Paul J Bushway

University of California, San Diego, San Diego, California, United States

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Publications (16)132.86 Total impact

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    ABSTRACT: The elucidation of factors that activate the regeneration of the adult mammalian heart is of major scientific and therapeutic importance. Here we found that epicardial cells contain a potent cardiogenic activity identified as follistatin-like 1 (Fstl1). Epicardial Fstl1 declines following myocardial infarction and is replaced by myocardial expression. Myocardial Fstl1 does not promote regeneration, either basally or upon transgenic overexpression. Application of the human Fstl1 protein (FSTL1) via an epicardial patch stimulates cell cycle entry and division of pre-existing cardiomyocytes, improving cardiac function and survival in mouse and swine models of myocardial infarction. The data suggest that the loss of epicardial FSTL1 is a maladaptive response to injury, and that its restoration would be an effective way to reverse myocardial death and remodelling following myocardial infarction in humans.
    No preview · Article · Sep 2015 · Nature
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    ABSTRACT: Cardiomyopathy is the leading cause of death worldwide. Despite progress in medical treatments, heart transplantation is one of the only current options for those with infarcted heart muscle. Stem cell differentiation technology may afford cell-based therapeutics that may lead to the generation of new, healthy heart muscle cells from undifferentiated stem cells. Our approach is to use small molecules to stimulate stem cell differentiation. Herein, we describe a novel class of 1,5-disubstituted benzimidazoles that induce differentiation of stem cells into cardiac cells. We report on the evaluation in vitro for cardiomyocyte differentiation and describe structure-activity relationship results that led to molecules with drug-like properties. The results of this study show the promise of small molecules to direct stem cell lineage commitment, to probe signaling pathways and to develop compounds for the stimulation of stem cells to repair damaged heart tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.
    No preview · Article · Aug 2015 · Bioorganic & medicinal chemistry
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    ABSTRACT: Chemical genomics has the unique potential to expose novel mechanisms of complex cellular biology through screening of small molecules in in vitro assays of a biological phenotype of interest, followed by target identification. In the case of disease-specific assays, the cellular proteins identified might constitute novel drug targets, and the small molecules themselves might be developed as drug leads. In cardiovascular biology, a chemical genomics approach to study the formation of cardiomyocyte, vascular endothelial, and smooth muscle lineages might contribute to therapeutic regeneration. Here, we describe methods used to develop high content screening assays implementing multipotent cardiovascular progenitors derived from human pluripotent stem cells and have identified novel compounds that direct cardiac differentiation.
    No preview · Article · Jan 2015 · Methods in molecular biology (Clifton, N.J.)
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    ABSTRACT: Tight control over the segregation of endoderm, mesoderm, and ectoderm is essential for normal embryonic development of all species, yet how neighboring embryonic blastomeres can contribute to different germ layers has never been fully explained. We postulated that microRNAs, which fine-tune many biological processes, might modulate the response of embryonic blastomeres to growth factors and other signals that govern germ layer fate. A systematic screen of a whole-genome microRNA library revealed that the let-7 and miR-18 families increase mesoderm at the expense of endoderm in mouse embryonic stem cells. Both families are expressed in ectoderm and mesoderm, but not endoderm, as these tissues become distinct during mouse and frog embryogenesis. Blocking let-7 function in vivo dramatically affected cell fate, diverting presumptive mesoderm and ectoderm into endoderm. siRNA knockdown of computationally predicted targets followed by mutational analyses revealed that let-7 and miR-18 down-regulate Acvr1b and Smad2, respectively, to attenuate Nodal responsiveness and bias blastomeres to ectoderm and mesoderm fates. These findings suggest a crucial role for the let-7 and miR-18 families in germ layer specification and reveal a remarkable conservation of function from amphibians to mammals.
    Full-text · Article · Nov 2012 · Genes & development
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    ABSTRACT: A medium-throughput murine embryonic stem cell (mESC)-based high-content screening of 17000 small molecules for cardiogenesis led to the identification of a b-annulated 1,4-dihydropyridine (1,4-DHP) that inhibited transforming growth factor β (TGFβ)/Smad signaling by clearing the type II TGFβ receptor from the cell surface. Because this is an unprecedented mechanism of action, we explored the series' structure-activity relationship (SAR) based on TGFβ inhibition, and evaluated SAR aspects for cell-surface clearance of TGFβ receptor II (TGFBR2) and for biological activity in mESCs. We determined a pharmacophore and generated 1,4-DHPs with IC(50)s for TGFβ inhibition in the nanomolar range (e.g., compound 28, 170 nM). Stereochemical consequences of a chiral center at the 4-position was evaluated, revealing 10- to 15-fold more potent TGFβ inhibition for the (+)- than the (-) enantiomer. This stereopreference was not observed for the low level inhibition against Activin A signaling and was reversed for effects on calcium handling in HL-1 cells.
    No preview · Article · Nov 2012 · Journal of Medicinal Chemistry
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    ABSTRACT: This unit describes a robust protocol for producing multipotent Kdr-expressing mesoderm progenitor cells in serum-free conditions, and for functional genomics screening using these cells. Kdr-positive cells are able to differentiate into a wide array of mesodermal derivatives, including vascular endothelial cells, cardiomyocytes, hematopoietic progenitors, and smooth muscle cells. The efficient generation of such progenitor cells is of particular interest because it permits subsequent steps in cardiovascular development to be analyzed in detail, including deciphering the mechanisms that direct differentiation. In addition, the oligonucleotide transfection protocol used to functionally screen siRNA and miRNA libraries is a powerful tool to reveal networks of genes, signaling proteins, and miRNAs that control the diversification of cardiovascular lineages from multipotent progenitors. Technical limitations, troubleshooting, and potential applications of these methods are discussed. Curr. Protoc. Stem Cell Biol. 23:1F.13.1-1F.13.13. © 2012 by John Wiley & Sons, Inc.
    No preview · Article · Nov 2012 · Current protocols in stem cell biology

