Chemokine stromal derived factor 1 (SDF-1) is involved in trafficking of hematopoietic stem cells (HSCs) from the bone marrow (BM) to peripheral blood (PB) and has been found to enhance postischemia angiogenesis. This study was aimed at investigating whether SDF-1 plays a role in differentiation of BM-derived c-kit ؉ stem cells into endothelial progenitor cells (EPCs) and in ischemia-induced trafficking of stem cells from PB to ischemic tissues. We found that SDF-1 enhanced EPC number by promoting ␣ 2 , ␣ 4 , and ␣ 5 integrinmediated adhesion to fibronectin and collagen I. EPC differentiation was reduced in mitogen-stimulated c-kit ؉ cells, while cytokine withdrawal or the overexpression of the cyclin-dependent kinase (CDK) inhibitor p16 INK4 restored such differentiation, suggesting a link between control of cell cycle and EPC differentiation. We also analyzed the time course of SDF-1 expression in a mouse model of hind-limb ischemia. Shortly after femoral artery dissection, plasma SDF-1 levels were up-regulated, while SDF-1 expression in the bone marrow was down-regulated in a timely fashion with the increase in the percentage of PB progenitor cells. An increase in ischemic tissue expression of SDF-1 at RNA and protein level was also observed. Finally, using an in vivo assay such as injection of matrigel plugs, we found that SDF IntroductionIt has been shown that endothelial progenitor cells (EPCs) play a role in vascular repair following ischemic injury. 1 EPCs give rise to endothelial-like cells in culture, growing as spindleshaped cells attaching to culture dishes coated with extracellular matrix (ECM) components. 2 However, the mechanisms driving EPC differentiation are largely unknown. Stromal-derived factor 1 (SDF-1) regulates adhesion/chemotaxis of bone marrow hematopoietic progenitor cells through activation/regulation of specific integrin molecules. [3][4][5] This factor is, therefore, suggested to play a major role in successful hematopoietic stem cell (HSC) engraftment in the bone marrow. 6 In vivo gene inactivation of SDF-1 and its receptor C-X-C chemokine receptor 4 in mice led to early embryonic lethality due to abnormal cerebellar, gastrointestinal vasculature, and hematopoiesis development. [7][8][9] A role for SDF-1 in HSC/EPC recruitment from bone marrow (BM) to peripheral blood (PB) has been proposed, based on the evidence that granulocyte colony stimulating factor (G-CSF)-mediated HSC/EPC mobilization causes an imbalance between the expression of BM SDF-1 and CXCR4 in HSCs, 10 and that SDF-1␣ adenovirus gene transfer enhances the number of circulating HSCs/EPCs. [11][12][13] Recently, overexpression of SDF-1 in ischemic tissues has been found to enhance EPC recruitment from PB and to induce neoangiogenesis. 14,15 In this paper, we show that SDF-1 increases EPC number through enhancement of (BM) c-kit ϩ stem cell adhesion onto extracellular matrix components by integrin receptors. Further, we show that treatment of c-kit ϩ cells with mitogenic cytokines abolished SDF-1-mediated EPC differen...

No abstract

Pathological molecular mechanisms involved in myocardial remodeling contribute to alter the existing structure of the heart, leading to cardiac dysfunction. Among the complex signaling network that characterizes myocardial remodeling, the distinct processes are myocyte loss, cardiac hypertrophy, alteration of extracellular matrix homeostasis, fibrosis, defective autophagy, metabolic abnormalities, and mitochondrial dysfunction. Several pathophysiological stimuli, such as pressure and volume overload, trigger the remodeling cascade, a process that initially confers protection to the heart as a compensatory mechanism. Yet chronic inflammation after myocardial infarction also leads to cardiac remodeling that, when prolonged, leads to heart failure progression. Here, we review the molecular pathways involved in cardiac remodeling, with particular emphasis on those associated with myocardial infarction. A better understanding of cell signaling involved in cardiac remodeling may support the development of new therapeutic strategies towards the treatment of heart failure and reduction of cardiac complications. We will also discuss data derived from gene therapy approaches for modulating key mediators of cardiac remodeling.

