Exceptional aggressiveness of cerebral cavernous malformation disease associated with PDCD10 mutations.

PURPOSE: The phenotypic manifestations of cerebral cavernous malformation disease caused by rare PDCD10 mutations have not been systematically examined, and a mechanistic link to Rho kinase-mediated hyperpermeability, a potential therapeutic target, has not been established. METHODS: We analyzed PDCD10 small interfering RNA-treated endothelial cells for stress fibers, Rho kinase activity, and permeability. Rho kinase activity was assessed in cerebral cavernous malformation lesions. Brain permeability and cerebral cavernous malformation lesion burden were quantified, and clinical manifestations were assessed in prospectively enrolled subjects with PDCD10 mutations. RESULTS: We determined that PDCD10 protein suppresses endothelial stress fibers, Rho kinase activity, and permeability in vitro. Pdcd10 heterozygous mice have greater lesion burden than other Ccm genotypes. We demonstrated robust Rho kinase activity in murine and human cerebral cavernous malformation vasculature and increased brain vascular permeability in humans with PDCD10 mutation. Clinical phenotype is exceptionally aggressive compared with the more common KRIT1 and CCM2 familial and sporadic cerebral cavernous malformation, with greater lesion burden and more frequent hemorrhages earlier in life. We first report other phenotypic features, including scoliosis, cognitive disability, and skin lesions, unrelated to lesion burden or bleeding. CONCLUSION: These findings define a unique cerebral cavernous malformation disease with exceptional aggressiveness, and they inform preclinical therapeutic testing, clinical counseling, and the design of trials.Genet Med 17 3, 188-196.

Fasudil decreases lesion burden in a murine model of cerebral cavernous malformation disease.

BACKGROUND AND PURPOSE: Cerebral cavernous malformations (CCMs) are characterized by grossly dilated capillaries, associated with vascular leak and hemorrhage, and occur in sporadic or inherited (autosomal-dominant) forms with mutations in 1 of 3 gene loci (CCM 1, 2 or 3). We previously reported that the CCM1 protein (KRIT1) localizes to endothelial cell-cell junctions and loss of KRIT1 leads to junctional instability associated with activation of RhoA and its effector Rho kinase. Although Rho kinase inhibition has been proposed as potential therapy for CCM, there has been no demonstration of a therapeutic effect on CCM lesion genesis in vivo. METHODS: Our recently generated a model of CCM1 disease (Ccm1(+/-)Msh2(-/-)) was treated with the Rho kinase inhibitor fasudil (100 mg/kg/day administered in drinking water from weaning to 5 months of age), or placebo, and blindly assessed CCM lesion burden by systematic survey of animals' brains. For comparison, we also assessed therapeutic effect in previously described Ccm2(+/-)Trp53(-/-) mice treated with the same dose and duration of fasudil and placebo. RESULTS: Fasudil-treated Ccm1(+/-)Msh2(-/-) mice had a significantly decreased prevalence of CCM lesions compared with placebo controls. Lesions in treated animals were smaller and less likely associated with hemorrhage, inflammation, and endothelial proliferation and exhibited decreased expression of Rho kinase activation biomarkers. A therapeutic effect was also documented in Ccm2(+/-)Trp53(-/-) mice. CONCLUSIONS: This represents the first report of therapeutic benefit of pharmacological therapy in development and progression of CCMs and indicates that Rho kinase activation is a critical step in CCM lesion genesis and maturation.

A mechanism of Rap1-induced stabilization of endothelial cell--cell junctions.

Activation of Rap1 small GTPases stabilizes cell--cell junctions, and this activity requires Krev Interaction Trapped gene 1 (KRIT1). Loss of KRIT1 disrupts cardiovascular development and causes autosomal dominant familial cerebral cavernous malformations. Here we report that native KRIT1 protein binds the effector loop of Rap1A but not H-Ras in a GTP-dependent manner, establishing that it is an authentic Rap1-specific effector. By modeling the KRIT1-Rap1 interface we designed a well-folded KRIT1 mutant that exhibited a ~40-fold-reduced affinity for Rap1A and maintained other KRIT1-binding functions. Direct binding of KRIT1 to Rap1 stabilized endothelial cell-cell junctions in vitro and was required for cardiovascular development in vivo. Mechanistically, Rap1 binding released KRIT1 from microtubules, enabling it to locate to cell--cell junctions, where it suppressed Rho kinase signaling and stabilized the junctions. These studies establish that the direct physical interaction of Rap1 with KRIT1 enables the translocation of microtubule-sequestered KRIT1 to junctions, thereby supporting junctional integrity and cardiovascular development.

