Cerebral cavernous malformations proteins inhibit Rho kinase to stabilize vascular integrity - PubMed (original) (raw)

Cerebral cavernous malformations proteins inhibit Rho kinase to stabilize vascular integrity

Rebecca A Stockton et al. J Exp Med. 2010.

Abstract

Endothelial cell-cell junctions regulate vascular permeability, vasculogenesis, and angiogenesis. Familial cerebral cavernous malformations (CCMs) in humans result from mutations of CCM2 (malcavernin, OSM, MGC4607), PDCD10 (CCM3), or KRIT1 (CCM1), a Rap1 effector which stabilizes endothelial cell-cell junctions. Homozygous loss of KRIT1 or CCM2 produces lethal vascular phenotypes in mice and zebrafish. We report that the physical interaction of KRIT1 and CCM2 proteins is required for endothelial cell-cell junctional localization, and lack of either protein destabilizes barrier function by sustaining activity of RhoA and its effector Rho kinase (ROCK). Protein haploinsufficient Krit1(+/-) or Ccm2(+/-) mouse endothelial cells manifested increased monolayer permeability in vitro, and both Krit1(+/-) and Ccm2(+/-) mice exhibited increased vascular leak in vivo, reversible by fasudil, a ROCK inhibitor. Furthermore, we show that ROCK hyperactivity occurs in sporadic and familial human CCM endothelium as judged by increased phosphorylation of myosin light chain. These data establish that KRIT1-CCM2 interaction regulates vascular barrier function by suppressing Rho/ROCK signaling and that this pathway is dysregulated in human CCM endothelium, and they suggest that fasudil could ameliorate both CCM disease and vascular leak.

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Figures

Figure 1.

Figure 1.

KRIT1 inhibits RhoA and its effector ROCK to reduce MLC phosphorylation and permeability in vitro. (A) HUVECs treated with control or KRIT1 siRNA were evaluated for RhoA activity. KRIT1-depleted cells had a 3.5-fold increase in GTP-RhoA compared with control, reversible by cotransfection with WT KRIT1 bearing a knockdown-resistant silent mutation. Error bars are means and SE of n = 6. *, P < 0.001 compared with vector-only control siRNA. (B) Total RhoA is shown by immunoblotting, and equal protein content per sample is shown by actin loading. KRIT1 siRNA efficacy is shown by immunoprecipitated KRIT1 content. 1, control IP with mouse IgG; 2, control siRNA; 3, KRIT1 siRNA; 4, control siRNA + KRIT1 complementary DNA (cDNA); 5, KRIT1 siRNA + KRIT1 cDNA. (C) KRIT1 depletion increased MLC phosphorylation and f-actin stress fiber content. Reconstitution with WT KRIT1 prevented pMLC increase and reduced stress fibers. Treatment of KRIT1-depleted cells with ROCK inhibitor H-1152 also prevented MLC phosphorylation and stress fiber formation, indicating that ROCK acts downstream of KRIT1. Bar, 50 µm. (D) siRNA efficacy is shown by Western blot probed for KRIT1. 1, control IgG IP; 2, control siRNA; 3, KRIT1 siRNA; 4, KRIT1 siRNA+ KRIT1 cDNA; 5, KRIT1 siRNA + H-1152. (E) KRIT1 depletion increases HUVEC monolayer permeability in Transwell assays. The increase is reversible by H-1152 treatment, indicating that KRIT1 functions to inhibit ROCK-mediated monolayer leak. Error bars are means ± SE of n = 6. *, P < 0.001 compared with control siRNA

Figure 2.

Figure 2.

KRIT1 protein haploinsufficiency increases ROCK-mediated vascular leak in vivo. (A) _Krit_1+/− mice have one-half the amount of KRIT1 protein in lung and brain as Krit1+/+ mice. 1, control mouse IgG IP; 2, Krit1+/+ lung; 3, Krit1+/+ brain; 4, Krit1+/− lung; 5, Krit1+/− brain lysates. (B) Densitometry of blots in A shows a 50% reduction in KRIT1 in Krit1+/− lysates. Error bars are means ± SE (n = 3). *, P < 0.03 compared with Krit1+/+. (C) Krit1+/− mice had significantly greater basal and LPS-induced Evan’s blue dye leak in brain. Error bars are means and SE of n = 6 mice. *, P < 0.01; **, P < 0.04 compared with vehicle-treated Krit1+/+ mice. Fasudil reduced leak in the Krit1+/− mice. (D) Lungs were collected from animals treated as in C and evaluated for dye extravasation. Basal and LPS-induced lung leak were also reversed by fasudil treatment. Error bars are means ± SE (n = 6). *, P < 0.01; **, P < 0.03 compared with Krit1+/+. (E) Brain edema (difference in wet weight vs. dry weight) was also tested under the same conditions. Brains were weighed, desiccated, and reweighed. Means and SE of data from six animals are shown. Brain edema was increased in Krit1+/− mice and was reduced by fasudil treatment. Error bars are means ± SE (n = 6). *, P < 0.01 compared with Krit1+/+. (F) Krit1+/− mice also had increased pulmonary edema, which was diminished by fasudil treatment. Error bars are means ± SE (n = 6). *, P < 0.01 compared with Krit1+/+.

