No abstract

A20 is a stress response gene in endothelial cells (ECs). A20 serves a dual cytoprotective function, protecting from tumor necrosis factor (TNF)-mediated apoptosis and inhibiting inflammation via blockade of the transcription factor nuclear factor-B (NF-B). In this study, we evaluated the molecular basis of the cytoprotective function of A20 in EC cultures and questioned whether its protective effect extends beyond TNF to other apoptotic and necrotic stimuli. Our data demonstrate that A20 targets the TNF apoptotic pathway by inhibiting proteolytic cleavage of apical caspases 8 and 2, executioner caspases 3 and 6, Bid cleavage, and release of cytochrome c, thus preserving mitochondrion integrity. A20 also protects from Fas/CD95 and significantly blunts natural killer cell-mediated EC apoptosis by inhibiting caspase 8 activation. In addition to protecting ECs from apoptotic stimuli, A20 safeguards ECs from complement-mediated necrosis. These data demonstrate, for the first time, that the cytoprotective effect of A20 in ECs is not limited to TNF-triggered apoptosis. Rather, A20 affords broad EC protective functions by effectively shutting down cell death pathways initiated by inflammatory and immune offenders. IntroductionA20 is a zinc finger protein originally identified as a tumor necrosis factor (TNF)-responsive gene in endothelial cells (ECs). 1 A20 is expressed in multiple cell types in response to a variety of stimuli that activate the transcription factor nuclear factor-B (NF-B), including interleukin 1 (IL-1), lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), H 2 O 2 , and CD40 ligand. [2][3][4][5][6][7] We and others have demonstrated that A20, initially described as an antiapoptotic gene, is also a potent inhibitor of the transcription factor NF-B. 7-9 A20-null mice fail to terminate TNF-induced NF-B activation, develop severe inflammation and cachexia, and die prematurely, indicating the importance of A20 in the hierarchy of anti-inflammatory defense processes. 6,10 Elucidating the molecular basis and binding partner(s) that determine the inhibitory effect of A20 upon NF-B activation is the focus of ongoing research. 8 A20 has been shown to interact with components of the NF-B signaling cascade upstream of inhibitor of NF-B (IB), including TNF receptor-associated factors (TRAFs) 11,12 TRAF-1, TRAF-2, and TRAF-6; A20 binding inhibitors of NF-B; and the signalosome IB kinase-␥ (IKK␥)/NF-B essential modulator (NEMO) unit. [13][14][15] Further work is required to test the relevance of these interactions in blocking NF-B activation in response to stimuli other than TNF. 16 In contrast to its so far universal inhibitory effect on NF-B activation, the antiapoptotic activity of A20 remains controversial and appears to be specific to cell type and stimulus. Overexpression of A20 protects human breast carcinoma MCF-7 cells, murine fibrosarcoma WEHI 164, and murine embryonic NIH3T3, but not Hela and lung epithelial A459 cells from TNF-mediated apoptosis. 9,[17][18][19] In cultures derived from prima...

Vascular smooth muscle cell (VSMC) proliferation and migration contribute significantly to atherosclerosis, postangioplasty restenosis, and transplant vasculopathy. Forkhead transcription factors belonging to the FoxO subfamily have been shown to inhibit growth and cell cycle progression in a variety of cell types. We hypothesized that forkhead proteins may play a role in VSMC biology. Under in vitro conditions, platelet-derived growth factor (PDGF)-BB, tumor necrosis factor-␣, and insulin-like growth factor 1 stimulated phosphorylation of FoxO in human coronary artery smooth muscle cells via MEK1/2 and/or phosphatidylinositol 3-kinasedependent signaling pathways. PDGF-BB, tumor necrosis factor-␣, and insulin-like growth factor 1 treatment resulted in the nuclear exclusion of FoxO, whereas PDGF-BB alone down-regulated the FoxO target gene, p27 kip1 , and enhanced cell survival and progression through the cell cycle. These effects were abrogated by overexpression of a constitutively active, phosphorylation-resistant mutant of the FoxO family member, TM-FKHRL1. The anti-proliferative effect of TM-FKHRL1 was partially reversed by small interfering RNA against p27 kip1 . In a rat balloon carotid arterial injury model, adenovirus-mediated gene transfer of FKHRL1 caused an increase in the expression of p27 kip1 in the VSMC and inhibition of neointimal hyperplasia. These data suggest that FoxO activity inhibits VSMC proliferation and activation and that this signaling axis may represent a therapeutic target in vasculopathic disease states.

