Intracerebral hemorrhage (ICH) is the second most common and deadliest form of stroke. Currently, no pharmacologic treatment strategies exist for this devastating disease. Following the initial mechanical injury suffered at hemorrhage onset, secondary brain injury proceeds through both direct cellular injury and inflammatory cascades, which trigger infiltration of granulocytes and monocytes, activation of microglia, and disruption of the blood–brain barrier with resulting cerebral edema. The complement cascade has been shown to play a central role in the pathogenesis of secondary injury following ICH, although the specific mechanisms responsible for the proximal activation of complement remain incompletely understood. Cerebral injury following cleavage of complement component (C3) proceeds through parallel but interrelated pathways of anaphylatoxin-mediated inflammation and direct toxicity secondary to membrane attack complex-driven erythrocyte lysis. Complement activation also likely plays an important physiologic role in recovery following ICH. As such, a detailed understanding of the variation in functional effects of complement activation over time is critical to exploiting this target as an exciting translational strategy for intracerebral hemorrhage.

AimsThe mesenchymal cushions lining the early embryonic heart undergo complex remodelling to form the membranous ventricular septum as well as the atrioventricular and semilunar valves in later life. Disruption of this process underlies the most common congenital heart defects. Here, we identified a novel role for Slit-Robo signalling in the development of the murine membranous ventricular septum and cardiac valves.Methods and resultsExpression of Robo1 and Robo2 receptors and their ligands, Slit2 and Slit3, was present in or adjacent to all cardiac cushions/valves. Loss of Robo1 or both Robo1 and Robo2 resulted in membranous ventricular septum defects at birth, a defect also found in Slit3, but not in Slit2 mutants. Additionally, Robo1;Robo2 double mutants showed thickened immature semilunar and atrioventricular valves as well as highly penetrant bicuspid aortic valves. Slit2 mutants recapitulated the semilunar phenotype, whereas Slit3 mutants displayed thickened atrioventricular valves. Bicuspid aortic cushions were already observed at E12.5 in the Robo1;Robo2 double mutants. Expression of Notch- and downstream Hey and Hes genes was down-regulated in Robo1 mutants, suggesting that reduced Notch signalling in mice lacking Robo might underlie the defects. Luciferase assays confirmed regulation of Notch signalling by Robo.ConclusionCardiac defects in mutants for Robo or Slit range from membranous ventricular septum defects to bicuspid aortic valves. These ligands and receptors have unique functions during development of specific cardiac cushion derivatives, and the Slit-Robo signalling pathway likely enforces its role by regulating Notch signalling, making these mutants a valuable new model to study cardiac valve formation.

Rationale: The Slit–Roundabout (Robo) signaling pathway has pleiotropic functions during Drosophila heart development. However, its role in mammalian heart development is largely unknown. Objective: To analyze the role of Slit–Robo signaling in the formation of the pericardium and the systemic venous return in the murine heart. Methods and Results: Expression of genes encoding Robo1 and Robo2 receptors and their ligands Slit2 and Slit3 was found in or around the systemic venous return and pericardium during development. Analysis of embryos lacking Robo1 revealed partial absence of the pericardium, whereas Robo1/2 double mutants additionally showed severely reduced sinus horn myocardium, hypoplastic caval veins, and a persistent left inferior caval vein. Mice lacking Slit3 recapitulated the defects in the myocardialization, alignment, and morphology of the caval veins. Ligand binding assays confirmed Slit3 as the preferred ligand for the Robo1 receptor, whereas Slit2 showed preference for Robo2. Sinus node development was mostly unaffected in all mutants. In addition, we show absence of cross-regulation with previously identified regulators Tbx18 and Wt1 . We provide evidence that pericardial defects are created by abnormal localization of the caval veins combined with ectopic pericardial cavity formation. Local increase in neural crest cell death and impaired neural crest adhesive and migratory properties underlie the ectopic pericardium formation. Conclusions: A novel Slit–Robo signaling pathway is involved in the development of the pericardium, the sinus horn myocardium, and the alignment of the caval veins. Reduced Slit3 binding in the absence of Robo1, causing impaired cardiac neural crest survival, adhesion, and migration, underlies the pericardial defects.