Figure 3.

Figure 3.

Krit1+/− mouse endothelial cells have increased in vivo and in vitro pMLC and in vitro leak. (A) Endothelial cells were isolated from Krit1+/+ and Krit1+/− mouse brain, lung, liver, and kidney as described in Materials and methods, lysed, and Western blotted for immunoprecipitated KRIT1 protein and for pMLC in whole cell lysate. 1, brain; 2, lung; 3, liver; 4, kidney. Endothelial cells from Krit1+/− mice had similar KRIT1 protein expression in all organs but ∼1/2 the amount seen in Krit1+/+ mice. Endothelial cells from all organs in Krit1+/− mice exhibited increased pMLC content in vitro. (B) Isolated pulmonary endothelial cells grown on FN-coated coverslips were also stained for pMLC and f-actin content. Krit1+/− cells exhibited an increase in both. Bar, 50 µm. (C) In vitro permeability assay of isolated pulmonary endothelial cells. Krit1+/− endothelial monolayers were twofold more permeable than WT. Permeability was reduced by the ROCK inhibitor H-1152. Error bars are means ± SE (n = 6). *, P < 0.001; **, P < 0.05 compared with vehicle-treated Krit1+/+.

Figure 4.

Figure 4.

KRIT1 haploinsufficiency increases ROCK activity in human cerebral lesion endothelium. (A) Human CCM cerebral lesions were compared with nonlesion control brain tissue. Slides were processed as described in Materials and methods, stained with anti-pMLC as an index of ROCK activation, and counterstained with hematoxylin. Control antibody stains are with rabbit IgG. The left and center columns are stained for pMLC, and the right column is stained with control antibody. Bars, 500 µm. The CCM lesions are clusters of distended capillaries lacking SMC underlayer. Arrows indicate cavern endothelial cells; asterisks mark lumen of a typical expanded lesion vessel. The control non-CCM brain tissue panel shows an arteriole in cross section, exhibiting pMLC in SMC subendothelial layer, but little in endothelial cells (arrow) facing vessel lumen. Sporadic lesion #1 is KRIT1 protein deficient (

Fig. S3

). Familial lesion #2 is CCM2 deficient (

Fig. S3

) and has equally robust endothelial pMLC staining. (B) Magnified view of familial lesion #2 endothelium.

Figure 5.

Figure 5.

CCM2 depletion increases RhoA and ROCK activity in vitro. (A) HUVECs were treated with CCM2 or control siRNA, with or without WT CCM2 bearing a silent mutation resistant to knockdown. Depletion of CCM2 produced a four- to sixfold increase in GTP-RhoA, reversible by cotransfection with WT CCM2. Error bars are means ± SE (n = 6). *, P < 0.001 compared with control siRNA. (B) Total RhoA is equal in all treatments. Equal sample protein is shown by GAPDH. CCM2 knockdown and reconstitution efficacy are shown by immunoprecipitated CCM2. 1, vector + control siRNA; 2, vector + CCM2 siRNA; 3, control siRNA + WT CCM2 cDNA; 4, CCM2 siRNA + WT CCM2 cDNA; 5, control rabbit IgG IP. (C) CCM2 depletion by siRNA increases pMLC and stress fiber content of HUVEC, reversible by treatment with ROCK inhibitor H-1152. Bar, 50 µm. (D) As previously seen with KRIT1, CCM2 presence is required for suppression of ROCK-mediated monolayer permeability. Error bars are means ± SE (n = 4). *, P < 0.001 compared with vehicle-treated control siRNA.

Figure 6.

Figure 6.

