Mouse Models for Graft Arteriosclerosis

Graft arteriosclerois (GA),  also called allograft vasculopathy, is a pathologic lesion that develops over  months to years in transplanted organs ...

Xenotransplantation from pig to human being is viewed as a potential solution for the acute organ shortage. However, consequent xenorejection induced by Gal alpha 1,3 Gal (Gal, Gal antigen) prevents xenotransplantation from clinical application. Thus, the most attracting attempt to prevent xenorejection is the elimination of Gal. Our study suggested that compared with the human alpha 1,2 fucosyltransferase (FT) gene and porcine antisense alpha 1,3 galactosyltransferase gene, sequence-specific siRNA targeting Gal were capable of suppressing Gal expression markedly, and therefore, significantly inhibiting xenoreactivity and the complement activation with human serum in PIEC cells. We also demonstrated the concordant inhibitory effect of siRNA and human FT gene on Gal and corresponding functions, which implied a practical significance of combined transgenic strategy. The successful application of vector-based dsRNA-GT may extend the list of available modalities in the abrogation of xenorejection in xenotransplantation.

Effect of RNA interference on Gal alpha 1,3 Gal expression in PIEC cells.

Xenotransplantation from pigs to human beings is viewed as a potential solution for the acute organ shortage. However, consequent xenorejection induced by Gal alpha 1,3 Gal (a Gal, Gal antigen) prevents xenotransplantation from clinical application. Thus, the most attracting attempt to prevent xenorejection is the elimination of Gal. Our study suggested that compared with the human alpha 1,2 fucosyltransferase (FT) gene and the porcine antisense alpha 1,3 galactosyltransferase gene, sequence-specific siRNA targeting Gal was capable of suppressing Gal expression markedly, and therefore, significantly inhibiting xenoreactivity and the complement activation with human serum in PIEC cells. We also demonstrated the concordant inhibitory effect of siRNA and the human FT gene on Gal and corresponding functions, which implied a practical significance of combined transgenic strategy. The successful application of vector-based dsRNA-GT may extend the list of available modalities in the abrogation of xenorejection in xenotransplantation.

Focal cerebral ischemia causes apoptosis in neural cells during the postischemia period. TNF is critically involved in such neuronal apoptosis mediated by caspase pathways. A20 can inhibit TNF-induced apoptosis in many cell types. However, little work has been carried out in central nervous system. In the present study, gene transfer of A20 resulted in reduction of infarct volume and improvement of neurological deficit in ischemia rats. Results of flow cytometry, TUNEL and DNA fragmentation assay all indicated A20 could inhibit TNF-induced apoptosis both in primary rat hippocampal neurons and SH-SY5Y cells. Moreover, we found A20 targeted the TNF apoptotic pathway by inhibiting proteolytic cleavage of caspase 8 and 3 in SH-SY5Y cells. These data demonstrated A20 could effectively protect neurons from postischemic apoptosis and may function partly on death receptor caspase pathway. Gene transfer of A20 may be a promising approach to gene therapy for cerebral ischemia in the future.

Neuroprotective effect of A20 on TNF-induced postischemic apoptosis.

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 stress-induced 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-DeltaPH), 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.

AIP1 is critical in transducing IRE1-mediated endoplasmic reticulum stress response.

Previously we have shown that AIP1 (apoptosis signal-regulating kinase [ASK]1-interacting protein 1), a novel member of the Ras-GAP protein family, facilitates dephosphorylation of ASK1 at pSer967 and subsequently 14-3-3 release from ASK1, leading to enhanced ASK1-JNK signaling. However, the phosphatase(s) responsible for ASK1 dephosphorylation at pSer967 has not been identified. In the present study, we identified protein phosphatase (PP)2A as a potential phosphatase in vascular endothelial cells (ECs). Tumor necrosis factor (TNF)-induced dephosphorylation of ASK1 pSer967 in ECs was blocked by PP2A inhibitor okadaic acid. Overexpression of PP2A catalytic subunit induced dephosphorylation of ASK1 pSer967 and activation of c-Jun N-terminal kinase (JNK). In contrast, a catalytic inactive form of PP2A or PP2A small interfering RNA blunted TNF-induced dephosphorylation of ASK1 pSer967 and activation of JNK without effects on NF-kappaB activation. Whereas AIP1, via its C2 domain, binds to ASK1, PP2A binds to the GAP domain of AIP1. Endogenous AIP1-PP2A complex can be detected in the resting state, and TNF induces a complex formation of AIP1-PP2A with ASK1. Furthermore, TNF-induced association of PP2A with ASK1 was diminished in AIP1-knockdown ECs, suggesting a critical role of AIP1 in recruiting PP2A to ASK1. TNF-signaling molecules TRAF2 and RIP1, known to be in complex with AIP1 and activate AIP1 by phosphorylating AIP1 at Ser604, are critical for TNF-induced ASK1 dephosphorylation. Finally, PP2A and AIP1 cooperatively induce activation of ASK1-JNK signaling and EC apoptosis, as demonstrated by both overexpression and small interfering RNA knockdown approaches. Taken together, our data support a critical role of PP2A-AIP1 complex in TNF-induced activation of ASK1-JNK apoptotic signaling.

