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.

No abstract

We have previously shown that ASK1-interacting protein 1 (AIP1) transduces tumor necrosis factor-induced ASK1-JNK signaling. Because endoplasmic reticulum (ER) stress activates ASK1-JNK signaling cascade, we investigated the role of AIP1 in ER stress-induced signaling. We created AIP1-deficient mice (AIP1-KO) from which mouse embryonic fibroblasts and vascular endothelial cells were isolated. AIP1-KO cells show dramatic reductions in ER stress-induced, but not oxidative stress-induced, ASK1-JNK activation and cell apoptosis. The ER stressinduced IRE1-JNK/XBP-1 axis, but not the PERK-CHOP1 axis, is blunted in AIP1-KO cells. ER stress induced formation of an AIP1-IRE1 complex, and the PH domain of AIP1 is critical for the IRE1 interaction. Furthermore, reconstitution of AIP1-KO cells with AIP1 wild type, not an AIP1 mutant with a deletion of the PH domain (AIP1-⌬PH), restores ER stress-induced IRE1-JNK/XBP-1 signaling. AIP1-IRE1 association facilitates IRE1 dimerization, a critical step for activation of IRE1 signaling. More importantly, AIP1-KO mice show impaired ER stress-induced IRE1-dependent signaling in vivo. We conclude that AIP1 is essential for transducing the IRE1-mediated ER stress response.

Small ubiquitin-like modifier (SUMO) modification of proteins (SUMOylation) and deSUMOylation have emerged as important regulatory mechanisms for protein function. SENP1 (SUMO-specific protease) deconjugates SUMOs from modified proteins. We have created SENP1 knockout (KO) mice based on a Cre–loxP system. Global deletion of SENP1 (SENP1 KO) causes anemia and embryonic lethality between embryonic day 13.5 and postnatal day 1, correlating with erythropoiesis defects in the fetal liver. Bone marrow transplantation of SENP1 KO fetal liver cells to irradiated adult recipients confers erythropoiesis defects. Protein analyses show that the GATA1 and GATA1-dependent genes are down-regulated in fetal liver of SENP1 KO mice. This down-regulation correlates with accumulation of a SUMOylated form of GATA1. We further show that SENP1 can directly deSUMOylate GATA1, regulating GATA1-dependent gene expression and erythropoiesis by in vitro assays. Moreover, we demonstrate that GATA1 SUMOylation alters its DNA binding, reducing its recruitment to the GATA1-responsive gene promoter. Collectively, we conclude that SENP1 promotes GATA1 activation and subsequent erythropoiesis by deSUMOylating GATA1.

Background ASK1-interacting protein-1 (AIP1), a Ras GTPase-activating protein family member, is highly expressed in endothelial cells (EC) and vascular smooth muscle cells (VSMC). The role of AIP1 in VSMC and VSMC-proliferative disease is not known. We employed mouse graft arteriosclerosis models characterized by VSMC accumulation and intimal expansion to determine the function of AIP1. Methods and Results In a single minor histocompatibility antigen (male to female)-dependent aorta transplantation model, AIP1 deletion in the graft augmented neointima formation, an effect reversed in AIP1/ interferon-γ receptor (IFN-γR) doubly deficient aorta donors. In a syngeneic aortic transplantation model in which WT or AIP1-KO mouse aortas were transplanted into IFN-γ receptor deficient recipient and neointima formation induced by intravenous administration of adenovirus encoding a mouse IFN-γ transgene, donor grafts from AIP1-KO enhanced IFN-γ -induced VSMC proliferation and neointima formation. Mechanistically, knockout or knockdown of AIP1 in VSMC significantly enhanced IFN-γ-induced JAK-STAT signaling and IFN-γ-dependent VSMC migration and proliferation, two critical steps in neointima formation. Furthermore, AIP1 specifically binds to JAK2 and inhibits its activity. Conclusion AIP1 functions as a negative regulator in IFN-γ-induced intimal formation, in part, by downregulating IFN-γ-JAK2-STAT1/3-dependent migratory and proliferative signaling in VSMC.

Human amniotic epithelial cells (hAECs), derived from the innermost layer of the term placenta closest to the fetus, have been shown to be potential seed cells for allogeneic cell therapy. Previous studies have shown a certain therapeutic effect of hAECs. However, no appropriate isolation and culture system for hAECs has been developed for clinical applications. In the present study, we established a serum-free protocol for hAEC isolation and cultivation, in which better cell growth was observed compared with that in a traditional culture system with serum. In addition to specific expression of cell surface markers (CD29, CD166 and CD90), characterization of the biological features of hAECs revealed expression of the pluripotent markers SSEA4, OCT4 and NANOG, which was greater than that in human mesenchymal stem cells, whereas very low levels of HLA-DR and HLA-DQ were detected, suggesting the weak immunogenicity of hAECs. Intriguingly, CD90+ hAECs were identified as a unique population with a powerful immunoregulatory capacity. In a systemic safety evaluation, intravenous administration of hAEC did not result in hemolytic, allergy, toxicity issues or, more importantly, tumorigenicity. Finally, the therapeutic effect of hAECs was demonstrated in mice with radiation-induced damage. The results revealed a novel function of hAECs in systemic injury recovery. Therefore, the current study provides an applicable and safe strategy for hAEC cell therapy administration in the clinical setting.

