Background-Inflammation plays a pivotal role in atherosclerosis. In addition to being a risk marker for cardiovascular disease, much recent data suggest that C-reactive protein (CRP) promotes atherogenesis via effects on monocytes and endothelial cells. The metabolic syndrome is associated with significantly elevated levels of CRP. Plasminogen activator inhibitor-1 (PAI-1), a marker of atherothrombosis, is also elevated in the metabolic syndrome and in diabetes, and endothelial cells are the major source of PAI-1. However, there are no studies examining the effect of CRP on PAI-1 in human aortic endothelial cells (HAECs). Methods and Results-Incubation of HAECs with CRP results in a time-and dose-dependent increase in secreted PAI-1 antigen, PAI-1 activity, intracellular PAI-1 protein, and PAI-1 mRNA. CRP stabilizes PAI-1 mRNA. Inhibitors of endothelial NO synthase, blocking antibodies to interleukin-6 and an endothelin-1 receptor blocker, fail to attenuate the effect of CRP on PAI-1. CRP additionally increased PAI-1 under hyperglycemic conditions. Conclusions-This study makes the novel observation that CRP induces PAI-1 expression and activity in HAECs and thus has implications for both the metabolic syndrome and atherothrombosis. Key Words: inflammation Ⅲ endothelium Ⅲ thrombosis I nflammation plays a critical role in all stages of atherosclerosis from the nascent lesion to acute coronary syndromes. 1 C-reactive protein (CRP) is a prototypic marker of inflammation and has been shown in several prospective studies to predict cardiovascular events (CVEs). [2][3][4][5][6] Although CRP is clearly a risk marker, data are evolving to suggest that CRP also promotes atherogenesis. [7][8][9][10][11][12][13][14] To date, it has been shown in monocytes that CRP induces the production of inflammatory cytokines and promotes monocyte chemotaxis and tissue factor expression. [7][8][9] In endothelial cells, CRP increases the expression of cell adhesion molecules, chemokines, and endothelin-1 (ET-1), decreases endothelial NO synthase (eNOS) expression and activity, and augments monocyte-endothelial cell adhesion. 10 -14 Also, it is present in the foam cells in atherosclerotic lesions and colocalizes with activated fragments of the complement system. 7 Plasminogen activator inhibitor-1 (PAI-1) has a molecular mass of 50 000 and belongs to the superfamily of serine protease inhibitors. 15 It is a marker of impaired fibrinolysis and atherothrombosis. 15-22 PAI-1 is a key regulator of fibrinolysis by inhibiting tissue plasminogen activator (tPA). Decreased fibrinolysis, primarily attributable to increased PAI-1 activity, has been demonstrated in patients with coronary artery disease (CAD), and there is considerable evidence for elevated PAI-1 levels in CAD, but its status as a factor is still unclear. The role of PAI-1 as a CAD risk marker was first described by Hamsten et al 17 in survivors of myocardial infarction. Increased PAI-1 levels have been shown to enhance thrombosis, and antibodies directed against PAI-1 prevented the p...

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is crucial in controlling cellular activities in response to extracellular cytokines. Dysfunctions of the JAK/ STAT pathway result in various hematopoietic and immune disorders. The central events in regulating this pathway are tyrosine phosphorylation and dephosphorylation of the signaling components, which are carried out by protein tyrosine kinases and protein tyrosine phosphatases (PTP), respectively. Here, we review recent advances in the regulatory roles of PTPs, in particular, SHP2 phosphatase, in the JAK/STAT signaling pathway. INTRODUCTIONIntracellular signal transduction pathways are essential for connecting extracellular cytokine stimulations to appropriate cellular responses, such as proliferation, differentiation, and apoptosis. One of the most important pathways activated by cytokines is the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, which was initially discovered in the studies on gene induction by interferon (IFN) (1). It is now clear that protein phosphorylation and dephosphorylation play a central role in controlling the activity of the JAK/STAT pathway. The majority of phosphorylation and dephosphorylation occur at tyrosine residues of the component proteins, which are carried out by protein tyrosine kinases and protein tyrosine phosphatases (PTP), respectively (2-4). This short article summarizes latest insights into how PTPs, including SHP2, SHP1, CD45, PTP1B, T-cell PTP (TC-PTP), PTPRT, and PTPBL, regulate the JAK/STAT pathway.In the current model of the JAK/STAT signaling pathway, the engagement between a cytokine and its cell surface receptor results in receptor oligomerization and subsequent activation of receptor-associated JAK tyrosine kinases. Activated JAKs phosphorylate specific tyrosine resides in the cytoplasmic domain of the receptor which in turn serves as the docking sites for cytoplasmic transcription factors known as STATs. STATs are therefore recruited to the phosphorylated receptor and subsequently phosphorylated by JAKs. The phosphorylated STATs then dimerize, leave the receptor, and translocate to the nucleus where they activate gene transcription (5,6). In mammalian cells, four JAK members (JAK1, JAK2, JAK3, and -terminus (7,8), and a SRC homology 2 (SH2) domain. The SH2 domain is required for STAT activation and dimerization (9,10) while the amino (N-) terminal region of STAT is involved in the formation of STAT tetramers (11) and tyrosine dephosphorylation (12). Genetic knockout studies have revealed various but specific functions of JAKs and STATs. One who is interested in details about the structures and functions of JAKs and STATs could look at the reviews (2,13-15). The following sections of this review will discuss how PTPs regulate JAK/STAT signaling with a focus on the SHP2 phosphatase, since the role of SHP2 in this context is more complicated than other PTPs. REGULATION OF THE JAK/STAT PATHWAY BY PTPS SHP2Structure, regulati...

