Objective-Coagulation factor XI is proposed as therapeutic target for anticoagulation. However, it is still unclear whether the antithrombotic properties of factor XI inhibitors influence atherosclerotic disease and atherothrombosis. Our aim is to investigate whether factor XI antisense oligonucleotides could prevent thrombus formation on acutely ruptured atherosclerotic plaques. Approach and Results-Atherosclerotic plaques in the carotid arteries of Apoe −/− mice were acutely ruptured using ultrasound. The subsequent thrombus formation was visualized and quantified by intravital microscopy and immunohistochemistry. Mice were pretreated with either factor XI antisense or nonsense oligonucleotides (50 mg/kg) to lower factor XI plasma levels. A tail bleeding assay was used to determine the safety. On plaque rupture, initial platelet adhesion and platelet plug formation were not impaired in animals treated with factor XI antisense oligonucleotides. However, the ensuing thrombus formation and fibrin deposition were significantly lower after 5 to 10 minutes (P<0.05) in factor XI antisense oligonucleotide-treated animals without inducing a bleeding tendency. Furthermore, thrombi from antisense-treated animals were less stable than thrombi from placebo-treated animals. Moreover, macrophage infiltration and collagen deposition were lower in the carotid arteries of factor XI antisense-treated animals. No neutrophils were present. Conclusions-Factor

TNF plays a key role in immune-mediated inflammatory diseases including rheumatoid arthritis (RA) and spondyloarthritis (SpA). It remains incompletely understood how TNF can lead to different disease phenotypes such as destructive peripheral polysynovitis in RA versus axial and peripheral osteoproliferative inflammation in SpA. We observed a marked increase of transmembrane (tm) versus soluble (s) TNF in SpA versus RA together with a decrease in the enzymatic activity of ADAM17. In contrast with the destructive polysynovitis observed in classical TNF overexpression models, mice overexpressing tmTNF developed axial and peripheral joint disease with synovitis, enthesitis, and osteitis. Histological and radiological assessment evidenced marked endochondral new bone formation leading to joint ankylosis over time. SpA-like inflammation, but not osteoproliferation, was dependent on TNF-receptor I and mediated by stromal tmTNF overexpression. Collectively, these data indicate that TNF can drive distinct inflammatory pathologies. We propose that tmTNF is responsible for the key pathological features of SpA.

Coagulation factor XI (FXI) is a promising target for anticoagulation, because of its major role in thrombosis and relatively minor role in haemostasis. This implies that inhibition of FXI can prevent thrombosis without causing bleeding. It was our aim to investigate the antithrombotic properties of two novel inhibitory anti-human FXI antibodies (αFXI-175 and αFXI-203). The in vitro properties of both antibodies were analysed using standard clotting assays and calibrated automated thrombography. For the in vivo model we used FXI knockout mice, in which FXI plasma levels were restored with purified human FXI. Thrombosis was induced by applying ferric chloride to the vena cava inferior, after which time to occlusion was analysed. A tail bleeding assay was used to investigate the safety of both antibodies. Using calibrated automated thrombography, both antibodies inhibited thrombin generation initiated via the intrinsic pathway. In contrast, upon tissue factor (TF)-initiated thrombin generation, αFXI-203 did not inhibit thrombin generation, while αFXI-175 inhibited thrombin generation only at low concentrations of TF. In the murine thrombosis model, the vena cava inferior remained patent for 25 minutes (min) in mice treated with αFXI-175 and for 12.5 min in αFXI-203 treated animals, which was significantly longer than in placebo-treated animals (5 min, p<0.05). Neither antibody caused severe blood loss in a tail bleeding assay. In conclusion, the two inhibitory antibodies against FXI prevented cessation of blood flow in a murine thrombosis model without inducing a bleeding tendency.

To cite this article: Vergouwen MDI, Knaup VL, Roelofs JJTH, de Boer OJ, Meijers JCM. Effect of recombinant ADAMTS-13 on microthrombosis and brain injury after experimental subarachnoid hemorrhage. J Thromb Haemost 2014; 12: 943-7.Summary. Background: A common complication after aneurysmal subarachnoid hemorrhage (SAH) is delayed cerebral ischemia (DCI), which is associated with vasospasm and other mechanisms such as microthrombosis. ADAMTS-13 activity plays a role in the prevention of thrombus formation in the cerebral microvasculature. Previously, we observed that patients with DCI have lower levels of ADAMTS-13. Objectives: To examine whether recombinant human ADAMTS-13 (rADAMTS-13) reduces cerebral microthrombus formation and brain injury in an experimental mouse model of SAH including wild-type and ADAMTS-13 À/À mice. Methods: Experimental SAH was induced with the prechiasmatic blood injection model. The following experimental groups were investigated: (i) C57BL/6J mice (n = 10); (ii) C57BL/6J mice (n = 10) treated with rADAMTS-13 20 min after SAH; (iii) ADAMTS-13 À/À mice (n = 10); and (iv) AD-AMTS-13 À/À mice (n = 10) treated with rADAMTS-13 20 min after SAH. Mice were killed at 48 h. Results are presented as means with standard errors of the mean. Results: Infusion with rADAMTS-13 reduced the extent of microthrombosis by~50% in both wild-type mice (mean fibrinogen area: 0.28% AE 0.09% vs. 0.15% AE 0.04%; P = 0.20) and ADAMTS-13 À/À mice (mean fibrinogen area: 0.32% AE 0.05% vs. 0.16% AE 0.03%; P = 0.016). In addition, rADAMTS-13 reduced brain injury by > 60% in both wild-type mice (mean microglia area: 0.65% AE 0.18% vs. 0.18% AE 0.04%; P = 0.013) and ADAMTS-13 À/À mice (mean microglia area: 1.24% AE 0.36% vs. 0.42% AE 0.13%; P = 0.077). Conclusions: Our results support the further study of rADAMTS-13 as a treatment option for the prevention of microthrombosis and brain injury after SAH.

