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.

FIGURE 1.

RhoA abundance and activity are increased in shCCM1, -2, or -3 endothelial cells. A, Western blot showing that the abundance of RhoA in shCCM1, -2, or -3 cells is increased 23-, 37-, and 28-fold relative to WT cells. B, WT, shCCM1, -2, or -3 cells were infected with a FRET-based RhoA biosensor, where activation of RhoA leads to FRET. The measured FRET signal has been pseudocolored, where blue indicates low FRET and low RhoA activity, and red/white indicates high FRET and high RhoA activity. RhoA activity is increased at the cell edge (defined as 1.5-μm width at the edge of the cell), the cytoplasm, and the nucleus of shCCM1, -2, or -3 endothelial cells. C, enlarged view of a representative control and shCCM2 knockdown cell showing active RhoA at the cell edge, cytoplasm, and nucleus. D, bar graph showing the -fold change in FRET intensity for the cytoplasm, nucleus, and cell edge of shCCM1, -2, or -3 relative to WT control cells. Cytoplasm -fold FRET increase values are: shCCM1 cells = 1.62 (p value 4 × 10⁻¹¹), shCCM2 cells = 1.16 (p value 0.001), shCCM3 cells = 1.23 (p value 0.0003). Nuclear -fold FRET increases are: shCCM1 cells = 2.33 (p value 4.8 × 10⁻¹¹), shCCM2 cells = 1.16 (p value 0.002), shCCM3 cells = 1.23 (p value 0.0002). Cell edge -fold FRET increases are: shCCM1 cells = 1.48 (p value 5 × 10⁻⁹), shCCM2 cells = 1.16 (p value 0.002), shCCM3 cells = 1.20 (p value 0.002). Data represent the mean ± S.E. for a minimum of 25 cells in two independent experiments. Error bars, ***, p < 0.001, **, p < 0.02, *, p < 0.05.

FIGURE 2.

Western blots showing an increase in phospho-MLC2 in shCCM1, -2, or -3 endothelial cells, which is lost upon shRNA knockdown of ROCK2. The bar graph shows the densitometric quantitation of band intensity. The abundance of phospho-MLC2 (P-MLC2) in shCCM1, -2, or -3 cells increases 1.7-, 3.0-, and 1.6-fold, respectively, relative to control pLKO.1 cells (the graph represents an average of three independent experiments). Upon shRNA knockdown of ROCK2, this abundance decreases below detectable levels, 50 and 90%, respectively, for shCCM1, -2, or -3 relative to pLKO.1. Error bars, ***, p < 0.001, **, p < 0.02, *, p < 0.05. Line indicates two separate blots.

FIGURE 3.

ROCK inhibitor Y-27632 and ROCK2 shRNA rescue tube formation in shCCM1, -2, or -3 endothelial cells. A, knockdown of CCM1, -2, or -3 results in loss of tube formation. The image is a nine-panel montage of ×10 frames, and the bar represents 300 μm. B, tube formation in shCCM1 cells can be rescued with Y-27632 (+Y) or shROCK2 (+R). Frames are from supplemental Movie 1 taken at 0, 1, 3, 5, and 15 h after the start of treatment with Y-27632. The bar represents 100 μm. The image is a single ×10 field. C, tube formation rescue in shCCM2 cells by shROCK2 or Y-27632. D, tube formation rescue in shCCM3 cells by shROCK2 or Y-27632. E, the bar graph represents the mean tube area of pLKO.1 and shCCM1, -2, and -3 cells, normalized to pLKO.1. The data are the means ± S.E. of six frames from at least three independent experiments. Error bars, ***, p < 0.001, **, p < 0.02, *, p < 0.05.