Hemophilia A is caused by mutations within the Factor VIII (FVIII) gene that lead to depleted protein production and inefficient blood clotting. Several attempts at gene therapy have failed for various reasons-including immune rejection. The recent generation of induced pluripotent stem (iPS) cells from somatic cells by the ectopic expression of 3 transcription factors, Oct4, Sox2, and Klf4, provides a means of circumventing the immune rejection barrier. To date, iPS cells appear to be indistinguishable from ES cells and thus provide tremendous therapeutic potential. Here we prepared murine iPS cells from tail-tip fibroblasts and differentiated them to both endothelial cells and endothelial progenitor cells by using the embryoid body differentiation method. These iPS cells express major ES cell markers such as Oct4, Nanog, SSEA-1, alkaline phosphatase, and SALL4. Endothelial/endothelial progenitor cells derived from iPS cells expressed cell-specific markers such as CD31, CD34, and Flk1 and secreted FVIII protein. These iPS-derived cells were injected directly into the liver of irradiated hemophilia A mice. At various times after transplantation (7-90 days) hemophilia A mice and their control mice counterparts were challenged by a tail-clip bleeding assay. Nontransplanted hemophilia A mice died within a few hours, whereas transplanted mice survived for more than 3 months. Plasma FVIII levels increased in transplanted hemophilia A mice during this period to 8% to 12% of wild type and corrected the hemophilia A phenotype. Our studies provide additional evidence that iPS cell therapy may be able to treat human monogenetic disorders in the future.endothelial cell precursors ͉ Factor VIII ͉ Oct4 ͉ Sox2 ͉ Klf4

induced pluripotent stem cells ͉ epigenetic regulation ͉ Oct4 ͉ Nanog ͉ Sox2

Increasing studies suggest that SALL4 may play vital roles in leukemogenesis and stem cell phenotypes. We have mapped the global gene targets of SALL4 using chromatin immunoprecipitation followed by microarray hybridization and identified more than 2000 highconfidence, SALL4-binding genes in the human acute promyelocytic leukemic cell line, NB4. Analysis of SALL4-binding sites reveals that genes involved in cell death, cancer, DNA replication/repair, and cell cycle were highly enriched (P < .05).These genes include 38 important apoptosis-inducing genes (TNF, TP53, PTEN, CARD9, CARD11, CYCS, LTA) and apoptosis-inhibiting genes (Bmi-1, BCL2, XIAP, DAD1, TEGT). Real-time polymerase chain reaction has shown that expression levels of these genes changed significantly after SALL4 knockdown, which ubiquitously led to cell apoptosis.

Activation of hepatic stellate cells (HSCs) is the dominant event in liver fibrosis. The early events in the organization of HSC activation have been termed initiation. Initiation encompasses rapid changes in gene expression and phenotype that render the cells responsive to cytokines and other local stimuli. Cellular responses following initiation are termed perpetuation, which encompasses those cellular events that amplify the activated phenotype through enhanced growth factor expression and responsiveness. Multiple cells and cytokines play a part in the regulation of HSC activation. HSC activation consists of discrete phenotype responses, mainly proliferation, contractility, fibrogenesis, matrix degradation, chemotaxis and retinoid loss. Currently, antifibrotic therapeutic strategies include inhibition of HSC proliferation or stimulation of HSC apoptosis, downregulation of collagen production or promotion of its degradation, administration of cytokines, and infusion of mesenchymal stem cells. In this review, we summarize the latest advances in our understanding of the mechanisms of HSC activation and possible antifibrotic therapeutic strategies.

A common Shp2 mutation leads to myeloproliferative disease and malignant acute leukemia in stem cells and committed progenitors, associated with Shp2 maintaining chromosomal stability

Derivation of patient-specific induced pluripotent stem cells (iPSCs) opens a new avenue for future applications of regenerative medicine. However, before iPSCs can be used in a clinical setting, it is critical to validate their in vivo fate following autologous transplantation. Thus far, preclinical studies have been limited to small animals and have yet to be conducted in large animals that are physiologically more similar to humans. In this study, we report the first autologous transplantation of iPSCs in a large animal model through the generation of canine iPSCs (ciPSCs) from the canine adipose stromal cells and canine fibroblasts of adult mongrel dogs. We confirmed pluripotency of ciPSCs using the following techniques: (i) immunostaining and quantitative PCR for the presence of pluripotent and germ layer-specific markers in differentiated ciPSCs; (ii) microarray analysis that demonstrates similar gene expression profiles between ciPSCs and canine embryonic stem cells; (iii) teratoma formation assays; and (iv) karyotyping for genomic stability. Fate of ciPSCs autologously transplanted to the canine heart was tracked in vivo using clinical positron emission tomography, computed tomography, and magnetic resonance imaging. To demonstrate clinical potential of ciPSCs to treat models of injury, we generated endothelial cells (ciPSC-ECs) and used these cells to treat immunodeficient murine models of myocardial infarction and hindlimb ischemia.