Claudia Tersteeg

Myocardial Ischemia/Reperfusion Injury Is Mediated by Leukocytic Toll-Like Receptor-2 and Reduced by Systemic Administration of a Novel Anti–Toll-Like Receptor-2 Antibody

Background— Reperfusion therapy for myocardial infarction is hampered by detrimental inflammatory responses partly via Toll-like receptor (TLR) activation. Targeting TLR signaling may optimize reperfusion therapy and enhance cell survival and heart function after myocardial infarction. Here, we evaluated the role of TLR2 as a therapeutic target using a novel monoclonal anti-TLR2 antibody. Method and Results— Mice underwent 30 minutes of ischemia followed by reperfusion. Compounds were administered 5 minutes before reperfusion. Cardiac function and dimensions were assessed at baseline and 28 days after infarction with 9.4-T mouse magnetic resonance imaging. Saline and IgG isotype treatment resulted in 34.5±3.3% and 31.4±2.7% infarction, respectively. Bone marrow transplantation experiments between wild-type and TLR2-null mice revealed that final infarct size is determined by circulating TLR2 expression. A single intravenous bolus injection of anti-TLR2 antibody reduced infarct size to 18.9±2.2% (P=0.001). Compared with saline-treated mice, anti-TLR2–treated mice exhibited less expansive remodeling (end-diastolic volume 68.2±2.5 versus 76.8±3.5 &mgr;L; P=0.046) and preserved systolic performance (ejection fraction 51.0±2.1% versus 39.9±2.2%, P=0.009; systolic wall thickening 3.3±6.0% versus 22.0±4.4%, P=0.038). Anti-TLR2 treatment significantly reduced neutrophil, macrophage, and T-lymphocyte infiltration. Furthermore, tumor necrosis factor-&agr;, interleukin-1&agr;, granulocyte macrophage colony-stimulating factor, and interleukin-10 were significantly reduced, as were phosphorylated c-jun N-terminal kinase, phosphorylated p38 mitogen-activated protein kinase, and caspase 3/7 activity levels. Conclusions— Circulating TLR2 expression mediates myocardial ischemia/reperfusion injury. Antagonizing TLR2 just 5 minutes before reperfusion reduces infarct size and preserves cardiac function and geometry. Anti-TLR2 therapy exerts its action by reducing leukocyte influx, cytokine production, and proapoptotic signaling. Hence, monoclonal anti-TLR2 antibody is a potential candidate as an adjunctive for reperfusion therapy in patients with myocardial infarction.

Mechanisms of Autoantibody-Induced Pathology

Autoantibodies are frequently observed in healthy individuals. In a minority of these individuals, they lead to manifestation of autoimmune diseases, such as rheumatoid arthritis or Graves’ disease. Overall, more than 2.5% of the population is affected by autoantibody-driven autoimmune disease. Pathways leading to autoantibody-induced pathology greatly differ among different diseases, and autoantibodies directed against the same antigen, depending on the targeted epitope, can have diverse effects. To foster knowledge in autoantibody-induced pathology and to encourage development of urgently needed novel therapeutic strategies, we here categorized autoantibodies according to their effects. According to our algorithm, autoantibodies can be classified into the following categories: (1) mimic receptor stimulation, (2) blocking of neural transmission, (3) induction of altered signaling, triggering uncontrolled (4) microthrombosis, (5) cell lysis, (6) neutrophil activation, and (7) induction of inflammation. These mechanisms in relation to disease, as well as principles of autoantibody generation and detection, are reviewed herein.

Plasmin Cleavage of von Willebrand Factor as an Emergency Bypass for ADAMTS13 Deficiency in Thrombotic Microangiopathy