AIP1 recruits phosphatase PP2A to ASK1 in tumor necrosis factor-induced ASK1-JNK activation.

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.

Endothelial-specific expression of mitochondrial thioredoxin promotes ischemia-mediated arteriogenesis and angiogenesis.

Previously we have shown that tyrosine 718 of ASK1 when phosphorylated is critical for SOCS1 binding and SOCS1-mediated degradation of ASK1. However, the kinase and phosphatase responsible for phosphorylation and dephosphorylation of ASK1 at Tyr-718 are unknown. In this study, we identified JAK2 and SHP2 as a Tyr-718-specific kinase and phosphatase, respectively. Interferon-gamma (IFN-gamma) induced degradation of ASK1 in normal but not in SOCS1-KO endothelial cells (EC). IFN-gamma-induced tyrosine phosphorylation of ASK1 at Tyr-718 was blocked by a JAK2-specific inhibitor. IFN-gamma enhanced the association between JAK2 and ASK1, and the ASK1-JAK2 complex was labile and was stabilized by the proteasomal inhibitor MG132. Furthermore, JAK2, but not JAK1, directly bound to and phosphorylated ASK1 at Tyr-718, leading to an enhanced association of ASK1 with SOCS1 and subsequent ASK1 degradation. Next, we showed that overexpression of the SH2-containing protein-tyrosine phosphatase-2 (SHP2) augmented, whereas a phosphatase-inactive mutant of SHP2 inhibited, TNF-induced ASK1 dephosphorylation. SHP2 associated with ASK1 in response to tumor necrosis factor in EC. An SHP-2 substrate-trapping mutant formed a complex with tyrosine-phosphorylated ASK1, suggesting that ASK1 is a direct SHP2 substrate. Moreover, SHP2 wild type, but not a catalytically inactive mutant, dissociated SOCS1 from ASK1. IFN-gamma-induced ASK1 Tyr(P)-718 was enhanced in mouse EC deficient in SHP2 (SHP2-KO). In contrast, tumor necrosis factor-induced dephosphorylation of ASK1 at Tyr(P)-718 and activation of ASK1-JNK signaling, as well as EC apoptosis, are significantly reduced in SHP2-KO EC. Our data suggest that JAK2-SOCS1 and SHP2 reciprocally regulate ASK1 phosphorylation and stability in response to cytokines.

JAK2 and SHP2 reciprocally regulate tyrosine phosphorylation and stability of proapoptotic protein ASK1.

In metastatic prostate cancer (PCa) cells, imbalance between cell survival and death signals such as constitutive activation of phosphatidylinositol 3-kinase (PI3K)-Akt and inactivation of apoptosis-stimulated kinase (ASK1)-JNK pathways is often detected. Here, we show that DAB2IP protein, often down-regulated in PCa, is a potent growth inhibitor by inducing G(0)/G(1) cell cycle arrest and is proapoptotic in response to stress. Gain of function study showed that DAB2IP can suppress the PI3K-Akt pathway and enhance ASK1 activation leading to cell apoptosis, whereas loss of DAB2IP expression resulted in PI3K-Akt activation and ASK1-JNK inactivation leading to accelerated PCa growth in vivo. Moreover, glandular epithelia from DAB2IP(-/-) animal exhibited hyperplasia and apoptotic defect. Structural functional analyses of DAB2IP protein indicate that both proline-rich (PR) and PERIOD-like (PER) domains, in addition to the critical role of C2 domain in ASK1 activity, are important for modulating PI3K-Akt activity. Thus, DAB2IP is a scaffold protein capable of bridging both survival and death signal molecules, which implies its role in maintaining cell homeostasis.