Objective-Thioredoxin-2 (Trx2), a major antioxidant protein in mitochondria, enhances nitric oxide bioavailability and inhibits ASK1-dependent apoptosis in endothelial cells (ECs). However, the in vivo role of Trx2 in angiogenesis has not been defined. Here we used EC-specific transgenesis of Trx2 (Trx2-TG) in mice to determine the in vivo function of Trx2 in arteriogenesis and angiogenesis. Methods and Results-In a femoral artery ligation model, Trx2-TG mice had enhanced capacity in limb perfusion recovery and ischemic reserve capacity compared to the nontransgenic littermates. Ischemia-initiated arteriogenesis in the upper limb was augmented in Trx2-TG mice. Trx2-TG mice also showed significantly enhanced capillary formation and maturation in the lower limb. In nontransgenic limb, ischemia specifically induced a downregulation of Trx2 protein, leading to increased oxidative stress, ASK1 activation, and EC apoptosis. In contrast, Trx2-TG maintained a constitutive level of Trx2, reducing the ischemia-induced deleterious responses. We then defined the mechanism by which Trx2 increases angiogenesis using ECs isolated from Trx2-TG mice. Trx2-TG ECs showed increased NO and NO-dependent migration. In addition, these cells were more resistant to oxidative stress-induced activation of ASK1 signaling and apoptosis. Moreover, Trx2-augmented EC survival is NO-independent. To define the relative contributions of Trx2-increased NO and Trx2-reduced ASK1 apoptotic activity to angiogenesis in vivo, we examined Trx2 effects on ischemia-induced angiogenesis in eNOS-deficient mice. The eNOS deletion caused severe impairment in the functional flow recovery in response to ischemia. Trx2 expression in eNOS-KO mice still dramatically inhibited ischemia-induced ASK1 and EC apoptosis, leading to an enhanced functional flow recovery. Conclusion-These in vivo and in vitro data support that Trx2 maintains EC function by two parallel pathwaysscavenging ROS to increase NO bioavailability and inhibiting ASK1 activity to enhance EC survival, facilitating ischemia-mediated arteriogenesis and angiogenesis. Key Words: thioredoxin Ⅲ angiogenesis Ⅲ ischemia Ⅲ apoptosis A ngiogenesis, a process of new blood vessel formation, contributes to various physiological processes and pathological settings. 1 While excessive angiogenesis links atherosclerosis, cancer, and diabetic retinopathy, defects in angiogenesis directly contribute to myocardial infarction and peripheral arterial disease. Recent reports suggest that reactive oxygen species (ROS) can positively or negatively regulate angiogenesis. Although physiological levels of ROS are required for angiogenesis, excess amount of ROS generated during inflammation and ischemic response may inhibit reparative vascular remodeling by inducing endothelial dysfunction and apoptosis. ROS-producing systems in vascular endothelial cells (ECs) are numerous including various NADPH oxidases, xanthine oxidase, the uncoupling of NO synthase as well as mitochondria. [2][3][4][5] The NADPH oxidases have been considered...

Two groups of frequency sweeping modes are observed and interpreted in the HL-2 A plasmas with qmin ∼ 1. The tokamak simulation code calculations indicate the presence of a reversed shear q-profile during the existence of these modes. The mode frequencies lie in between TAE and BAE frequencies, i.e. ωBAE < ω < ωTAE, and these modes are highly localized near qmin, i.e. r/a ∼ 0.25. A group of modes characterized by down-sweeping frequency with qmin decrease due to qmin > 1 and nqmin − m > 0, and another group of modes characterized by up-sweeping frequency with qmin drop, owing to qmin < 1 and nqmin − m < 0 before sawtooth crash. The kinetic Alfvén eigenmode code analysis supports that the down-sweeping modes are kinetic reverse shear Alfvén eigenmodes (KRSAEs), and the up-sweeping modes are RSAEs, which exist in the ideal or kinetic MHD limit. In addition, the down- and up-sweeping RSAEs both have fast nonlinear frequency behaviour in the process of slow frequency sweeping, i.e. producing pitch-fork phenomena. These studies provide valuable constraint conditions for the q-profile measurements.