A novel mouse model of cerebral cavernous malformations based on the two-hit mutation hypothesis recapitulates the human disease.

Cerebral cavernous malformations (CCMs) are vascular lesions of the central nervous system appearing as multicavernous, blood-filled capillaries, leading to headache, seizure and hemorrhagic stroke. CCM occurs either sporadically or as an autosomal dominant disorder caused by germline mutation of one of the three genes: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10. Surgically resected human CCM lesions have provided molecular and immunohistochemical evidence for a two-hit (germline plus somatic) mutation mechanism. In contrast to the equivalent human genotype, mice heterozygous for a Ccm1- or Ccm2-null allele do not develop CCM lesions. Based on the two-hit hypothesis, we attempted to improve the penetrance of the model by crossing Ccm1 and Ccm2 heterozygotes into a mismatch repair-deficient Msh2(-/-) background. Ccm1(+/-)Msh2(-/-) mice exhibit CCM lesions with high penetrance as shown by magnetic resonance imaging and histology. Significantly, the CCM lesions range in size from early-stage, isolated caverns to large, multicavernous lesions. A subset of endothelial cells within the CCM lesions revealed somatic loss of CCM protein staining, supporting the two-hit mutation mechanism. The late-stage CCM lesions displayed many of the characteristics of human CCM lesions, including hemosiderin deposits, immune cell infiltration, increased endothelial cell proliferation and increased Rho-kinase activity. Some of these characteristics were also seen, but to a lesser extent, in early-stage lesions. Tight junctions were maintained between CCM lesion endothelial cells, but gaps were evident between endothelial cells and basement membrane was defective. In contrast, the Ccm2(+/-)Msh2(-/-) mice lacked cerebrovascular lesions. The CCM1 mouse model provides an in vivo tool to investigate CCM pathogenesis and new therapies.

Cerebral cavernous malformations proteins inhibit Rho kinase to stabilize vascular integrity.

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.

Localized alpha4 integrin phosphorylation directs shear stress-induced endothelial cell alignment.

Vascular endothelial cells respond to laminar shear stress by aligning in the direction of flow, a process which may contribute to atheroprotection. Here we report that localized alpha4 integrin phosphorylation is a mechanism for establishing the directionality of shear stress-induced alignment in microvascular endothelial cells. Within 5 minutes of exposure to a physiological level of shear stress, endothelial alpha4 integrins became phosphorylated on Ser(988). In wounded monolayers, phosphorylation was enhanced at the downstream edges of cells relative to the source of flow. The shear-induced alpha4 integrin phosphorylation was blocked by inhibitors of cAMP-dependent protein kinase A (PKA), an enzyme involved in the alignment of endothelial cells under prolonged shear. Moreover, shear-induced localized activation of the small GTPase Rac1, which specifies the directionality of endothelial alignment, was similarly blocked by PKA inhibitors. Furthermore, endothelial cells bearing a nonphosphorylatable alpha4(S(988)A) mutation failed to align in response to shear stress, thus establishing alpha4 as a relevant PKA substrate. We thereby show that shear-induced PKA-dependent alpha4 integrin phosphorylation at the downstream edge of endothelial cells promotes localized Rac1 activation, which in turn directs cytoskeletal alignment in response to shear stress.

Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability.

The angiogenic sprout has been compared to the growing axon, and indeed, many proteins direct pathfinding by both structures. The Roundabout (Robo) proteins are guidance receptors with well-established functions in the nervous system; however, their role in the mammalian vasculature remains ill defined. Here we show that an endothelial-specific Robo, Robo4, maintains vascular integrity. Activation of Robo4 by Slit2 inhibits vascular endothelial growth factor (VEGF)-165-induced migration, tube formation and permeability in vitro and VEGF-165-stimulated vascular leak in vivo by blocking Src family kinase activation. In mouse models of retinal and choroidal vascular disease, Slit2 inhibited angiogenesis and vascular leak, whereas deletion of Robo4 enhanced these pathologic processes. Our results define a previously unknown function for Robo receptors in stabilizing the vasculature and suggest that activating Robo4 may have broad therapeutic application in diseases characterized by excessive angiogenesis and/or vascular leak.

KRIT-1/CCM1 is a Rap1 effector that regulates endothelial cell cell junctions.