  • No preview · Conference Paper · Aug 2012
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    ABSTRACT: The cellular signals controlling the formation of cardi-omyocytes, vascular smooth muscle, and endothe-lial cells from stem cell-derived mesoderm are poorly understood. To identify these signals, a mouse em-bryonic stem cell (ESC)-based differentiation assay was screened against a small molecule library result-ing in a 1,4-dihydropyridine inducer of type II TGF-b receptor (TGFBR2) degradation-1 (ITD-1). ITD an-alogs enhanced proteasomal degradation of TGFBR2, effectively clearing the receptor from the cell surface and selectively inhibiting intracellular signaling (IC 50 $0.4–0.8 mM). ITD-1 was used to eval-uate TGF-b involvement in mesoderm formation and cardiopoietic differentiation, which occur sequen-tially during early development, revealing an essen-tial role in both processes in ESC cultures. ITD-1 selectively enhanced the differentiation of uncom-mitted mesoderm to cardiomyocytes, but not to vascular smooth muscle and endothelial cells. ITD-1 is a highly selective TGF-b inhibitor and reveals an unexpected role for TGF-b signaling in controlling cardiomyocyte differentiation from multipotent cardiovascular precursors.
    Full-text · Article · Aug 2012 · Cell Stem Cell
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    ABSTRACT: The cellular signals controlling the formation of cardiomyocytes, vascular smooth muscle, and endothelial cells from stem cell-derived mesoderm are poorly understood. To identify these signals, a mouse embryonic stem cell (ESC)-based differentiation assay was screened against a small molecule library resulting in a 1,4-dihydropyridine inducer of type II TGF-β receptor (TGFBR2) degradation-1 (ITD-1). ITD analogs enhanced proteasomal degradation of TGFBR2, effectively clearing the receptor from the cell surface and selectively inhibiting intracellular signaling (IC(50) ~0.4-0.8 μM). ITD-1 was used to evaluate TGF-β involvement in mesoderm formation and cardiopoietic differentiation, which occur sequentially during early development, revealing an essential role in both processes in ESC cultures. ITD-1 selectively enhanced the differentiation of uncommitted mesoderm to cardiomyocytes, but not to vascular smooth muscle and endothelial cells. ITD-1 is a highly selective TGF-β inhibitor and reveals an unexpected role for TGF-β signaling in controlling cardiomyocyte differentiation from multipotent cardiovascular precursors.
    Full-text · Article · Aug 2012 · Cell stem cell
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    ABSTRACT: Hepatocyte nuclear factor (HNF)4α is a central regulator of gene expression in cell types that play a critical role in metabolic homeostasis, including hepatocytes, enterocytes, and pancreatic β cells. Although fatty acids were found to occupy the HNF4α ligand-binding pocket and were proposed to act as ligands, there is controversy about both the nature of HNF4α ligands as well as the physiological role of the binding. Here, we report the discovery of potent synthetic HNF4α antagonists through a high-throughput screen for effectors of the human insulin promoter. These molecules bound to HNF4α with high affinity and modulated the expression of known HNF4α target genes. Notably, they were found to be selectively cytotoxic to cancer cell lines in vitro and in vivo, although in vivo potency was limited by suboptimal pharmacokinetic properties. The discovery of bioactive modulators for HNF4α raises the possibility that diseases involving HNF4α, such as diabetes and cancer, might be amenable to pharmacologic intervention by modulation of HNF4α activity.
    Full-text · Article · Jul 2012 · Chemistry & biology
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    Paul J Bushway · Behrad Azimi · Susanne Heynen-Genel
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    ABSTRACT: The standard (STD) 5 × 5 hybrid median filter (HMF) was previously described as a nonparametric local backestimator of spatially arrayed microtiter plate (MTP) data. As such, the HMF is a useful tool for mitigating global and sporadic systematic error in MTP data arrays. Presented here is the first known HMF correction of a primary screen suffering from systematic error best described as gradient vectors. Application of the STD 5 × 5 HMF to the primary screen raw data reduced background signal deviation, thereby improving the assay dynamic range and hit confirmation rate. While this HMF can correct gradient vectors, it does not properly correct periodic patterns that may present in other screening campaigns. To address this issue, 1 × 7 median and a row/column 5 × 5 hybrid median filter kernels (1 × 7 MF and RC 5 × 5 HMF) were designed ad hoc, to better fit periodic error patterns. The correction data show periodic error in simulated MTP data arrays is reduced by these alternative filter designs and that multiple corrective filters can be combined in serial operations for progressive reduction of complex error patterns in a MTP data array.