Mesenchymal stem cells (MSCs) are adult multipotent cells currently employed in several clinical trials due to their immunomodulating, angiogenic and repairing features. The adipose tissue is certainly considered an eligible source of MSCs. Recently, putative adipose tissue derived MSCs (ADMSCs) have been isolated from the mediastinal depots. However, very little is known about the properties, the function and the potential of human mediastinal ADMSCs (hmADMSCs). However, the lack of standardized methodologies to culture ADMSCs prevents comparison across. Herein for the first time, we report a detailed step by step description to optimize the isolation and the expansion methodology of hmADMSCs using a virally inactivated good manufacturing practice (GMP)-grade platelet lysate, highlighting the critical aspects of the procedure and providing useful troubleshooting suggestions. Our approach offers a reproducible system which could provide standardization across laboratories. Moreover, our system is time and cost effective, and it can provide a reproducible source of adipose stem cells to enable future studies to unravel new insights regard this promising stem cell population.

Background Little clinical research on new‐generation heat‐not‐burn cigarettes ( HNBC ) in comparison with electronic vaping cigarettes ( EVC ) and traditional tobacco combustion cigarettes ( TC ) has been reported. We aimed to appraise the acute effects of single use of HNBC , EVC, and TC in healthy smokers. Methods and Results This was an independent, cross‐over, randomized trial in 20 TC smokers, with allocation to different cycles of HNBC , EVC , and TC . All participants used all types of products, with an intercycle washout of 1 week. End points were oxidative stress, antioxidant reserve, platelet activation, flow‐mediated dilation, blood pressure, and satisfaction scores. Single use of any product led to an adverse impact on oxidative stress, antioxidant reserve, platelet function, flow‐mediated dilation, and blood pressure. HNBC had less impact than EVC and TC on soluble Nox2‐derived peptide (respectively, P =0.004 and 0.001), 8‐iso‐prostaglandin F2α‐ III ( P =0.004 and <0.001), and vitamin E ( P =0.018 and 0.044). HNBC and EVC were equally less impactful than TCs on flow‐mediated dilation ( P =0.872 for HNBC versus EVC ), H 2 O 2 ( P =0.522), H 2 O 2 breakdown activity ( P =0.091), soluble CD 40 ligand ( P =0.849), and soluble P‐selectin ( P =0.821). The effect of HNBC and, to a lesser extent EVC , on blood pressure was less evident than that of TC , whereas HNBC appeared more satisfying than EVC (all P <0.05). Conclusions Acute effects of HNBC , EVC, and TC are different on several oxidative stress, antioxidant reserve, platelet function, cardiovascular, and satisfaction dimensions, with TCs showing the most detrimental changes in clinically relevant features. Clinical Trial Registration URL : http://www.clinicaltrials.gov . Unique identifier: ...

Key Words: HDAC7 Ⅲ ␤-catenin Ⅲ VEGF Ⅲ 14-3-3 Ⅲ cell cycle E ndothelial cells (ECs) are critical cellular components of blood vessels and act as selectively permeable barriers between blood and tissues. In standard physiological conditions they are compact, 1 growth inhibited, protected from apoptosis and retain full control of permeability. 2,3 In contrast, following injury, they become elongated, highly motile, and stimulate cell replication and replacement in response to growth factors. 4 Adherens junctions are formed by vascular endothelial (VE)-cadherin. VE-cadherin predominantly mediates cell contact and regulates angiogenesis by controlling EC adhesion, migration, proliferation, and survival via interactions with vascular endothelial growth factor (VEGF) receptors. 5,6 VEGF is involved in new vessel formation during embryogenesis and in proliferative diseases in adults by inducing differentiation in vascular structures and EC proliferation. 7 A number of signal transduction molecules are activated or modified in response to VEGF stimulation, such as phosphoinositide 3-kinase (PI 3-kinase) and its downstream substrates, serine/threonine kinase Akt/protein kinase B, phospholipase (PL)C␥, Src family tyrosine kinases, the Ras GTPase-activating protein, small adaptor molecule Nck, focal adhesion kinase C, extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase. 8,9 ␤-Catenin is a signaling molecule that promotes cell proliferation and growth by inducing gene transcription through activation of T-cell factor/lymphoid enhancer factor (TCF/LEF), 10 Id2 (inhibitor of DNA binding 2), 11 and follistatin 12 transcription factors. The phosphorylation of ␤-catenin by casein kinase 1 (CK1) at Ser45 and by glycogen Original received May 20, 2009; resubmission received November 13, 2009; revised resubmission received February 26, 2010; accepted February 26, 2010 20 However, the detailed mechanisms of how HDAC7 is involved in these key angiogenic processes is still unclear. In this study we demonstrate that HDAC7 controls EC growth through modulation of ␤-catenin and maintains ECs in a low proliferation stage. MethodsAn expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Cell CultureHuman umbilical vein endothelial cells (HUVECs) were isolated from the human umbilical cord and cultured on collagen I-coated Figure 1. Elevated HDAC7 suppresses EC proliferation via prevention of ␤-catenin translocation. A, Overexpression of HDAC7 decreases metabolism (MTT) and BrdUrd incorporation. HUVECs were uninfected (Ctl) or infected with Ad-HD7 or Ad-tTA. MTT and BrdUrd assays were performed 48 hour after infection. *PϽ0.05. B, Fluorescenceactivated cell-sorting analysis revealed that overexpression of HDAC7 in HUVECs prevents the G 1 /S phase transition. C and D, Overexpression of HDAC7 decreases cyclin D1, Id2, and TCF-1 expression at the RNA level by real-time PCR (*PϽ0.05) (C) or at the protein level by Western blot (D) 48 hours after infection. ␣-T...

