title: Rho kinase inhibition rescues the endothelial cell cerebral cavernous malformation phenotype.
abstract: Cerebral cavernous malformations (CCM) are vascular lesions causing seizures and stroke. Mutations causing inactivation of one of three genes, ccm1, -2, or -3, are sufficient to induce vascular endothelial cell defects resulting in CCM. Herein, we show that loss of expression of the CCM1, -2, or -3 proteins causes a marked increase in expression of the GTPase RhoA. Live cell imaging with a RhoA-specific biosensor demonstrates increased RhoA activity with loss of CCM1, -2, or -3, with an especially pronounced RhoA activation in both the cytosol and the nucleus with loss of CCM1 expression. Increased RhoA activation was associated with Rho kinase-dependent phosphorylation of myosin light chain 2. Functionally, loss of CCM1, -2, or -3 inhibited endothelial cell vessel-like tube formation and extracellular matrix invasion, each of which is rescued by chemical inhibition or short hairpin RNA knockdown of Rho kinase. The findings, for the first time, define a signaling network for CCM1, -2, and -3 in CCM pathology, whereby loss of CCM1, -2, or -3 protein expression results in increased RhoA activity, with the activation of Rho kinase responsible for endothelial cell dysregulation. The results define Rho kinase as a therapeutic target to rescue endothelial cells from loss of CCM protein function.
authors.affiliation: Department of Pharmacology and Lineberger Comprehensive Cancer Center, School of Dentistry, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: Asya L
authors.initials: AL
authors.lastname: Borikova
authors.forename: Christopher F
authors.initials: CF
authors.lastname: Dibble
authors.forename: Noah
authors.initials: N
authors.lastname: Sciaky
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Welch
authors.forename: Amy N
authors.initials: AN
authors.lastname: Abell
authors.forename: Sompop
authors.initials: S
authors.lastname: Bencharit
authors.forename: Gary L
authors.initials: GL
authors.lastname: Johnson
mesh_terms: D000577:Amides / Q000494:pharmacology; D051017:Apoptosis Regulatory Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D015374:Biosensing Techniques; D024722:Cardiac Myosins / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D020786:Hemangioma, Cavernous, Central Nervous System / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D000075927:KRIT1 Protein; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D018994:Myosin Light Chains / Q000378:metabolism; D010641:Phenotype; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology; D034622:RNA Interference; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D000577:Amides; D051017:Apoptosis Regulatory Proteins; C502544:CCM2 protein, human; D002352:Carrier Proteins; D000075927:KRIT1 Protein; C107928:KRIT1 protein, human; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D018994:Myosin Light Chains; C495390:PDCD10 protein, human; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011725:Pyridines; C107184:myosin light chain 2; C066737:RHOA protein, human; C108830:Y 27632; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases; D024722:Cardiac Myosins; D020742:rhoA GTP-Binding Protein