after brain injury by actingKinins are proinflammatory and vasoactive peptides that are released during tissue damage and may contribute to neuronal degeneration, inflammation, and edema formation after brain injury by acting on discrete bradykinin receptors, B1R and B2R. We studied the expression of B1R and B2R and the effect of their inhibition on lesion size, blood-brain barrier (BBB) disruption, and inflammatory processes after a focal cryolesion of the right parietal cortex in mice. B1R and B2R gene transcripts were significantly induced in the lesioned hemispheres of wild-type mice (P<0.05). The volume of the cortical lesions and neuronal damage at 24 h after injury in B1R(-/-) mice were significantly smaller than in wild-type controls (2.5+/-2.6 versus 11.5+/-3.9 mm(3), P<0.001). Treatment with the B1R antagonist R-715 1 h after lesion induction likewise reduced lesion volume in wild-type mice (2.6+/-1.4 versus 12.2+/-6.1 mm(3), P<0.001). This was accompanied by a remarkable reduction of BBB disruption and tissue inflammation. In contrast, genetic deletion or pharmacological inhibition of B2R had no significant impact on lesion formation or the development of brain edema. We conclude that B1R inhibition may offer a novel therapeutic strategy after acute brain injuries.

• Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo.

  Authors: Felicitas Müller, Nicola J Mutch, Wolfdieter A Schenk, Stephanie A Smith, Lucie Esterl, Henri M Spronk, Stefan Schmidbauer, William A Gahl, James H Morrissey, Thomas Renné

  Cell. 12/2009; 139(6):1143-56.

  Platelets play a central role in thrombosis, hemostasis, and inflammation. We show that activated platelets release inorganic polyphosphate (polyP), a polymer of 60-100 phosphate residues thatPlatelets play a central role in thrombosis, hemostasis, and inflammation. We show that activated platelets release inorganic polyphosphate (polyP), a polymer of 60-100 phosphate residues that directly bound to and activated the plasma protease factor XII. PolyP-driven factor XII activation triggered release of the inflammatory mediator bradykinin by plasma kallikrein-mediated kininogen processing. PolyP increased vascular permeability and induced fluid extravasation in skin microvessels of mice. Mice deficient in factor XII or bradykinin receptors were resistant to polyP-induced leakage. PolyP initiated clotting of plasma via the contact pathway. Ablation of intrinsic coagulation pathway proteases factor XII and factor XI protected mice from polyP-triggered lethal pulmonary embolism. Targeting polyP with phosphatases interfered with procoagulant activity of activated platelets and blocked platelet-induced thrombosis in mice. Addition of polyP restored defective plasma clotting of Hermansky-Pudlak Syndrome patients, who lack platelet polyP. The data identify polyP as a new class of mediator having fundamental roles in platelet-driven proinflammatory and procoagulant disorders.

• Differential VASP phosphorylation controls remodeling of the actin cytoskeleton.

  Authors: Peter M Benz, Constanze Blume, Stefanie Seifert, Sabine Wilhelm, Jens Waschke, Kai Schuh, Frank Gertler, Thomas Münzel, Thomas Renné

  Journal of cell science. 10/2009;

  Proteins of the Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family link signal transduction pathways to actin cytoskeleton dynamics. VASP is substrate of cAMP-dependent, cGMP-dependentProteins of the Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family link signal transduction pathways to actin cytoskeleton dynamics. VASP is substrate of cAMP-dependent, cGMP-dependent and AMP-activated protein kinases that primarily phosphorylate the sites S157, S239 and T278, respectively. Here, we systematically analyzed functions of VASP phosphorylation patterns for actin assembly and subcellular targeting in vivo and compared the phosphorylation effects of Ena/VASP family members. Methods used were the reconstitution of VASP-null cells with 'locked' phosphomimetic VASP mutants, actin polymerization of VASP mutants in vitro and in living cells, site-specific kinase-mediated VASP phosphorylation, and analysis of the endogenous protein with phosphorylation-status-specific antibodies. Phosphorylation at S157 influenced VASP localization, but had a minor impact on F-actin assembly. Phosphorylation of the S157-equivalent site in the Ena/VASP family members Mena and EVL had no effect on the ratio of cellular F-actin to G-actin. By contrast, VASP phosphorylation at S239 (and the equivalent site in Mena) or T278 impaired VASP-driven actin filament formation. The data show that VASP functions are precisely regulated by differential phosphorylation and provide new insights into cytoskeletal control by serine/threonine kinase-dependent signaling pathways.

• Blockade of Bradykinin Receptor B1 but Not Bradykinin Receptor B2 Provides Protection From Cerebral Infarction and Brain Edema.