DAB2IP coordinates both PI3K-Akt and ASK1 pathways for cell survival and apoptosis.

A single nucleotide polymorphism in the DAB2IP gene is associated with risk of aggressive prostate cancer (PCa), and loss of DAB2IP expression is frequently detected in metastatic PCa. However, the functional role of DAB2IP in PCa remains unknown. Here, we show that the loss of DAB2IP expression initiates epithelial-to-mesenchymal transition (EMT), which is visualized by repression of E-cadherin and up-regulation of vimentin in both human normal prostate epithelial and prostate carcinoma cells as well as in clinical prostate-cancer specimens. Conversely, restoring DAB2IP in metastatic PCa cells reversed EMT. In DAB2IP knockout mice, prostate epithelial cells exhibited elevated mesenchymal markers, which is characteristic of EMT. Using a human prostate xenograft-mouse model, we observed that knocking down endogenous DAB2IP in human carcinoma cells led to the development of multiple lymph node and distant organ metastases. Moreover, we showed that DAB2IP functions as a scaffold protein in regulating EMT by modulating nuclear beta-catenin/T-cell factor activity. These results show the mechanism of DAB2IP in EMT and suggest that assessment of DAB2IP may provide a prognostic biomarker and potential therapeutic target for PCa metastasis.

Role of DAB2IP in modulating epithelial-to-mesenchymal transition and prostate cancer metastasis.

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 as a messenger RNA whose abundance was induced 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 the vascular endothelium, smooth muscle cells, or neurons. Mice with global or endothelial cell-specific deletion of CCM3 exhibited defects in embryonic angiogenesis and died at an early embryonic stage. CCM3 deletion reduced vascular endothelial growth factor receptor 2 (VEGFR2) signaling in embryos and endothelial cells. In response to VEGF stimulation, CCM3 was recruited to and stabilized VEGFR2, and the carboxyl-terminal domain of CCM3 was required for the stabilization of VEGFR2. Indeed, the CCM3 mutants found in human patients lacking the carboxyl-terminal domain were labile and were unable to stabilize and activate VEGFR2. These results demonstrate that CCM3 promotes VEGFR2 signaling during vascular development.

Stabilization of VEGFR2 signaling by cerebral cavernous malformation 3 is critical for vascular development.

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.

SENP1-mediated GATA1 deSUMOylation is critical for definitive erythropoiesis.

Kleine Ubiquitin-&auml;hnliche-Modifizierer (SUMO) &Auml;nderung der Proteine (SUMOylation) und DeSUMOylation sind entstanden, als wichtige regulatorische Mechanismen f&uuml;r Proteinfunktion. SENP1 (SUMO-spezifische Protease) deconjugates SUMOs aus modifizierte Proteine. Wir haben SENP1 Knockout (KO) M&auml;use basiert auf einem Cre-LoxP-System geschaffen. Globale L&ouml;schung des SENP1 (SENP1 KO) verursacht An&auml;mie und embryonale T&ouml;dlichkeit zwischen embryonalen Tag 13,5 und postnatale Tag 1, Korrelation mit Erythropoese-M&auml;ngel in der fetalen Leber. Knochenmark-Transplantation von SENP1 KO fetalen Leberzellen an bestrahlten Erwachsenen Empf&auml;nger &uuml;bertr&auml;gt Erythropoese M&auml;ngel. Protein-Analysen zeigen, dass die Gene GATA1 und GATA1-abh&auml;ngige unten-geregelt in der fetalen Leber von M&auml;usen SENP1 KO. Diese Herunterregulation korreliert mit Akkumulation einer SUMOylated Form der GATA1. Weiter zeigen wir, dass SENP1 k&ouml;nnen direkt DeSUMOylate GATA1, Regulierung GATA1-abh&auml;ngigen Genexpression und Erythropoese durch in-vitro-Tests. Dar&uuml;ber hinaus zeigen wir, dass GATA1 SUMOylation seine DNA-Bindung &auml;ndert Verringerung ihrer Rekrutierung f&uuml;r den GATA1-responsive gen-F&ouml;rderer. Kollektiv, schlie&szlig;en wir, dass SENP1 DeSUMOylating GATA1 GATA1-Aktivierung und anschlie&szlig;ende Erythropoese f&ouml;rdert.

