The proposed nomogram with or without plasma EBV DNA resulted in more accurate prognostic prediction for NPC patients.

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: CCM1, CCM2, and CCM3. CCM3, also known as PDCD10 (programmed cell death 10), was initially identified by its mRNA induction by apoptotic stimuli in vitro. However, the in vivo function of CCM3 has not been determined. Here, we describe mice with a deletion of the CCM3 gene either ubiquitously or specifically in certain cell types, including the vascular endothelium, smooth muscle cells, and neurons. Mice with global or endothelial cell-specific deletion of CCM3 die at embryonic stage, exhibiting defects in embryonic angiogenesis. CCM3 deletion reduces VEGFR2 signaling in embryos and derived endothelial cells. CCM3 is recruited to and stabilizes VEGFR2 in response to stimulation by VEGF, and the C-terminal domain of CCM3 is required for the stabilization of VEGFR2. Indeed, the CCM3 mutants found in human patients with a deletion of the C-terminal domain were labile, and unable to stabilize and activate VEGFR2. These results demonstrate that CCM3 regulates vascular development by modulating VEGFR2 signaling.

We have previously shown that tumor necrosis factor (TNF) acts via its two receptors TNFR1 and TNFR2 to elicit distinct signaling pathways in vascular endothelial cells (ECs). Here we used a femoral artery ligation model to demonstrate that TNFR1-knockout (KO) mice had enhanced, whereas TNFR2-KO had reduced, capacity in clinical recovery, limb perfusion, and ischemic reserve capacity compared with the wildtype mice. Consistently, ischemia-initiated collateral growth (arteriogenesis) in the upper limb and capillary formation and vessel maturation (angiogenesis) in the lower limb were enhanced in TNFR1-KO but were reduced in TNFR2-KO mice. Furthermore, our results suggest that vascular proliferation, but not infiltration of macrophages and lymphocytes, accounted for the phenotypic differences between the TNFR1-KO and TNFR2-KO mice. In wild-type animals TNFR2 protein in vascular endothelium was highly up-regulated in response to ischemia, leading to increased TNFR2-specific signaling as determined by the formation TNFR2-TRAF2 complex and activation of TNFR2-specific kinase Bmx/Etk. In isolated murine ECs, activation of TNFR2 induced nuclear factor-Bdependent reporter gene expression, EC survival, and migration. In contrast, activation of TNFR1 caused inhibition of EC migration and EC apoptosis. These data demonstrate that TNFR1 and TNFR2 play differential roles in ischemia-mediated arteriogenesis and angiogenesis, partly because of their opposite effects on EC survival and migration.

No abstract

ASK1-interacting protein-1 (AIP1), a recently identified member of the Ras GTPase-activating protein family, is highly expressed in vascular ECs and regulates EC apoptosis in vitro. However, its function in vivo has not been established. To study this, we generated AIP1-deficient mice (KO mice). Although these mice showed no obvious defects in vascular development, they exhibited dramatically enhanced angiogenesis in 2 models of inflammatory angiogenesis. In one of these models, the enhanced angiogenesis observed in the KO mice was associated with increased VEGF-VEGFR2 signaling. Consistent with this, VEGF-induced ear, cornea, and retina neovascularization were greatly augmented in KO mice and the enhanced retinal angiogenesis was markedly diminished by overexpression of AIP1. In vitro, VEGF-induced EC migration was inhibited by AIP1 overexpression, whereas it was augmented by both AIP1 knockout and knockdown, with the enhanced EC migration caused by AIP1 knockdown being associated with increased VEGFR2 signaling. We present mechanistic data that suggest AIP1 is recruited to the VEGFR2-PI3K complex, binding to both VEGFR2 and PI3K p85, at a late phase of the VEGF response, and that this leads to inhibition of VEGFR2 signaling. Taken together, our data demonstrate that AIP1 functions as an endogenous inhibitor in VEGFR2-mediated adaptive angiogenesis in mice.

