Head of Westport, MA, United States
Head of Westport, MA, United States

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French A.,University of Oxford | Bravery C.,Consulting on Advanced Biologicals Ltd | Smith J.,University of Oxford | Chandra A.,Loughborough University | And 36 more authors.
Stem Cells Translational Medicine | Year: 2015

There is a need for physical standards (reference materials) to ensure both reproducibility and consistency in the production of somatic cell types from human pluripotent stem cell (hPSC) sources.We have outlined the need for reference materials (RMs) in relation to the unique properties and concerns surrounding hPSC-derived products and suggest in-house approaches to RMgeneration relevant to basic research, drug screening, and therapeutic applications. hPSCs have an unparalleled potential as a source of somatic cells for drug screening, disease modeling, and therapeutic application. Undefined variation and product variability after differentiation to the lineage or cell type of interest impede efficient translation and can obscure the evaluation of clinical safety and efficacy. Moreover, in the absence of a consistent population, data generated from in vitro studies could be unreliable and irreproducible. Efforts to devise approaches and tools that facilitate improved consistency of hPSC-derived products, both as development tools and therapeutic products, will aid translation. Standards exist in both written and physical form; however, because many unknown factors persist in the field, premature written standards could inhibit rather than promote innovation and translation. We focused on the derivation of physical standardRMs. We outline the need for RMs and assess the approaches to in-house RMgeneration for hPSC-derived products, a critical tool for the analysis and control of product variation that can be applied by researchers and developers. We then explore potential routes for the generation of RMs, including both cellular and non cellular materials and novel methods that might provide valuable tools to measure and account for variation. Multiparametric techniques to identify “signatures” for therapeutically relevant cell types, such as neurons and cardiomyocytes that can be derived from hPSCs, would be of significant utility, although physical RMs will be required for clinical purposes. © AlphaMed Press 2015.


Villarreal Jr. G.,Center for Excellence in Vascular Biology | Villarreal Jr. G.,Harvard-MIT Division of Health Sciences and Technology | Zhang Y.,Center for Excellence in Vascular Biology | Larman H.B.,Center for Excellence in Vascular Biology | And 3 more authors.
Biochemical and Biophysical Research Communications | Year: 2010

The Kruppel-like factor 2 (KLF2) and Kruppel-like factor 4 (KLF4) transcription factors have recently been shown to act as critical regulators of endothelial homeostasis. While several insights have been made into the signaling mechanisms orchestrating endothelial KLF2 expression, those governing the expression of KLF4 in the vascular endothelium remain largely unknown. Here, we show that diverse vasoprotective stimuli including an atheroprotective shear stress waveform, simvastatin, and resveratrol induce the expression of KLF4 in cultured human endothelial cells. We further demonstrate that the induction of KLF4 by resveratrol and atheroprotective shear stress occurs via a MEK5/MEF2-dependent signaling pathway. Since MEK5 activation is also critical for the expression of KLF2, we assessed the individual contribution of KLF4 and KLF2 to the global transcriptional activity triggered by MEK5 activation. Genome-wide transcriptional profiling of endothelial cells overexpressing KLF4, KLF2, or constitutively active MEK5 revealed that 59.2% of the genes regulated by the activation of MEK5 were similarly controlled by either KLF2 or KLF4. Collectively, our data identify a significant degree of mechanistic and functional conservation between KLF2 and KLF4, and importantly, provide further insights into the complex regulatory networks governing endothelial vasoprotection. © 2009 Elsevier Inc. All rights reserved.


Azcutia V.,Center for Excellence in Vascular Biology | Azcutia V.,Brigham and Women's Hospital | Routledge M.,Center for Excellence in Vascular Biology | Routledge M.,Imperial College London | And 17 more authors.
Molecular Biology of the Cell | Year: 2013

CD47 plays an important but incompletely understood role in the innate and adaptive immune responses. CD47, also called integrin-associated protein, has been demonstrated to associate in cis with β1 and β3 integrins. Here we test the hypothesis that CD47 regulates adhesive functions of T-cell α4β1 (VLA-4) and αLβ2 (LFA-1) in in vivo and in vitro models of inflammation. Intravital microscopy studies reveal that CD47 -/- Th1 cells exhibit reduced interactions with wild-type (WT) inflamed cremaster muscle microvessels. Similarly, murine CD47-/- Th1 cells, as compared with WT, showed defects in adhesion and transmigration across tumor necrosis factor-α (TNF-α)-activated murine endothelium and in adhesion to immobilized intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1) under flow conditions. Human Jurkat T-cells lacking CD47 also showed reduced adhesion to TNF-α-activated endothelium and ICAM-1 and VCAM-1. In cis interactions between Jurkat T-cell β2 integrins and CD47 were detected by fluorescence lifetime imaging microscopy. Unexpectedly, Jurkat CD47 null cells exhibited a striking defect in β1 and β2 integrin activation in response to Mn2+ or Mg2+/ethylene glycol tetraacetic acid treatment. Our results demonstrate that CD47 associates with β2 integrins and is necessary to induce high-affinity conformations of LFA-1 and VLA-4 that recognize their endothelial cell ligands and support leukocyte adhesion and transendothelial migration. © 2013 Azcutia et al.


PubMed | Center for Excellence in Vascular Biology
Type: Journal Article | Journal: Journal of immunology (Baltimore, Md. : 1950) | Year: 2012

At sites of inflammation, endothelial adhesion molecules bind leukocytes and transmit signals required for transendothelial migration (TEM). We previously reported that adhesive interactions between endothelial cell CD47 and leukocyte signal regulatory protein (SIRP) regulate human T cell TEM. The role of endothelial CD47 in T cell TEM in vivo, however, has not been explored. In this study, CD47/ mice showed reduced recruitment of blood T cells as well as neutrophils and monocytes in a dermal air pouch model of TNF--induced inflammation. Reconstitution of CD47/ mice with wild-type bone marrow cells did not restore leukocyte recruitment to the air pouch, indicating a role for endothelial CD47. The defect in leukocyte TEM in the CD47/ endothelium was corroborated by intravital microscopy of inflamed cremaster muscle microcirculation in bone marrow chimera mice. In an in vitro human system, CD47 on both HUVEC and T cells was required for TEM. Although previous studies showed CD47-dependent signaling required G(i)-coupled pathways, this was not the case for endothelial CD47 because pertussis toxin, which inactivates G(i), had no inhibitory effect, whereas G(i) was required by the T cell for TEM. We next investigated the endothelial CD47-dependent signaling events that accompany leukocyte TEM. Ab-induced cross-linking of CD47 revealed robust actin cytoskeleton reorganization and Src- and Pyk-2-kinase dependent tyrosine phosphorylation of the vascular endothelial-cadherin cytoplasmic tail. This signaling was pertussis toxin insensitive, suggesting that endothelial CD47 signaling is independent of G(i). These findings suggest that engagement of endothelial CD47 by its ligands triggers outside-in signals in endothelium that facilitate leukocyte TEM.

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