Role of Rho-Associated Kinase in the Pathophysiology of Cerebral Cavernous Malformations

Abstract

Cerebral cavernous malformations (CCMs) are vascular lesions characterized by a porous endothelium. The lack of a sufficient endothelial barrier can result in microbleeds and frank intracerebral hemorrhage. A primary mechanism for lesion development is a sequence variant in at least 1 of the 3 CCM genes (CCM1, CCM2, and CCM3), which influence various signaling pathways that lead to the CCM phenotype. A common downstream process associated with CCM gene loss of function involves overactivation of RhoA and its effector Rho-associated kinase (ROCK). In this study, we review RhoA/ROCK-related mechanisms involved in CCM pathophysiology as potential therapeutic targets. Literature searches were conducted in PubMed using combinations of search terms related to RhoA/ROCK and CCMs. In endothelial cells, CCM1, CCM2, and CCM3 proteins normally associate to form the CCM protein complex, which regulates the functions of a wide variety of protein targets (e.g., MAP3K3, SMURF1, SOK-1, and ICAP-1) that directly or indirectly increase RhoA/ROCK activity. Loss of CCM complex function and increased RhoA/ROCK activity can lead to the formation of stress fibers that contribute to endothelial junction instability. Other RhoA/ROCK-mediated pathophysiologic outcomes include a shift to a senescence-associated secretory phenotype (primarily mediated by ROCK2), which is characterized by endothelial cell migration, cell cycle arrest, extracellular matrix degradation, leukocyte chemotaxis, and inflammation. ROCK represents a potential therapeutic target, and direct (fasudil, NRL-1049) and indirect (statins) ROCK inhibitors have demonstrated various levels of efficacy in reducing lesion burden in preclinical models of CCM. Current (atorvastatin) and planned (NRL-1049) clinical studies will determine the efficacy of ROCK inhibitors for CCM in humans, for which no US Food and Drug Administration-approved or EU-approved pharmacologic treatment exists.

Figure 1. RhoA/ROCK Signaling in CCM

CCM protein complex regulation of RhoA/ROCK: Loss of function in CCM1, CCM2, or CCM3 proteins leads to activation of RhoA/ROCK. Stress fiber formation: ROCK activity increases phosphorylation status of MRLC, the formation of actomyosin, and stress fibers. RhoA isoprenylation and VEGF-A signaling: VEGF/VEGFR-2 enhances HSP90-dependent RhoA/ROCK activity, phosphorylation of JNK/FAK1, and endothelial cell migration and angiogenesis. Endothelial cell invasiveness, leukocyte chemotaxis, and inflammation: ROCK2 regulates the expression of cell cycle activators and inhibitors, leading to cell cycle arrest and senescence. RhoA/ROCK activity increases expression of SNAIL1 and SLUG to promote endothelial to mesenchymal transition and endothelial cell invasiveness, while cell adhesion proteins and chemokines are upregulated to promote leukocyte chemotaxis and inflammation. Endothelial cell junctions: Basal ROCK2 activity is required for normal endothelial intercellular junctions, and a loss of CCM1 at adherens junctions prevents ROCK2 recruitment, resulting in junctional instability. Abbreviations: B = binding; CCL = CC motif chemokine ligand; CCM = cerebral cavernous malformation; CDK1 = cyclin-dependent kinase 1; CKS1 = cyclin-dependent kinase regulatory subunit 1; CM = covalent modifications; CS = complex subunit; E2F1 = E2F transcription factor 1; ERK5 = extracellular signal–regulated protein kinase 5; FAK1 = focal adhesion kinase 1; GGTase-1 = geranylgeranyltransferase type 1; HEG1 = heart of glass; HSP90 = heat shock protein 90; ICAP-1 = integrin cytoplasmic domain–associated protein-1; IE = influence on expression; IL = interleukin; ITGB1 = β1 integrin; JNK = c-Jun N-terminal kinase; KLF2 = Kruppel-like factor 2; KLF4 = Kruppel-like factor 4; Krit1 = Krev1 interaction trapped protein 1; LIMK = LIM domain kinase 1; MAP2K5 = mitogen-activated protein kinase kinase 5; MAP3K3 = mitogen-activated protein kinase kinase kinase 3; MAPK8-10 = mitogen-activated protein kinases 8–10; MEF2A = myocyte enhancer factor 2A; MEF2C = myocyte enhancer factor 2C; MEK5 = mitogen/extracellular signal-regulated kinase kinase-5; MLCP = myosin light-chain phosphatase; MMP-9 = matrix metalloproteinase-9; MRLC = myosin regulatory light chain; PDCD10 = programmed cell death protein 10; RAP-1A = Ras-related protein Rap-1A; ROCK1 = Rho-associated kinase 1; ROCK2 = Rho-associated kinase 2; SLUG = zinc finger protein SNAI2; SMURF1 = SMAD specific E3 ubiquitin protein ligase 1; SNAIL = zinc finger protein SNAI1; SOK-1 = Ste-20 oxidant stress response kinase 1; TR = transcription regulation; VCAM1 = vascular cell adhesion molecule 1; VE-cadherin = vascular endothelial cadherin; VEGF = vascular endothelial growth factor; VEGFR-2 = vascular endothelial growth factor receptor 2. Symbols: +P, phosphorylation; -P, dephosphorylation; ?, unspecified interactions. Symbol colors: Green indicates positive/activation, red indicates negative/inhibition, and gray is unspecified. An X indicates disruption in disease. See eAppendix 1 (links.lww.com/NXG/A667) for full graphic key.

Figure 2. Effect of Rho-Associated Kinase (ROCK) Inhibition on Cavernous Cerebral Malformation Lesion and Bleeding

(A–B): Representative microcomputed tomography images illustrating the effect of treatment with placebo (A) or NRL-1049 (B), a selective ROCK2 inhibitor, on CCM lesions in Ccm3^+/−Trp53^−/− mice. (C–D): Representative Perls' Prussian blue staining, which detects nonheme iron, illustrating the effect of placebo (C) or NRL-1049 (D) on lesional bleeding. Bar, 500 μm. Adapted with permission from McKerracher, et al.