    Full-text · Article · Sep 2011 · Journal of Biomolecular Screening
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    ABSTRACT: Microtiter plate (MTP) assays often exhibit distortions, such as caused by edge-dependent drying and robotic fluid handling variation. Distortions vary by assay system but can have both systematic patterns (predictable from plate to plate) and random (sporadic and unpredictable) components. Random errors can be especially difficult to resolve by assay optimization alone, and postassay algorithms reported to date have smoothing effects that often blunt hits. We implemented a 5 x 5 bidirectional hybrid median filter (HMF) as a local background estimator to scale each data point to the MTP global background median and compared it with a recently described Discrete Fourier Transform (DFT) technique for correcting errors on computationally and experimentally generated MTP datasets. Experimental data were generated from a 384-well format fluorescent bioassay using cells engineered to express eGFP and DsRED. MTP arrays were produced with and without control treatments used to simulate hits in random wells. The HMF demonstrated the greatest improvements in MTP coefficients of variation and dynamic range (defined by the ratio of average hit amplitude to standard deviation, SD) for all synthetic and experimental MTPs examined. After HMF application to a MTP of eGFP signal from mouse insulinoma (MIN6) cells obtained by a plate-reader, the assay coefficient of variation (CV) decreased from 8.0% in the raw dataset to 5.1% and the hit amplitudes were reduced by only 1% while the DFT method increased the CV by 36.0% and reduced the hit amplitude by 21%. Thus, our results show that the bidirectional HMF provides superior corrections of MTP data distortions while at the same time preserving hit amplitudes and improving dynamic range. The software to perform hybrid median filter MTP corrections is available at http://bccg.burnham.org/HTS/HMF_Download_Page.aspx, password is pbushway.
    Full-text · Article · Mar 2010 · Assay and Drug Development Technologies
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    Erik Willems · Paul J Bushway · Mark Mercola
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    ABSTRACT: Debilitating cardiomyocyte loss underlies the progression to heart failure. Although there have been significant advances in treatment, current therapies are intended to improve or preserve heart function rather than regenerate lost myocardium. A major hurdle in implementing a cell-based regenerative therapy is the inefficient differentiation of cardiomyocytes from either endogenous or exogenous stem cell sources. Moreover, cardiomyocytes that develop in human embryonic stem cell (hESC) or human-induced pluripotent stem cell (hIPSC) cultures are comparatively immature, even after prolonged culture, and differences in their calcium handling, ion channel, and force generation properties relative to adult cardiomyocytes raise concerns of improper integration and function after transplantation. Thus, the discovery of natural and novel small molecule synthetic regulators of differentiation and maturation would accelerate the development of stem-cell-based myocardial therapies. Here, we document recent advances in defining natural signaling pathways that direct the multistep cardiomyogenic differentiation program and the development of small molecules that might be used to enhance differentiation as well as the potential characteristics of lead candidates for pharmaceutical stimulation of endogenous myocardial replacement.
    Full-text · Article · Apr 2009 · Pediatric Cardiology
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    Paul J Bushway · Mark Mercola · Jeffrey H Price
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    ABSTRACT: We evaluated the performance of two plate readers (the Beckman Coulter [Fullerton, CA] DTX and the PerkinElmer [Wellesley, MA] EnVision) and a plate imager (the General Electric [Fairfield, CT] IN Cell 1000 Analyzer) in a primary fluorescent cellular screen of 10,000 Molecular Libraries Screening Center Network library compounds for up- and down-regulation of vascular cell adhesion molecule (VCAM)-1, which has been shown to be up-regulated in atherothrombotic vascular disease and is a general indicator of chronic inflammatory disease. Prior to screening, imaging of a twofold, six-step titration of fluorescent cells in a 384-well test plate showed greater consistency, sensitivity, and dynamic range of signal detection curves throughout the detection range, as compared to the plate