  Authors: Madeleine Austinat, Stefan Braeuninger, João B Pesquero, Marc Brede, Michael Bader, Guido Stoll, Thomas Renné, Christoph Kleinschnitz

  Stroke; a journal of cerebral circulation. 12/2008;

  BACKGROUND AND PURPOSE: Brain edema is detrimental in ischemic stroke and its treatment options are limited. Kinins are proinflammatory peptides that are released during tissue injury. The effects ofBACKGROUND AND PURPOSE: Brain edema is detrimental in ischemic stroke and its treatment options are limited. Kinins are proinflammatory peptides that are released during tissue injury. The effects of kinins are mediated by 2 different receptors (B1 and B2 receptor [B1R and B2R]) and comprise induction of edema formation and release of proinflammatory mediators. METHODS: Focal cerebral ischemia was induced in B1R knockout, B2R knockout, and wild-type mice by transient middle cerebral artery occlusion. Infarct volumes were measured by planimetry. Evan's blue tracer was applied to determine the extent of brain edema. Postischemic inflammation was assessed by real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry. To analyze the effect of a pharmacological kinin receptor blockade, B1R and B2R inhibitors were injected. RESULTS: B1R knockout mice developed significantly smaller brain infarctions and less neurological deficits compared to wild-type controls (16.8+/-4.7 mm(3) vs 50.1+/-9.1 mm(3), respectively; P<0.0001). This was accompanied by a dramatic reduction of brain edema and endothelin-1 expression, as well as less postischemic inflammation. Pharmacological blockade of B1R likewise salvaged ischemic tissue (15.0+/-9.5 mm(3) vs 50.1+/-9.1 mm(3), respectively; P<0.01) in a dose-dependent manner, even when B1R inhibitor was applied 1 hour after transient middle cerebral artery occlusion. In contrast, B2R deficiency did not confer neuroprotection and had no effect on the development of tissue edema. CONCLUSIONS: These data demonstrate that blocking of B1R can diminish brain infarction and edema formation in mice and may open new avenues for acute stroke treatment in humans.

• Novel roles for factor XII-driven plasma contact activation system.

  Authors: Felicitas Müller, Thomas Renné

  Current opinion in hematology. 10/2008; 15(5):516-21.

  PURPOSE OF REVIEW: Blood coagulation is a tightly regulated process, involving vascular endothelium, platelets, and plasma coagulation factors. Formation of fibrin involves a series of sequentialPURPOSE OF REVIEW: Blood coagulation is a tightly regulated process, involving vascular endothelium, platelets, and plasma coagulation factors. Formation of fibrin involves a series of sequential proteolytic reactions, initiated by the 'extrinsic' and 'intrinsic' pathway of coagulation. As hereditary deficiency of factor XII, the protease that triggers the intrinsic pathway and the kallikrein-kinin system, is not associated with a bleeding disorder or other disease states, the physiological role of factor XII is unknown. RECENT FINDINGS: Patient studies, genetically altered mouse models, and plasma assays analyzed functions of the factor XII-driven contact activation system for coagulation and inflammation. This review focuses on articles, which report phenotypization of animals deficient in the contact system proteins factor XII, factor XI and high-molecular-weight kininogen, as well as novel links between factor XII and edema formation, discovery of new in-vivo activators of factor XII, and functions of the factor XII downstream protease factor XI. SUMMARY: Recent studies improved understanding of the factor XII-driven contact system in hemostasis, thrombosis, and inflammation. Studies in mouse models revealed that deficiency in contact system proteins protects from arterial thrombus formation, but does not affect hemostasis. Targeting contact system proteins offers new opportunities for safe anticoagulation associated with minimal bleeding risk. Furthermore, targeting factor XII activity provides an opportunity to treat edema formation.

• The calcium sensor STIM1 is an essential mediator of arterial thrombosis and ischemic brain infarction.

  Authors: David Varga-Szabo, Attila Braun, Christoph Kleinschnitz, Markus Bender, Irina Pleines, Mirko Pham, Thomas Renné, Guido Stoll, Bernhard Nieswandt

  The Journal of experimental medicine. 08/2008; 205(7):1583-91.