Background— Von Willebrand factor (VWF) multimer size is controlled through continuous proteolysis by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type I motif, member 13). This prevents spontaneous platelet agglutination and microvascular obstructions. ADAMTS13 deficiency is associated with thrombotic thrombocytopenic purpura, in which life-threatening episodes of microangiopathy damage kidneys, heart, and brain. Enigmatically, a complete ADAMTS13 deficiency does not lead to continuous microangiopathy. We hypothesized that plasmin, the key enzyme of the fibrinolytic system, serves as a physiological backup enzyme for ADAMTS13 in the degradation of pathological platelet–VWF complexes. Methods and Results— Using real-time microscopy, we determined that plasmin rapidly degrades platelet–VWF complexes on endothelial cells in absence of ADAMTS13, after activation by urokinase-type plasminogen activator or the thrombolytic agent streptokinase. Similarly, plasmin degrades platelet–VWF complexes in platelet agglutination studies. Plasminogen directly binds to VWF and its A1 domain in a lysine-dependent manner, as determined by enzyme-linked immunosorbent assay. Plasma levels of plasmin–&agr;2-antiplasmin complexes increase with the extent of thrombocytopenia in patients with acute episodes of thrombotic thrombocytopenic purpura, independent of ADAMTS13 activity. This indicates that plasminogen activation takes place during microangiopathy. Finally, we show that the thrombolytic agent streptokinase has therapeutic value for Adamts13−/− mice in a model of thrombotic thrombocytopenic purpura. Conclusions— We propose that plasminogen activation on endothelial cells acts as a natural backup for ADAMTS13 to degrade obstructive platelet–VWF complexes. Our findings indicate that thrombolytic agents may have therapeutic value in the treatment of microangiopathies and may be useful to bypass inhibitory antibodies against ADAMTS13 that cause thrombotic thrombocytopenic purpura.

Age and coumarin-type anticoagulation are associated with the occurrence of intraplaque hemorrhage, while statins are associated less with intraplaque hemorrhage: a large histopathological study in carotid and femoral plaques.

INTRODUCTION Intraplaque hemorrhage (IPH) is an important determinant of progression and destabilization of atherosclerotic plaque. We recently demonstrated that IPH is an independent predictor of future cardiovascular events after carotid endarterectomy. Thus far, it is unknown whether clinical patient characteristics, such as medication use, are associated with the occurrence of IPH. The purpose of this study was to examine the association of IPH with clinical patient characteristics. METHODS AND RESULTS 1070 consecutive patients who underwent a carotid (n=794) or femoral (n=276) endarterectomy were included. Endarterectomy specimens were subjected to histopathological examination. IPH was observed in 644/794 (81%) carotid and 175/276 (63%) femoral plaques. Carotid IPH was positively correlated with advanced age (69 years [IQR: 62-75] vs. 65 years [IQR: 57-73]; P=0.002) and coumarin-type anticoagulation use prior to operation (104/116 [90%] with coumarin derivatives vs. 540/678 [80%] without coumarin derivatives; P=0.01). Carotid IPH was less frequently observed in patients that used statins prior to endarterectomy (468/595 [79%] with statin vs. 176/199 [88%] without statin; P=0.002). In multivariate analysis, age, coumarin-type anticoagulation use and statin use were independently correlated with carotid IPH. No association was observed between femoral IPH and clinical patient characteristics. CONCLUSION Advanced age and coumarin-type anticoagulation use are associated with the occurrence of IPH, while statin use is associated with less IPH.

Potential for Recombinant ADAMTS13 as an Effective Therapy for Acquired Thrombotic Thrombocytopenic Purpura

Objective—The metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) regulates the size of von Willebrand factor multimers. A deficiency in ADAMTS13 activity is associated with the life-threatening disease thrombotic thrombocytopenic purpura (TTP). The vast majority of patients have acquired TTP, where circulating anti-ADAMTS13 autoantibodies are causative for the decreased ADAMTS13 activity. Current treatment consists of plasma exchange, but improved therapies are highly warranted. Approach and Results—We have developed a new rat model mimicking various aspects of acquired TTP to investigate the therapeutic efficacy of human recombinant ADAMTS13. A polyclonal antibody against ADAMTS13 completely blocked endogenous rat ADAMTS13 activity in Sprague–Dawley rats. When TTP was triggered using recombinant von Willebrand factor, the animals displayed severe TTP-like symptoms, such as thrombocytopenia, hemolytic anemia, and von Willebrand factor–rich thrombi in the kidneys and brain. Subsequent injection of 400, 800, or 1600 U/kg recombinant ADAMTS13 prevented full development of these symptoms. Analysis of plasma samples confirmed that recombinant ADAMTS13 was able to override circulating anti-ADAMTS13 inhibitory antibodies, resulting in restoration of ADAMTS13 activity and degradation of ultralarge von Willebrand factor multimers. Conclusions—Recombinant ADAMTS13 was shown to be effective in averting severe acquired TTP-like symptoms in rats and holds promising value for the treatment of this severe and life-threatening disease in humans.