readers. With the same 384-well test plate, the detection limits for fluorescent protein-labeled cells on the DTX and EnVision instruments were 2,250 and 560 fluorescent cells per well, respectively, as compared to 280 on the IN Cell 1000. During VCAM screening, sensitivity was critical for detection of antagonists, which reduced brightness of the primary immunofluorescence readout; inhibitor controls yielded Z' values of 0.41 and 0.16 for the IN Cell 1000 and EnVision instruments, respectively. The best 1% of small molecule inhibitors from all platforms were visually confirmed using images from the IN Cell 1000. The EnVision and DTX plate readers mutually identified approximately 57% and 21%, respectively, of the VCAM-1 inhibitors visually confirmed in the IN Cell best 1% of inhibitors. Furthermore, the plate reader hits were largely exclusive, with only 6% agreement across all platforms (three hits out of 47). Taken together, the imager outperformed the plate readers at hit detection in this bimodal assay because of superior sensitivity and had the advantage of speeding hit confirmation during post-acquisition analysis.
    Preview · Article · Sep 2008 · Assay and Drug Development Technologies
  • Paul J Bushway · Mark Mercola
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    ABSTRACT: Realizing the potential of stem cell biology requires the modulation of self-renewal and differentiation, both of which are incompletely understood. This chapter describes methods for the design, development, and implementation of cell-based screens of small molecules, genes and expressed proteins for modulation of stem and progenitor cell fate. These include the engineering of embryonic and other stem cells with gene promoter-reporter protein constructs and their application in automated screening. We discuss considerations of promoter reporter selection, assay development and implementation, and image acquisition, analysis, and data handling. Such black-box screens are useful for the identification of probes of developmental processes and should provide tools that will identify druggable targets for biochemical assays.
    No preview · Article · Feb 2006 · Methods in Enzymology
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    ABSTRACT: Unmasking the essential genes and pathways that control cardiac lineage decision has paramount importance not only for cardiac development but also for regenerative medicine, including the directed differentiation of adult progenitors or resident cardiac fibroblasts and stem cells to cardiac fates. Self repair is unquestionably insufficient to offset cell death in heart disease and this has triggered considerable efforts to restore myocardium with cardiac cells from either exogenous sources via transplantation or by stimulating endogenous regeneration. Although some reports cite improvement in cardiac performance other show equivocal results clearly showing that more research is needed to achieve adequate cardiac regeneration. microRNAs (miRs) exert cell-autonomous control over many biological processes, including heart formation and physiology, and they represent a powerful means to identify key genes and pathways regulating cardiovascular differentiation. Here we present the results of two studies using whole genome miR screens that led us to uncover two groups of miRs regulating cardiovascular differentiation. First, a systematic screen of a whole genome miR library (875 miRs) revealed that let-7 and miR-18 families increase mesoderm at the expense of endoderm in mouse embryonic stem cells. siRNA screening of computationally predicted targets revealed that the miRs exert their action by attenuating Nodal signaling, consistent with their expression overlapping Acvr1b in vertebrate embryos. Blocking let-7 function in vivo dramatically affected cell fate, converting presumptive mesoderm and ectoderm into endoderm. This is the first demonstration that miRs apportion germ-layer fate, and reveals a remarkable conservation of function from amphibians to mammals. Next, a second screen revealed that 47 families of miRs are able to modulate the fate of a cardiovascular progenitor population co-expressing globally Gata4, Mef2c and Tbx5, to differentiate into either cardiac or endothelial cells. Here, we will emphasize on the use of a powerful combinatorial method aiming at increasing the outcome of miR functional screening.
    No preview · Conference Paper ·

Publication Stats

184 Citations
132.86 Total Impact Points

Institutions

  • 2015
    • University of California, San Diego
      San Diego, California, United States
  • 2006-2015
    • Sanford Burnham Prebys Medical Discovery Institute
      • • Conrad Prebys Center for Chemical Genomics
      • • Muscle Development and Regeneration Research Program
      لا هویا, California, United States