Robert Shenkar, PhD

CARDIAC, THORACIC, VASCULAR | IMMUNOLOGY, INFLAMMATION, PATHOBIOLOGY | NEUROSCIENCE

Overview:

1. Focus on signaling and therapy in cerebral cavernous malformations.
2. Focus on biomarkers of cerebral cavernous malformations.
3. Focus on inflammation in cerebral cavernous malformations.

Several approaches are being used to conduct research on cerebral cavernous malformations, including murine models, magnetic resonance imaging, electron microscopy and confocal microscopy.

Disease of Interest:

Cerebral cavernous malformation (CCM) is a vascular disorder of the brain affecting 0.5% of the population. CCM disease can either be sporadic or familial, resulting from haploinsufficiency in one of three genes, KRIT1/CCM1, CCM2 or PDCD10/CCM3. Symptoms are CCM disease include seizures, neurological deficits and/or hemorrhagic stroke. There is presently no therapy to prevent the development or clinical progression of CCM lesions.

Current Projects:

1. ROCK Inhibition as Therapy for Cerebral Cavernous Malformation: We have shown that the loss of CCM proteins, KRIT1, CCM2 or PDCD10 activates RhoA and its downstream effector Rho kinase (ROCK). Increased vascular permeability in Krit1 and Ccm2 heterozygous mice is reversed by ROCK inhibition. ROCK activity is increased in human and mouse CCM endothelium. These observations led us to hypothesize that aberrant Rho activation causes CCM pathogenesis. We are currently conducting a cross sectional study on the effects of ROCK inhibition on CCM lesion burden. We are presently treating three CCM murine models (Krit1, Ccm2, Pdcd10) with the specific ROCK inhibitor fasudil, and the more pleiotropic ROCK inhibitors simvastatin and atorvastatin. After therapy, we are assessing the mouse brains for lesion burden, lesion area, iron deposition, inflammation, ultrastructure and endothelial junctional protein expression. In longitudinal studies we examine CCM lesions for growth and size, iron deposition and phenotypic markers after therapy in murine models after they are identified by magnetic resonance imaging in vivo, and the effects of therapy will be evaluated mechanistically through differential gene expression. Surgically resected human CCM lesions will be examined for differential expression and signaling pathways for ROCK related genes.

2. Biomarkers of Brain Permeability in Human Cerebral Cavernous Malformations: Our observations on the relationship between CCM proteins, endothelial permeability and ROCK activity suggest that both endothelial permeability and ROCK activity could be used as biomarkers for CCM disease. We are currently measuring vascular permeability in the brains of patients with CCM disease by dynamic contrast-enhanced quantitative perfusion magnetic resonance imaging. Because ROCK activity in leukocytes was shown to decrease by statins by our collaborator, Dr. James Liao, we are using leukocyte ROCK activity as a biomarker in patients with CCM disease. These techniques will allow us to assess brain vascular permeability and leukocyte ROCK activity as potential biomarkers for CCM disease severity in patients.

3. Inflammation in Cerebral Cavernous Malformation: My observation that immunoglobulins were among the most highly expressed genes in surgically resected human cerebral cavernous malformations, led our laboratory to show a robust presence of B, T and plasma cells in CCM lesions both from patients and murine models, and immune complexes in human lesions. Immunoglobulins from plasma cells in human CCM lesions were shown to be oligoclonal. The immunoglobulin variable region RNA isolated from plasma cells from human CCM lesions was sequenced and used to make recombinant antibodies, which may be used to identify the triggering antigen. We are presently pursuing the effect of B depletion on CCM lesions in murine models.