Cerebral cavernous malformation (CCM), a disease associated with defective endothelial junctions, result from autosomal dominant CCM1 mutations that cause loss of KRIT-1 protein function, though how the loss of KRIT-1 leads to CCM is obscure. KRIT-1 binds to Rap1, a guanosine triphosphatase that maintains the integrity of endothelial junctions. Here, we report that KRIT-1 protein is expressed in cultured arterial and venous endothelial cells and is present in cell-cell junctions. KRIT-1 colocalized and was physically associated with junctional proteins via its band 4.1/ezrin/radixin/moesin (FERM) domain. Rap1 activity regulated the junctional localization of KRIT-1 and its physical association with junction proteins. However, the association of the isolated KRIT-1 FERM domain was independent of Rap1. Small interfering RNA-mediated depletion of KRIT-1 blocked the ability of Rap1 to stabilize endothelial junctions associated with increased actin stress fibers. Thus, Rap1 increases KRIT-1 targeting to endothelial cell-cell junctions where it suppresses stress fibers and stabilizes junctional integrity.

Induction of vascular permeability: beta PIX and GIT1 scaffold the activation of extracellular signal-regulated kinase by PAK.

Increased permeability of blood vessels is an important component of inflammation, but in some circumstances it contributes to tissue injury and organ failure. Previous work showed that p21-activated kinase (PAK) is a critical regulator of endothelial cell-cell junctions through effects on myosin light chain phosphorylation and cell contractility. We now show that blocking PAK function inhibits fluid leak in a mouse model of acute lung injury. In cultured endothelial cells, induction of myosin light chain phosphorylation by PAK is mediated by mitogen-activated protein kinase kinase and extracellular signal-regulated kinase (Erk). Erk in lipopolysaccharide (LPS)-treated mouse lung is activated in a PAK-dependent manner in several cell types, most prominently vascular endothelium. Activation of Erk requires the integrity of the complex between PAK, PIX, and GIT1. Several means of disrupting this complex inhibit stimulation of vascular permeability in vitro. A cell-permeant peptide that blocks binding of PAK to PIX inhibits LPS-induced fluid leak in the mouse lung injury model. We conclude that the PAK-PIX-GIT1 complex is critical for Erk-dependent myosin phosphorylation and vascular permeability.

Matrix-specific p21-activated kinase activation regulates vascular permeability in atherogenesis.

Elevated permeability of the endothelium is thought to be crucial in atherogenesis because it allows circulating lipoproteins to access subendothelial monocytes. Both local hemodynamics and cytokines may govern endothelial permeability in atherosclerotic plaque. We recently found that p21-activated kinase (PAK) regulates endothelial permeability. We now report that onset of fluid flow, atherogenic flow profiles, oxidized LDL, and proatherosclerotic cytokines all stimulate PAK phosphorylation and recruitment to cell-cell junctions. Activation of PAK is higher in cells plated on fibronectin (FN) compared to basement membrane proteins in all cases. In vivo, PAK is activated in atherosclerosis-prone regions of arteries and correlates with FN in the subendothelium. Inhibiting PAK in vivo reduces permeability in atherosclerosis-prone regions. Matrix-specific PAK activation therefore mediates elevated vascular permeability in atherogenesis.

Blocking p21-activated kinase reduces lipopolysaccharide-induced acute lung injury by preventing polymorphonuclear leukocyte infiltration.

RATIONALE: Excessive recruitment of polymorphonuclear leukocytes (PMNs) to the lung promotes acute lung injury (ALI). Chemokine receptors and adhesion molecules initiate leukocyte-endothelial interactions, but mediators of PMN migration through the alveolo-capillary membrane remain to be identified. p21-Activated kinase (PAK) is an effector of small GTPases and has been implicated in cell migration. OBJECTIVES: To test the role of PAK in ALI. METHODS: An inhibitory PAK peptide was used to determine the role of PAK in cytoskeletal actin polymerization, cell adhesion, and oxidative burst. PMN migration was investigated in vitro and in a murine model of lipopolysaccharide-induced lung injury. MEASUREMENTS AND MAIN RESULTS: PMN migration into lung interstitium and alveolar space was suppressed by an inhibitory PAK peptide. Neutrophils that had taken up the inhibitory PAK peptide were unable to enter the alveolar space. CXCL2/3, an important PMN chemoattractant in murine lung injury, induced PAK phosphorylation in PMNs. Blocking PAK function inhibited chemotaxis, chemokine-induced cytoskeletal actin polymerization, and adhesion-induced oxidative burst. CONCLUSIONS: We conclude that neutrophil PAK is a critical mediator of PMN migration and may be an attractive target in ALI.

