Fluorescence Analysis of Reactive Oxygen Species (ROS) in Cellular Models of Cerebral Cavernous Malformation Disease.

Cerebral cavernous malformation (CCM) is a vascular disease of proven genetic origin, which may arise sporadically or can be inherited as an autosomal dominant condition with incomplete penetrance and highly variable expressivity. CCM disease exhibits a range of different phenotypes, including wide interindividual differences in lesion number, size, and susceptibility to intracerebral hemorrhage (ICH). Mutations of the KRIT1 gene account for over 50% of familial cases. Previously, we demonstrated that KRIT1 loss-of-function is associated with altered homeostasis of intracellular reactive oxygen species (ROS) and abnormal activation of redox-sensitive transcription factors, which collectively result in pro-oxidative, pro-inflammatory, and pro-angiogenic effects, suggesting a novel pathogenic mechanism for CCM disease. Consistently, these original discoveries have been confirmed and extended by subsequent findings showing mechanistic relationships between pleiotropic redox-dependent effects of KRIT1 loss-of-function and enhanced cell sensitivity to oxidative stress, which may eventually lead to cellular dysfunctions and CCM disease pathogenesis. In this chapter, we describe few basic methods used for qualitative and quantitative analysis of intracellular ROS in cellular models of CCM disease.