PI3K was investigated. Far Western blotting with a CRKL-SH3 glutathione S-transferase fusion protein showed that CRKL binds directly to p85 PI3K in vitro. However, although a small amount of CRKL was preassociated with p85 PI3K , the interaction was increased after SF stimulation, suggesting that the interactions of these three proteins are complex. We conclude that SF induces the formation of a signaling complex potentially containing CRKL and p120 CBL , both of which bind to c-Kit through p85 PI3K. These data suggest that one function of CRKL in normal cells might be to recruit signaling molecules such as CBL into a complex with PI3K. Such complexes could be important in propagating signals involving PI3K such as gene expression and adhesion.

No abstract

CRKL is an SH2-SH3-SH3 adapter protein that is a major substrate of the BCR/ABL oncogene. The function of CRKL in normal cells is unknown. In cells transformed by BCR/ABL we have previously shown that CRKL is associated with two focal adhesion proteins, tensin and paxillin, suggesting that CRKL could be involved in integrin signaling. In two hematopoietic cell lines, MO7e and H9, we found that CRKL rapidly associates with tyrosine-phosphorylated proteins after cross-linking of ␤1 integrins with fibronectin or anti-␤1 integrin monoclonal antibodies. The major tyrosinephosphorylated CRKL-binding protein in the megakaryocytic MO7e cells was identified as p120 CBL , the cellular homolog of the v-Cbl oncoprotein. However, in the lymphoid H9 cell line, the major tyrosine-phosphorylated CRKL-binding protein was p110HEF1 . In both cases, this binding was mediated by the CRKL SH2 domain. Interestingly, although both MO7e and H9 cells express p120 CBL and p110 HEF1 , ␤1 integrin cross-linking induces tyrosine phosphorylation of p120 CBL (but not p110 HEF1 ) in MO7e cells and of p110 HEF1 (but not p120 CBL ) in H9 cells. In both cell types, CRKL is constitutively complexed to C3G, SOS, and c-ABL through its SH3 domains, and the stoichiometry of these complexes does not change upon integrin ligation. Thus, in different cell types CRKL and its SH3-associated proteins may form different multimeric complexes depending on whether p120CBL or p110 HEF1 is tyrosine-phosphorylated after integrin ligation. The shift in association of CRKL and its SH3-associated proteins from p120 CBL to p110 HEF1 could contribute to different functional outcomes of "outside-in" integrin signaling in different cells.Integrins play a role in cell movement and apoptosis and also act as costimulatory molecules. The integrin receptors are ␣/␤ heterodimeric transmembrane proteins that mediate cell-cell or cell-extracellular matrix interactions. Activation of integrin receptors leads to the formation of focal adhesions where integrin cytoplasmic domains are connected with actin-containing cytoskeleton components, thereby providing a link between the extracellular environment and intracellular elements. Tyrosine phosphorylation of cellular proteins is an early event after integrin receptor stimulation and is believed to initiate a series of signaling events involving protein-protein interactions leading to changes in viability, proliferation, or other functions in various cells (1, 2). One tyrosine kinase that is localized to the focal adhesion and is activated after integrin ligation has been identified as p125 FAK (3). This kinase may have a negative regulatory role in the formation of focal adhesions (4). Also, another non-receptor tyrosine kinase (related adhesion focal tyrosine kinase) has been found to be partially associated with the actin cytoskeleton and is activated by integrins (5, 6). Recently, investigators have begun to identify the major cellular proteins that are tyrosine-phosphorylated after cross-linking of integrins by ligands. For exampl...

DE is a rare event that occurs more often with the Jetstream and DiamondBack 360 devices. In-stent and complex native lesions are at higher risk for DE. DE is typically reversible with endovascular techniques and has no effect on patency rates and limb salvage.

The endovascular management of CTO results in reasonable primary patency; moreover, secondary patency at 2 years is excellent. Endovascular therapy should be the first-line option for many patients with peripheral artery disease, including those with CLI, claudicants with poor bypass conduit, or patients at high medical risk for surgery. The presence of CTOs does not appear to change these recommendations. Although multiple reinterventions may be required, endovascular therapies can be considered a primary therapy for many patients with CTO.