  Platelet activation and aggregation are essential to limit posttraumatic blood loss at sites of vascular injury but also contributes to arterial thrombosis, leading to myocardial infarction andPlatelet activation and aggregation are essential to limit posttraumatic blood loss at sites of vascular injury but also contributes to arterial thrombosis, leading to myocardial infarction and stroke. Agonist-induced elevation of [Ca(2+)](i) is a central step in platelet activation, but the underlying mechanisms are not fully understood. A major pathway for Ca(2+) entry in nonexcitable cells involves receptor-mediated release of intracellular Ca(2+) stores, followed by activation of store-operated calcium (SOC) channels in the plasma membrane. Stromal interaction molecule 1 (STIM1) has been identified as the Ca(2+) sensor in the endoplasmic reticulum (ER) that activates Ca(2+) release-activated channels in T cells, but its role in mammalian physiology is unknown. Platelets express high levels of STIM1, but its exact function has been elusive, because these cells lack a normal ER and Ca(2+) is stored in a tubular system referred to as the sarcoplasmatic reticulum. We report that mice lacking STIM1 display early postnatal lethality and growth retardation. STIM1-deficient platelets have a marked defect in agonist-induced Ca(2+) responses, and impaired activation and thrombus formation under flow in vitro. Importantly, mice with STIM1-deficient platelets are significantly protected from arterial thrombosis and ischemic brain infarction but have only a mild bleeding time prolongation. These results establish STIM1 as an important mediator in the pathogenesis of ischemic cardio- and cerebrovascular events.

• Deficiency of bradykinin receptor B2 is not detrimental in experimental stroke.

  Authors: Christoph Kleinschnitz, Madeleine Austinat, Michael Bader, Thomas Renné, Guido Stoll

  Hypertension. 06/2008; 51(5):e41; author reply e42-3.

• Blocking of platelets or intrinsic coagulation pathway-driven thrombosis does not prevent cerebral infarctions induced by photothrombosis.

  Authors: Christoph Kleinschnitz, Stefan Braeuninger, Mirko Pham, Madeleine Austinat, Ingo Nölte, Thomas Renné, Bernhard Nieswandt, Martin Bendszus, Guido Stoll

  Stroke; a journal of cerebral circulation. 05/2008; 39(4):1262-8.

  BACKGROUND AND PURPOSE: Models of photochemically-induced thrombosis are widely used in cerebrovascular research. Photothrombotic brain infarctions can be induced by systemic application ofBACKGROUND AND PURPOSE: Models of photochemically-induced thrombosis are widely used in cerebrovascular research. Photothrombotic brain infarctions can be induced by systemic application of photosensitizing dyes followed by focal illumination of the cerebral cortex. Although the ensuing activation of platelets is well established, their contribution for thrombosis and tissue damage has not formally been proved. METHODS: Infarction to the cerebral cortex was induced in mice by Rose Bengal and a cold light source. To assess the functional role of platelets, animals were platelet-depleted by anti-GPIbalpha antibodies or treated with GPIIb/IIIa-blocking F(ab)(2) fragments. The significance of the plasmatic coagulation cascade was determined by using blood coagulation factor XII (FXII)-deficient mice or heparin. Infarct development and infarct volumes were determined by serial MRI and conventional and electron microscopy. RESULTS: There was no difference in development and final size of photothrombotic infarctions in mice with impaired platelet function. Moreover, deficiency of FXII, which initiates the intrinsic pathway of coagulation and is essential for thrombus formation, or blockade of FXa, the key protease during the waterfall cascade of plasmatic coagulation, by heparin likewise did not affect lesion development. CONCLUSIONS: Our data demonstrate that platelet activation, factor XII-driven thrombus formation, and plasmatic coagulation pathways downstream of FX are not a prerequisite for ensuing tissue damage in models of photothrombotic vessel injury indicating that other pathomechanisms are involved. We suggest that this widely used model does not depend on platelet- or plasmatic coagulation-derived thrombosis.

• Transmission of oxLDL-derived lipid peroxide radicals into membranes of vascular cells is the main inducer of oxLDL-mediated oxidative stress.

  Authors: Thomas E Hansen-Hagge, Elke Baumeister, Tanja Bauer, Daniel Schmiedeke, Thomas Renné, Christoph Wanner, Jan Galle

  Atherosclerosis. 05/2008; 197(2):602-11.