Rearrangement of the cytoskeleton in T cells plays a critical role in the organization of a complex signaling interface referred to as immunologic synapse (IS). Surprisingly, the contribution of antigen presenting cells, in particular dendritic cells (DCs), to the structure and function of the IS has not been investigated in as much detail. We have used a natural model of cytoskeletal dysfunction caused by deficiency of the Wiskott-Aldrich syndrome protein (WASp) to explore the contribution of the DC cytoskeleton to IS formation and to T-cell priming. In an antigen-specific system, T-DC contacts were found to be less stable when DCs alone lacked WASp, and associated with multiple defects of IS structure. As a consequence, DCs were unable to support normal IL-12 secretion, and events downstream of TCR signaling were abrogated, including increased calcium flux, microtubule organizing center (MTOC) polarization, phosphorylation of ZAP-70, and T-cell proliferation. Formation of an effective signaling interface is therefore dependent on active cytoskeletal rearrangements in DCs even when T cells are functionally competent. Deficiency of DC-mediated activities may contribute significantly to the varied immunodysregulation observed in patients with WAS, and also in those with limited myeloid reconstitution after allogeneic hematopoietic stem cell transplantation. (Blood. 2011;118(9):2492-2501) IntroductionAn effective immune response is reliant on optimal T-cell activation by dendritic cells (DC), mediated through highly organized signaling complexes or supramolecular activation clusters (SMAC) assembled within the immunologic synapse (IS). 1 The SMAC classically consists of an accumulation of receptors and signaling molecules spatially organized into concentric rings with MHC class II-TCR in the center (cSMAC), surrounded by ICAM-1-LFA-1 (pSMAC) and CD45 on the periphery (dSMAC). While many studies have investigated IS structures using lipid bilayers loaded with ligands or antibodies directed against T-cell SMAC components, surprisingly little is known about the contribution of DCs. It is likely that both partners in the conjugate have an active role in organization of the IS as there is a requirement for contactdependent signaling in each direction, and disruption of the DC cytoskeleton has previously been shown to abrogate IS formation. [2][3][4] Regulation of the actin cytoskeleton is tightly controlled by several molecules including the Rho family of small guanosine triphosphatases (GTPases). Of these Rac1 and Rac2 have been implicated in T-DC IS formation, through initiation and consolidation of cell contact. 4 We and others have shown that T-cell activation by DCs is also dependent on expression of the Cdc42 effector Wiskott-Aldrich syndrome protein (WASp). 5,6 Deficiency of WASp results in the primary X-linked immunodeficiency Wiskott-Aldrich syndrome (WAS), which is characterized by complex multilineage immune dysfunction and microthrombocytopenia. 7,8 While WASp is required in T cells for normal intrinsic fun...

No abstract