Different stages of intraplaque hemorrhage are associated with different plaque phenotypes: a large histopathological study in 794 carotid and 276 femoral endarterectomy specimens.

BACKGROUND AND PURPOSE Intraplaque hemorrhage (IPH) is an important determinant of progression and destabilization of atherosclerotic plaque. We recently demonstrated that IPH is an independent predictor of cardiovascular events. IPH has become more clinically relevant since magnetic resonance imaging (MRI) technique is able to visualize IPH in vivo. Different stages of IPH have been described. However, etiology of the different stages is not known and it is unclear if these detected different stages are all associated with the vulnerable plaque phenotype. METHODS AND RESULTS 1070 patients who underwent a carotid (n=794) or femoral (n=276) endarterectomy were included. Histopathological presence of IPH was determined and divided into 3 types: recent, organized and amorphous IPH. Carotid IPH was observed in 644/794 (81%) plaques, divided into 14 (2%) recent, 70 (11%) organized and 560 (87%) amorphous. Femoral IPH was observed in 175/276 (63%) plaques, divided into 2 (1%) recent, 89 (51%) organized and 84 amorphous (48%). Overall presence of carotid IPH was associated with a large lipid core, no or minor staining of smooth muscle cells, no or minor calcification and high microvessel density. Overall presence of femoral IPH was associated with moderate to heavy staining of macrophages. Plaques with organized IPHs revealed more macrophages, a larger lipid core, less smooth muscle cells, less calcification and higher microvessel density than plaques with amorphous IPHs. CONCLUSIONS IPH is a significant characteristic of carotid and femoral atherosclerotic plaque and can be classified into different types. Organized IPH is associated with unstable and amorphous IPH with stable plaque characteristics.

FLow-Induced PRotrusions (FLIPRs): A Platelet-Derived Platform for the Retrieval of Microparticles by Monocytes and Neutrophils

Rationale: Platelets are the most important cells in the primary prevention of blood loss after injury. In addition, platelets are at the interface between circulating leukocytes and the (sub)endothelium regulating inflammatory responses. Objective: Our aim was to study the dynamic process that leads to the formation of procoagulant and proinflammatory platelets under physiological flow. Methods and Results: In the present study, we describe the formation of extremely long, negatively charged membrane strands that emerge from platelets adhered under flow. These flow-induced protrusions (FLIPRs) are formed in vitro on different physiological substrates and are also detected in vivo in a mouse carotid injury model. FLIPRs are formed downstream the adherent and activated platelets and reach lengths of 250 μm. FLIPR formation is shear-dependent and requires cyclophilin D, calpain, and Rac1 activation. It is accompanied by a disassembly of the F-actin and microtubule organization. Monocytes and neutrophils roll over FLIPRs in a P-selectin/P-selectin glycoprotein ligand-1–dependent manner, retrieving fragments of FLIPRs as microparticles on their surface. Consequently, monocytes and neutrophils become activated, as demonstrated by increased CD11b expression and L-selectin shedding. Conclusions: The formation of long platelet membrane extensions, such as the ones presented in our flow model, may pave the way to generate an increased membrane surface for interaction with monocytes and neutrophils. Our study provides a mechanistic model for platelet membrane transfer and the generation of monocyte/neutrophil–microparticle complexes. We propose that the formation of FLIPRs in vivo contributes to the well-established proinflammatory function of platelets and platelet-derived microparticles. # Novelty and Significance {#article-title-41}Rationale: Platelets are the most important cells in the primary prevention of blood loss after injury. In addition, platelets are at the interface between circulating leukocytes and the (sub)endothelium regulating inflammatory responses. Objective: Our aim was to study the dynamic process that leads to the formation of procoagulant and proinflammatory platelets under physiological flow. Methods and Results: In the present study, we describe the formation of extremely long, negatively charged membrane strands that emerge from platelets adhered under flow. These flow-induced protrusions (FLIPRs) are formed in vitro on different physiological substrates and are also detected in vivo in a mouse carotid injury model. FLIPRs are formed downstream the adherent and activated platelets and reach lengths of 250 &mgr;m. FLIPR formation is shear-dependent and requires cyclophilin D, calpain, and Rac1 activation. It is accompanied by a disassembly of the F-actin and microtubule organization. Monocytes and neutrophils roll over FLIPRs in a P-selectin/P-selectin glycoprotein ligand-1–dependent manner, retrieving fragments of FLIPRs as microparticles on their surface. Consequently, monocytes and neutrophils become activated, as demonstrated by increased CD11b expression and L-selectin shedding. Conclusions: The formation of long platelet membrane extensions, such as the ones presented in our flow model, may pave the way to generate an increased membrane surface for interaction with monocytes and neutrophils. Our study provides a mechanistic model for platelet membrane transfer and the generation of monocyte/neutrophil–microparticle complexes. We propose that the formation of FLIPRs in vivo contributes to the well-established proinflammatory function of platelets and platelet-derived microparticles.