  Oxidatively modified LDL is generally accepted to be an important elicitor of pro-mitotic, pro-inflammatory, and atherogenic effects in vascular cells. The uptake of oxLDL and concomitant activationOxidatively modified LDL is generally accepted to be an important elicitor of pro-mitotic, pro-inflammatory, and atherogenic effects in vascular cells. The uptake of oxLDL and concomitant activation of the O(2)*-producing NAD(P)H oxidase and/or oxLDL as a self-contained emitter of O(2)* are believed to trigger these malfunctions. The following observations allowed reinvestigating the mode of oxLDL-induced stress: (1) we observed that artery smooth muscle primary cells internalize fluorescently labelled oxidized or acetylated LDL considerably less efficient than endothelial cells. (2) Both types of cells, however, displayed an oxLDL concentration dependent level of oxidative stress as monitored by the oxidation of carboxy-H2DCFDA to fluorescent carboxy-DCF. A dose dependent decrease of dihydroethidine oxidation to oxyethidine implied an oxLDL-induced depletion of the cellular energy pool. The release of O(2)* by exogenous oxLDL, as postulated above, did not sufficiently explain intracellular stress because the fluorescence was only marginally blocked by antioxidative enzymes (SOD, catalase) or substances (L-NAME, DMSO, DMHP, DMTU). We were able to reveal a third mode of oxLDL-induced stress by showing with the help of a fluorescent, oxidizable lipid analogue (BODIPY 581/591 C(11)) that oxLDL-derived lipid peroxides and radicals migrate into cellular membranes giving rise to a chronic inoculation of the vascular cells with oxidative chain reactions. The novel data may help to design adequate therapeutic strategies against oxLDL-induced cardiovascular diseases.

• Prostaglandin-induced VASP phosphorylation controls alpha II-spectrin breakdown in apoptotic cells.

  Authors: Peter M Benz, Stephan M Feller, Albert Sickmann, Ulrich Walter, Thomas Renné

  International immunopharmacology. 03/2008; 8(2):319-24.

  In pathological conditions, the inflammatory mediator prostaglandin E2 (PGE2) has been shown to induce apoptosis through a cAMP-dependent pathway. However, underlying mechanisms have remainedIn pathological conditions, the inflammatory mediator prostaglandin E2 (PGE2) has been shown to induce apoptosis through a cAMP-dependent pathway. However, underlying mechanisms have remained illusive. Irrespective whether apoptosis is induced by the intrinsic or extrinsic pathway, the cysteine protease caspase-3 becomes activated and cleaves many key proteins including spectrins. Cleavage of the plasma membrane-associated spectrins leads to cell shrinkage, membrane blebbing, the formation of apoptotic bodies, and irreversible cell death. Recently, we identified a novel interaction between alpha II-spectrin and vasodilator-stimulated phosphoprotein (VASP), which is abrogated by the cAMP-dependent protein kinase (PKA)-mediated phosphorylation of VASP. In the present study we investigated whether VASP binding to alpha II-spectrin affects spectrin breakdown in PGE2-induced apoptosis. PGE2 dose- and time-dependently triggered VASP phosphorylation. Following induction of apoptosis, caspase-3-mediated alpha II-spectrin breakdown and membrane blebbing were markedly delayed in wild-type as compared to VASP-deficient endothelial cells. This suggests that VASP binding to alpha II-spectrin attenuates alpha II-spectrin cleavage in apoptotic cells and that PGE2-induced VASP phosphorylation regulates this process. Our findings may therefore provide the molecular basis for PGE2-induced apoptosis in pathological events.

• Cytoskeleton assembly at endothelial cell-cell contacts is regulated by alphaII-spectrin-VASP complexes.

  Authors: Peter M Benz, Constanze Blume, Jan Moebius, Chris Oschatz, Kai Schuh, Albert Sickmann, Ulrich Walter, Stephan M Feller, Thomas Renné

  The Journal of cell biology. 02/2008; 180(1):205-19.

  Directed cortical actin assembly is the driving force for intercellular adhesion. Regulated by phosphorylation, vasodilator-stimulated phosphoprotein (VASP) participates in actin fiber formation. WeDirected cortical actin assembly is the driving force for intercellular adhesion. Regulated by phosphorylation, vasodilator-stimulated phosphoprotein (VASP) participates in actin fiber formation. We screened for endothelial proteins, which bind to VASP, dependent on its phosphorylation status. Differential proteomics identified alphaII-spectrin as such a VASP-interacting protein. alphaII-Spectrin binds to the VASP triple GP(5)-motif via its SH3 domain. cAMP-dependent protein kinase-mediated VASP phosphorylation at Ser157 inhibits alphaII-spectrin-VASP binding. VASP is dephosphorylated upon formation of cell-cell contacts and in confluent, but not in sparse cells, alphaII-spectrin colocalizes with nonphosphorylated VASP at cell-cell junctions. Ectopic expression of the alphaII-spectrin SH3 domain at cell-cell contacts translocates VASP, initiates cortical actin cytoskeleton formation, stabilizes cell-cell contacts, and decreases endothelial permeability. Conversely, the permeability of VASP-deficient endothelial cells (ECs) and microvessels of VASP-null mice increases. Reconstitution of VASP-deficient ECs rescues barrier function, whereas alphaII-spectrin binding-deficient VASP mutants fail to restore elevated permeability. We propose that alphaII-spectrin-VASP complexes regulate cortical actin cytoskeleton assembly with implications for vascular permeability.