CCM3 mutations are associated with cerebral cavernous malformation (CCM), a disease affecting 0.1-0.5% of the human population. CCM3 (PDCD10, TFAR15) is thought to form a CCM complex with CCM1 and CCM2; however, the molecular basis for these interactions is not known. We have determined the 2.5 Å crystal structure of CCM3. This structure shows an all ␣-helical protein containing two domains, an N-terminal dimerization domain with a fold not previously observed, and a C-terminal focal adhesion targeting (FAT)-homology domain. We show that CCM3 binds CCM2 via this FAThomology domain and that mutation of a highly conserved FAK-like hydrophobic pocket (HP1) abrogates CCM3-CCM2 interaction. This CCM3 FAT-homology domain also interacts with paxillin LD motifs using the same surface, and partial CCM3 co-localization with paxillin in cells is lost on HP1 mutation. Disease-related CCM3 truncations affect the FAT-homology domain suggesting a role for the FAT-homology domain in the etiology of CCM. Cerebral cavernous malformation (CCM)4 is a common vascular lesion that affects the central nervous system vasculature with a prevalence of 0.1-0.5% in the human population (1, 2) (OMIM 116860, 603284, 603285). CCMs manifest as thinwalled, dilated blood vessels lined by a monolayer of endothelial cells that lack tight junctions. The clinical effects of these lesions include seizures, headaches, and stroke in midlife and are often associated with focal hemorrhage (1, 2). These lesions can occur sporadically or as a familial form attributable to mutations in three different genes: CCM1, CCM2, and CCM3.A majority of mutations in CCM genes result in truncations of their protein products, CCM1 (Krev/Rap1 Interacting Trapped 1; KRIT1) (3, 4), CCM2 (malcavernin, MGC4607, osmosensing scaffold for mitogen-activated protein kinase kinase kinase-3; OSM) (5, 6), and CCM3 (programmed cell death 10; PDCD10, TF-1 cell apoptosis-related protein 15; TFAR15) (7,8). These mutations are inherited in an autosomal dominant fashion (9) with acquisition of CCM lesions hypothesized to be due to a two-hit mechanism (10 -12). Expression of the CCM proteins is required for both development and maintenance of endothelial cells in the vasculature; they are required for normal vasculogenesis (13) and global deletion of CCM1 renders mice nonviable (14), a result also seen in global or endothelial-specific deletion of CCM2 (15) and CCM3 (16). Overall, the clinical and in vivo data point to an essential role for the CCM proteins in endothelial cells that makes them critical for vasculogenesis and survival.CCM1, CCM2, and CCM3 interact with one another and play roles in multiple signaling pathways with CCM2 acting as a hub that directly interacts with both CCM3 (17) and CCM1 (18,19). The structural characteristics of the CCM proteins have not been directly assessed but have been inferred by molecular modeling and homology studies that suggest three regions in CCM1 (NPXY-rich, ankyrin repeat, and FERM domains) (20) and a predicted phosphotyrosine binding (PTB) dom...

Cerebral cavernous malformations (CCMs) are vascular malformations that affect the central nervous system and result in cerebral hemorrhage, seizure and stroke. CCM arises from loss-of-function mutations in one of three genes: CCM1, CCM2 and CCM3 (PDCD10). CCM3 mutations in human often result in a more severe form of the disease, and CCM3 knockout mice show severe phenotypes with yet-to-be defined mechanisms. We have recently reported that CCM3 regulates UNC13 family-mediated exocytosis. Here we investigate endothelial cells (EC) exocytosis in CCM disease progression. We find that CCM3 suppresses UNC13B/VAMP3-dependent exocytosis of angiopoietin-2 (ANGPT2) in brain endothelial cells. CCM3 ablation in EC augments exocytosis and secretion of ANGPT2, correlating with destabilized EC junctions, enlarged lumen formation, and endothelial cell-pericyte dissociations. UNC13B deficiency that blunts ANGPT2 secretion from EC or an ANGPT2 neutralization antibody normalizes the defects caused by CCM3 deficiency. More importantly, ANGPT2 neutralization antibody treatment or UNC13B deficiency blunts the CCM lesion phenotypes, including disruption of EC junctions, vessel dilation and pericyte dissociation, in the brains and retinas caused by endothelial cell-specific CCM3 inactivation. Our study reveals that enhanced secretion of ANGPT2 in endothelial cells contributes to the progression of the CCM disease, providing a novel therapeutic approach to treat this devastating pathology.

The function of the mitochondrial antioxidant system thioredoxin (Trx2) in vasculature is not understood. By using endothelial cell (EC)-specific transgenesis of the mitochondrial form of the thioredoxin gene in mice (Trx2 TG), we show the critical roles of Trx2 in regulating endothelium functions. Trx2 TG mice have increased total antioxidants, reduced oxidative stress, and increased nitric oxide (NO) levels in serum compared with their control littermates. Consistently, aortas from Trx2 TG mice show reduced vasoconstriction and enhanced vasodilation. By using ECs isolated from Trx2 TG mice, we further show that Trx2 increases the capacities of ECs in scavenging reactive oxygen species generated from mitochondria, resulting in increases in NO bioavailability in ECs. More importantly, Trx2 improves EC function and reduces atherosclerotic lesions in the apolipoprotein E-deficient mouse model. Our data provide the first evidence that Trx2 plays a critical role in preserving vascular EC function and prevention of atherosclerosis development, in part by reducing oxidative stress and increasing NO bioavailability.