Tracking down contact activation – from coagulation in vitro to inflammation in vivo

The contact system is a volatile and versatile enzyme system in blood plasma that responds to the presence of nonphysiological surface materials by spontaneous generation of enzymatic activity. In subsequent steps, it can trigger blood coagulation and is responsible for the generation of the proinflammatory peptide bradykinin. The physiological role of the contact system is presently unknown, but it is commonly used to trigger coagulation in a diagnostic setting. In this three‐part review, we will first describe the molecular mechanisms that drive contact activation on nonphysiological materials. Next, we will summarize and compare a number of bioassays, which are commonly used to investigate the contact system in health and disease. Finally, we will discuss recent findings from both fundamental and clinical studies on the contributions of contact system to cardiovascular, infectious, and inflammatory disease.

A fibronectin-fibrinogen-tropoelastin coating reduces smooth muscle cell growth but improves endothelial cell function

Reendothelialization of the stent surface after percutaneous coronary intervention (PCI) is known to be an important determinant of clinical outcome. We compared the effects of biological stent coatings, fibronectin, fibrinogen and tropoelastin, on human umbilical vein endothelial cell (HUVEC) and vascular smooth muscle cell (VSMC) characteristics. Umbilical cord arterial segments were cultured on coated surfaces and VSMC outgrowth (indicating proliferation and migration) was measured after 12 days. mRNA was isolated from HUVEC and VSMC cultured on these coatings and gene expression was profiled by QPCR. Procoagulant properties of HUVEC were determined by an indirect chromogenic assay which detects tissue factor activity. The varying stent coatings influence VSMC outgrowth: 31.2 ± 4.0 mm2 on fibronectin, 1.6 ± 0.3 mm2 on tropoelastin and 8.1 ± 1.5 mm2 on a mixture of fibronectin/fibrinogen/tropoelastin, although HUVEC migration remains unaffected. Culturing HUVEC on tropoelastin induces increased expression of VCAM‐1 (13.1 ± 4.4 pg/ml), ICAM‐1 (5.1 ± 1.3 pg/ml) and IL‐8 (11.6 ± 3.1 pg/ml) compared to fibronectin (0.7 ± 0.2, 0.8 ± 0.2, 2.3 ± 0.5 pg/ml, respectively), although expression levels on fibronectin/fibrinogen/tropoelastin remain unaltered. No significant differences in VCAM‐1, ICAM‐1 and IL‐8 mRNA expression are found in VSMC. Finally, HUVEC cultured on tropoelastin display a fivefold increased tissue factor activity (511.6 ± 26.7%), compared to cells cultured on fibronectin (100 ± 3.9%) or fibronectin/fibrinogen/tropoelastin (76.3 ± 25.0%). These results indicate that tropoelastin inhibits VSMC migration but leads to increased inflammatory and procoagulant markers on endothelial cells. Fibronectin/fibrinogen/tropoelastin inhibits VSMCs while compensating the inflammatory and procoagulant effects. These data suggest that coating a mixture of fibronectin/fibrinogen/tropoelastin on a stent may promote reendothelialization, while keeping unfavourable processes such as restenosis and procoagulant activity limited.

The role of platelet and endothelial GARP in thrombosis and hemostasis

Background Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. Objectives To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. Methods Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. Results Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. Conclusions Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.