CCM2 protein haploinsufficiency increases ROCK-mediated vascular leak. (A) Lung and brain lysates of Ccm2+/+ and Ccm2+/− mice evaluated for CCM2 protein content show a 50% protein reduction in Ccm2+/−. 1, control mouse IgG IP; 2, Ccm2+/+ lung; 3, Ccm2+/+ brain; 4, Ccm2+/− lung; 5, Ccm2+/− brain lysates. (B) Densitometry shows one-half CCM2 protein expression in heterozygous mouse lung and brain compared with WT. Error bars are means ± SE (n = 3). *, P < 0.01 compared with same organ of Ccm2+/+ mouse. (C) Ccm2+/+ and Ccm2+/− mice were evaluated for basal and LPS-induced cerebral vascular leak as described in Fig. 2. Ccm2+/− mice had significantly greater basal and induced leaks, reversible by ROCK inhibition with fasudil. Error bars are means ± SE (n = 6). *, P < 0.01; **, P < 0.03 compared with vehicle-treated Ccm2+/+. (D) Similarly, pulmonary vascular leak was increased in Ccm2+/− mice and was also reduced by fasudil. Error bars are means ± SE (n = 6). *, P <0.01; **, P < 0.04 compared with vehicle-treated Ccm2+/+. (E and F). Brain and lung edema in Ccm2+/− mice were increased, reversible by fasudil treatment. Error bars are means ± SE (n = 6). *, P < 0.01; **, P < 0.02 compared with vehicle-treated Ccm2+/+ for both panels.

Figure 7.

Figure 7.

Ccm2+/− mouse endothelial cells exhibit increased pMLC in vivo and in vitro. (A) Endothelial cells isolated from Ccm2+/+ and Ccm2+/− mouse brain, lung, liver, and kidney were lysed and probed for CCM2 protein and pMLC content. 1, brain; 2, lung; 3, liver; 4, kidney. Endothelial cells from all organs have equivalent expression of CCM2 protein; however, Ccm2+/− endothelial cells express 50% as much CCM2 as those from Ccm2+/+ mice. Ccm2+/− mouse endothelial cells exhibited increased pMLC content compared with WT. (B) Pulmonary endothelial cells were probed for pMLC and actin structure in vitro. Increased MLC phosphorylation and stress fiber content are seen in Ccm2+/− cells. Bar, 50 µm. (C) Ccm2+/− endothelial cells exhibit increased monolayer permeability reversible by treatment with ROCK inhibitor H-1152. Error bars are means ± SE (n = 6). *, P < 0.001; **, P < 0.05 compared with vehicle-treated Ccm2+/+ cells.

Figure 8.

Figure 8.

Physical interaction of CCM2 and KRIT1 is required for their endothelial junctional localization and for inhibition of RhoA→ROCK-mediated permeability. HUVECs were transfected with siRNA to deplete endogenous CCM2 and reconstituted with either WT CCM2 or CCM2-F217A, a mutation which abrogates CCM2 binding to KRIT1. Both WT CCM2 and CCM2-F217A constructs bear silent mutations conferring resistance to CCM2 siRNA. (A) Endogenous CCM2 and KRIT1 protein are localized to HUVEC endothelial cell–cell junctions in cells treated with control siRNA. CCM2 depletion by siRNA causes KRIT1 loss from junctions. Reconstitution of depleted endogenous CCM2 by transfection with CCM2-F217A does not restore junctional localization either to the mutant CCM2 or to endogenous KRIT1. Reexpressed WT CCM2 is junctional and permits endogenous KRIT1 junctional localization. (B) CCM2-F217A reconstitution of endogenous CCM2 depletion fails to suppress RhoA activity, whereas reconstitution with WT CCM2 limits RhoA activity. Error bars are means ± SE (n = 4). *, P < 0.01 compared with vector-treated control siRNA. (C) Transfection and knockdown efficacy for A and B is shown by Western blotting for CCM2 and total RhoA content. Equal loading is shown by staining for GAPDH. 1, vector; 2, vector + CCM2 siRNA; 3, CCM2 cDNA; 4, CCM2 cDNA+ CCM2 siRNA; 5, CCM2-F217A cDNA; 6, CCM2-F217A cDNA + CCM2 siRNA; 7, control rabbit IgG IP. (D) CCM2 depletion causes increased pMLC and actin stress fiber content, reversible by reconstitution with WT CCM2 but not by CCM2-F217A, indicating a requirement for CCM2-KRIT1 physical interaction for suppressing ROCK activity. Bar, 50 µm. (E) CCM2 depletion-induced hyperpermeability is not reversible by CCM2-F217A reconstitution. Error bars are means ± SE (n = 6). *, P < 0.001 compared with control siRNA.

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