Previous studies showed that complement activation is associated with poor functional outcome after aneurysmal subarachnoid hemorrhage (SAH). We investigated whether complement activation is underlying brain injury after aneurysmal SAH (n = 7) and if it is an appropriate treatment target. We investigated complement expression in brain tissue of aneurysmal SAH patients (n = 930) and studied the role of common genetic variants in C3 and C5 genes in outcome. We analyzed plasma levels (n = 229) to identify the functionality of a single nucleotide polymorphism (SNP) associated with outcome. The time course of C5a levels was measured in plasma (n = 31) and CSF (n = 10). In an SAH mouse model, we studied the extent of microglia activation and cell death in wild-type mice, mice lacking the C5a receptor, and in mice treated with C5-specific antibodies (n = 15 per group). Brain sections from aneurysmal SAH patients showed increased presence of complement components C1q and C3/C3b/iC3B compared to controls. The complement component 5 (C5) SNP correlated with C5a plasma levels and poor disease outcome. Serial measurements in CSF revealed that C5a was > 1400-fold increased 1 day after aneurysmal SAH and then gradually decreased. C5a in plasma was 2-fold increased at days 3-10 after aneurysmal SAH. In the SAH mouse model, we observed a ≈ 40% reduction in both microglia activation and cell death in mice lacking the C5a receptor, and in mice treated with C5-specific antibodies. These data show that C5 contributes to brain injury after experimental SAH, and support further study of C5-specific antibodies as novel treatment option to reduce brain injury and improve prognosis after aneurysmal SAH.

Conclusion: mTOR blockade inhibits IL-17A and TNFα production by PBMCs, and osteogenic differentiation of FLS from patients with SpA in vitro. In the HLA-B27 transgenic rat model of SpA, rapamycin inhibits arthritis and spondylitis development and severity, reduces articular bone erosions, decreases pathologic new bone formation and suppresses IL-17A expression. These results may support efforts to evaluate the efficacy of targeting the mTOR pathway in SpA patients.

IntroductionInsulin like growth factor (IGF)-I can act on a variety of cells involved in cartilage and bone repair, yet IGF-I has not been studied extensively in the context of inflammatory arthritis. The objective of this study was to investigate whether IGF-I overexpression in the osteoblast lineage could lead to increased reparative or pathological bone formation in rheumatoid arthritis and/or spondyloarthritis respectively.MethodsMice overexpressing IGF-I in the osteoblast lineage (Ob-IGF-I+/-) line 324–7 were studied during collagen induced arthritis and in the DBA/1 aging model for ankylosing enthesitis. Mice were scored clinically and peripheral joints were analysed histologically for the presence of hypertrophic chondrocytes and osteocalcin positive osteoblasts.Results90–100% of the mice developed CIA with no differences between the Ob-IGF-I+/- and non-transgenic littermates. Histological analysis revealed similar levels of hypertrophic chondrocytes and osteocalcin positive osteoblasts in the ankle joints. In the DBA/1 aging model for ankylosing enthesitis 60% of the mice in both groups had a clinical score 1<. Severity was similar between both groups. Histological analysis revealed the presence of hypertrophic chondrocytes and osteocalcin positive osteoblasts in the toes in equal levels.ConclusionOverexpression of IGF-I in the osteoblast lineage does not contribute to an increase in repair of erosions or syndesmophyte formation in mouse models for destructive and remodeling arthritis.

Background: The pathogenesis of delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH) remains unclear. It has been hypothesized that an inflammatory response in the subarachnoid space and microthrombosis contribute to DCI. Previous studies showed that plasma levels of complement factors are associated with DCI and functional outcome after aSAH. We performed an autopsy study to investigate the relationship between complement activation, microthrombosis and DCI. Methods: In autopsy material from 7 patients who died from DCI, we isolated 9 areas of cerebral infarction and 15 areas without infarction. From autopsy material of 5 controls (myocardial infarction: n=3; heart failure: n=1; pulmonary embolism: n=1) we isolated 15 corresponding areas. We performed immunohistochemistry to investigate the presence of C1q, C3c, and fibrin(ogen) (which is strongly correlated with microthrombosis). Optical density measurements were used for quantitative assessments. Optical density values were compared between areas with infarction, without infarction, and controls, and analyzed with a Mann-Whitney U test. Spearman correlation tests were performed to investigate the relationships of C1q and C3c with fibrin(ogen). Results: Optical density values of C1q, C3c, and fibrin(ogen) were higher in areas of infarction than in areas without infarction or controls (Table 1, p<0.001 for all comparisons). Correlations were found between C1q and fibrin(ogen) (r=0.73, p<0.001) and C3c with fibrin(ogen) (r=0.74, p<0.001). Conclusion: Our results show that areas of cerebral infarction in patients with aSAH have more complement activation and microthrombosis than areas without infarction or control brains. A strong correlation was found between complement activation and microthrombosis. Future studies are needed to investigate if drugs targeting complement activation decrease the risk of DCI and improve outcome after aSAH.