Part I: Overview of Cerebral Cavernous Malformations
1. From Genes and Mechanisms to Molecular-targeted Therapies: the Long Climb to the Cure of Cerebral Cavernous Malformation (CCM) Disease
Saverio Francesco Retta, Federica Finetti, Andrea Perrelli, and Lorenza Trabalzini
2. Incidence, Prevalence, and Clinical Presentation of Cerebral Cavernous Malformations
Kelly D. Flemming
3. Natural History, Clinical, and Surgical Management of Cavernous Malformations
Giovanni G. Vercelli, Fabio Cofano, Filippo Veneziani Santonio, Francesca Vincitorio, Francesco Zenga, and Diego Garbossa
Part II Diagnosis and Treatment of Cerebral Cavernous Malformations
4. Molecular Genetics Screening of CCM patients, An Overview
Elisabeth Tournier-Lasserve
5. Next Generation Sequecing (NGS) Strategies for Genetic Testing and Diagnosis of Cerebral Cavernous Malformation (CCM) Disease
Valerio Benedetti, Elisa Pellegrino, Alfredo Brusco, Roberto Piva, and Saverio Francesco Retta
6. Genome-wide Genotyping of Cerebral Cavernous Malformation Type 1 Individuals to Identify Genetic Modifiers of Disease Severity
Helene Choquet and Helen Kim
7. Clinical Imaging of Cerebral Cavernous Malformations: Computed Tomography and Magnetic Resonance Imaging
Marc Mabray and Blaine Hart
8. Neuroradiology: Differential Diagnosis, Follow-Up, and Reporting
Alessandra Splendiani, Federico Bruno, and Alfonso Cerase
9. Surgical Management of Brain Cavernous Malformations
Marco M. Fontanella, Luca Zanin, Alessandro Fiorindi, Giannantonio Spena, Federico Nicolosi, Francesco Belotti, Pierpaolo Panciani, Claudio Cornali, and Francesco Doglietto
Part III: Animal and Cellular Models of Cebral Cavernous Malformations
10. Generation of CCM Phenotype by a Human Microvascular Endothelial Model
Simona Delle Monache and Saverio Francesco Retta
11. Isolation and Purification of Mouse Brain Endothelial Cells to Study Cerebral Cavernous Malformation Disease
Preston Hale, Shady Ibrahim Soliman, Hao Sun, and Miguel Alejandro Lopez-Ramirez
12. Production of KRIT1-knockout and KRIT1-knockin Mouse Embryonic Fibroblasts as Cellular Models of CCM Disease
Luca Goitre, Claudia Fornelli, Alessia Zotta, Andrea Perrelli, and Saverio Francesco Retta
13. CRISPR/Cas9-mediated Generation of Human Endothelial Cell Knockout Models of CCM Disease
Konrad Schwefel, Stefanie Spiegler, Christiane D. Much, Ute Felbor, and Matthias Rath
14. Dissection of the Role of CCM Genes in Tubulogenesis using the Drosophila
Tracheal System as a Model
Alondra B. Schweizer Burguete and Amin S. Ghabrial
15. Generation and Analysis of CCM Phenotypes in C. elegans
Evelyn Popiel and William Brent Derry
16. Generation of Transgenic Lines of Zebrafish Expressing Fluorescently-tagged CCM Proteins to Study Their Function and Subcellular Localization within the Vasculature
Stefan Donat, and Salim Abdelilah-Seyfried
17. Vertebrate Models to Investigate CCM Pathogenesis: The Zebrafish and Mouse Model
Johnathan Abou-Fadel and Jun Zhang
18. Generation of Cerebral Cavernous Malformation in Neonatal Mouse Models using Inducible Cre-LoxP Strategy
Jaesung P. Choi and Xiangjian Zheng
19. Isolation of Cerebral Endothelial Cells from CCM1/KRIT1 Null Mouse Brain
Nicholas Nobiletti and Angela J. Glading
Part IV: Cellular and Molecular Mechanisms of Cerebral Cavernous Malformations
20. Identification of the KRIT1 Protein by LexA-based Yeast Two-Hybrid System
Ilya G. Serebriiskii, Mohamed Elmekawy, and Erica A. Golemis
21. Crystallographic Studies of the Cerebral Cavernous Malformations Proteins
Oriana S. Fisher, Xiaofeng Li, Weizhi Liu, Rong Zhang, and Titus J. Boggon
22. Microscopy Techniques to Investigate CCM Pathogenesis
Johnathan Abou-Fadel and Jun Zhang
23. Preparation and Analysis of Protein Extracts to Investigate CCM Pathogenesis
Johnathan Abou-Fadel and Jun Zhang
24. Systems Wide Analysis of CCM Signaling Complex (CSC) Alterations in CCM Deficient Models using Omics Approaches
Johnathan Abou-Fadel and Jun Zhang
25. Study of Molecular Interactions of CCM Proteins by using a GAL4-based Yeast Two-hybrid Screening
Federica Finetti and Lorenza Trabalzini
26. Study of CCM Microvascular Endothelial Phenotype by an in vitro Tubule Differentiation Model
Simona Delle Monache and Saverio Francesco Retta
27. Bidimentional in vitro Angiogenic Assays to Study CCM Pathogenesis: Endothelial Cell Proliferation and Migration
Federica Finetti and Lorenza Trabalzini
28. Measurement of Endothelial Barrier Function in Mouse Models of Cerebral Cavernous Malformations using Intravital Microscopy
Angela J. Glading
29. Immunofluorescence of Cell-Cell and Cell-Extracellular Matrix Adhesive Defects in in vitro Endothelial CCM Model. Juxtacrine Role of Mutant Extracellular Matrix on Wild-Type Endothelial Cells
Sandra Manet, Daphne Vannier, Anne-Pascale Bouin, Justyna Lisowska, Corinne Albiges-Rizo, and Eva Faurobert
30. Detection of p62/SQSTM1 Aggregates in Cellular Models of CCM Disease by Immunofluorescence
Saverio Marchi, Saverio Francesco Retta, and Paolo Pinton
31. Notch Signaling in Familial Cerebral Cavernous Malformations and Immunohistochemical Detection of cleaved Notch1 Intracellular Domain
Sana S. Hasan and Andreas Fischer
32. Measuring the Kinase Activity of GCKIII Proteins in vitro
Juan Zalvide, Cristina Almenglo, Sara Vazquez, Mar Garcia-Colomer, Miriam Sartages, and Celia M. Pombo
33. Spectrophotometric Method for Determining Glyoxalase 1 Activity in Cerebral Cavernous Malformation (CCM) Disease
Cinzia Antognelli, Vincenzo Nicola Talesa, and Saverio Francesco Retta
34. Fluorescence Analysis of Reactive Oxygen Species (ROS) in Cellular Models of Cerebral Cavernous Malformation Disease
Andrea Perrelli and Saverio Francesco Retta
35. Small RNA Library Preparation for Profiling Small Non-coding RNAs in Patients Affected by Cavernous Malformations
Souvik Kar, Robert Geffers, Amir Samii, and Helmut Bertalanffy
36. Affinity Purification and Preparation of Peptides for Mass Spectrometry from C. elegans
Evelyn Popiel and William Brent Derry