Fibroblast adhesion to micro- and nano-heterogeneous topography using diblock copolymers and homopolymers.

Polymeric substrates of different surface chemistry and length scales were found to have profound influence on cell adhesion. The adhesion of fibroblasts on surfaces of oxidized polystyrene (PS), on surfaces modified with random copolymers of PS and poly(methyl methacrylate) [P(S-r-MMA)] with topographic features, and chemically patterned surfaces that varied in lateral length scales from nanometers to microns were studied. Surfaces with heterogeneous topographies were generated from thin film mixtures of a block copolymer, PS-b-MMA, with homopolymers of PS and PMMA. The two homopolymers macroscopically phase separated and, with the addition of diblock copolymer, the size scales of the phases decreased to nanometer dimensions. Cell spreading area analysis showed that a thin film of oxidized PS surface promoted adhesion whereas a thin film of P(S-r-MMA) surface did not. Fibroblast adhesion was examined on surfaces in which the lateral length scale varied from 60 nm to 6 microm. It was found that, as the lateral length scale between the oxidized PS surfaces decreased, cell spreading area and degree of actin stress fiber formation increased. In addition, scanning electron microscopy was used to evaluate the location of filopodia and lamellipodia. It was found that most of the filopodia and lamellipodia interacted with the oxidized PS surfaces. This can be attributed to both chemical and topographic surface interactions that prevent cells from interacting with the P(S-r-MMA) at the base of the topographic features.

p21-activated kinase regulates endothelial permeability through modulation of contractility.

Endothelial cells lining the vasculature have close cell-cell associations that maintain separation of the blood fluid compartment from surrounding tissues. Permeability is regulated by a variety of growth factors and cytokines and plays a role in numerous physiological and pathological processes. We examined a potential role for the p21-activated kinase (PAK) in the regulation of vascular permeability. In both bovine aortic and human umbilical vein endothelial cells, PAK is phosphorylated on Ser141 during the activation downstream of Rac, and the phosphorylated subfraction translocates to endothelial cell-cell junctions in response to serum, VEGF, bFGF, TNFalpha, histamine, and thrombin. Blocking PAK activation or translocation prevents the increase in permeability across the cell monolayer in response to these factors. Permeability correlates with myosin phosphorylation, formation of actin stress fibers, and the appearance of paracellular pores. Inhibition of myosin phosphorylation blocks the increase in permeability. These data suggest that PAK is a central regulator of endothelial permeability induced by multiple growth factors and cytokines via an effect on cell contractility. PAK may therefore be a suitable drug target for the treatment of pathological conditions where vascular leak is a contributing factor, such as ischemia and inflammation.

5-lipoxygenase and cyclooxygenase regulate wound closure in NIH/3T3 fibroblast monolayers.

Wound healing involves multiple cell signaling pathways, including those regulating cell-extracellular matrix adhesion. Previous work demonstrated that arachidonate oxidation to leukotriene B(4) (LTB(4)) by 5-lipoxygenase (5-LOX) signals fibroblast spreading on fibronectin, whereas cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E(2) (PGE(2)) formation facilitates subsequent cell migration. We investigated arachidonate metabolite signaling in wound closure of perturbed NIH/3T3 fibroblast monolayers. We found that during initial stages of wound closure (0-120 min), all wound margin cells spread into the wound gap perpendicularly to the wound long axis. At regular intervals, between 120 and 300 min, some cells elongated to project across the wound and meet cells from the opposite margin, forming distinct cell bridges spanning the wound that act as foci for later wound-directed cell migration and resulting closure. 5-LOX inhibition by AA861 demonstrated a required LTB(4) signal for initial marginal cell spreading and bridge formation, both of which must precede wound-directed cell migration. 5-LOX inhibition effects were reversible by exogenous LTB(4). Conversely, COX inhibition by indomethacin reduced directed migration into the wound but enhanced early cell spreading and bridge formation. Exogenous PGE(2) reversed this effect and increased cell migration into the wound. The differential effects of arachidonic acid metabolites produced by LOX and COX were further confirmed with NIH/3T3 fibroblast cell lines constitutively over- and underexpressing the 5-LOX and COX-2 enzymes. These data suggest that two competing oxidative enzymes in arachidonate metabolism, LOX and COX, differentially regulate sequential aspects of fibroblast wound closure in vitro.