SENP1-vermittelte GATA1 DeSUMOylation ist entscheidend f&uuml;r die endg&uuml;ltige Erythropoese.

ASK1-interacting protein-1 (AIP1), a Ras GTPase-activating protein family member, is highly expressed in endothelial cells and vascular smooth musccells (VSMCs). The role of AIP1 in VSMCs and VSMC proliferative disease is not known.

AIP1 prevents graft arteriosclerosis by inhibiting interferon-Î³-dependent smooth muscle cell proliferation and intimal expansion.

Acetylcholinesterase (AChE) expression is pivotal during apoptosis. Indeed, AChE inhibitors partially protect cells from apoptosis. Insulin-dependent diabetes mellitus (IDDM) is characterized in part by pancreatic Î²-cell apoptosis. Here, we investigated the role of AChE in the development of IDDM and analyzed protective effects of AChE inhibitors. Multiple low-dose streptozotocin (MLD-STZ) administration resulted in IDDM in a mouse model. Western blot analysis, cytochemical staining, and immunofluorescence staining were used to detect AChE expression in MIN6 cells, primary Î² cells, and apoptotic pancreatic Î² cells of MLD-STZ-treated mice. AChE inhibitors were administered intraperitoneally to the MLD-STZ mice for 30 days. Blood glucose, plasma insulin, and creatine levels were measured, and glucose tolerance tests were performed. The effects of AChE inhibitors on MIN6 cells were also evaluated. AChE expression was induced in the apoptotic MIN6 cells and primary Î² cells in vitro and pancreatic islets in vivo when treated with STZ. Induction and progressive accumulation of AChE in the pancreatic islets were associated with apoptotic Î² cells during IDDM development. The administration of AChE inhibitors effectively decreased hyperglycemia and incidence of diabetes, and restored plasma insulin levels and plasma creatine clearance in the MLD-STZ mice. AChE inhibitors partially protected MIN6 cells from the damage caused by STZ treatment. Induction and accumulation of AChE in pancreatic islets and the protective effects of AChE inhibitors on the onset and development of IDDM indicate a close relationship between AChE and IDDM.

Article	Year
Effect of RNA interference on Gal alpha 1,3 Gal expression in PIEC cells. DNA and cell biology\| PubMed ID: 15767784	2005
Effect of RNA interference on Gal alpha 1,3 Gal expression in PIEC cells. DNA and cell biology\| PubMed ID: 15812240	2005
Neuroprotective effect of A20 on TNF-induced postischemic apoptosis. Neurochemical research\| PubMed ID: 16474993	2006
AIP1 is critical in transducing IRE1-mediated endoplasmic reticulum stress response. The Journal of biological chemistry\| PubMed ID: 18281285	2008
AIP1 recruits phosphatase PP2A to ASK1 in tumor necrosis factor-induced ASK1-JNK activation. Circulation research\| PubMed ID: 18292600	2008
Endothelial-specific expression of mitochondrial thioredoxin promotes ischemia-mediated arteriogenesis and angiogenesis. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 19150880	2009
JAK2 and SHP2 reciprocally regulate tyrosine phosphorylation and stability of proapoptotic protein ASK1. The Journal of biological chemistry\| PubMed ID: 19287004	2009
DAB2IP coordinates both PI3K-Akt and ASK1 pathways for cell survival and apoptosis. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19903888	2009
Role of DAB2IP in modulating epithelial-to-mesenchymal transition and prostate cancer metastasis. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 20080667	2010
Stabilization of VEGFR2 signaling by cerebral cavernous malformation 3 is critical for vascular development. Science signaling\| PubMed ID: 20371769	2010
SENP1-mediated GATA1 deSUMOylation is critical for definitive erythropoiesis. The Journal of experimental medicine\| PubMed ID: 20457756	2010
AIP1 prevents graft arteriosclerosis by inhibiting interferon-Î³-dependent smooth muscle cell proliferation and intimal expansion. Circulation research\| PubMed ID: 21700930	2011
Acetylcholinesterase is associated with apoptosis in Î² cells and contributes to insulin-dependent diabetes mellitus pathogenesis. Acta biochimica et biophysica Sinica\| PubMed ID: 22236578	2012

Luyang Yu

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