Shear stress induces endothelial-to-mesenchymal transition via the transcription factor Snail

Blood flow influences atherosclerosis by generating wall shear stress, which alters endothelial cell (EC) physiology. Low shear stress induces dedifferentiation of EC through a process termed endothelial-to-mesenchymal transition (EndMT). The mechanisms underlying shear stress-regulation of EndMT are uncertain. Here we investigated the role of the transcription factor Snail in low shear stress-induced EndMT. Studies of cultured EC exposed to flow revealed that low shear stress induced Snail expression. Using gene silencing it was demonstrated that Snail positively regulated the expression of EndMT markers (Slug, N-cadherin, α-SMA) in EC exposed to low shear stress. Gene silencing also revealed that Snail enhanced the permeability of endothelial monolayers to macromolecules by promoting EC proliferation and migration. En face staining of the murine aorta or carotid arteries modified with flow-altering cuffs demonstrated that Snail was expressed preferentially at low shear stress sites that are predisposed to atherosclerosis. Snail was also expressed in EC overlying atherosclerotic plaques in coronary arteries from patients with ischemic heart disease implying a role in human arterial disease. We conclude that Snail is an essential driver of EndMT under low shear stress conditions and may promote early atherogenesis by enhancing vascular permeability

A Loss of Function Screen of Identified Genome-Wide Association Study Loci Reveals New Genes Controlling Hematopoiesis

The formation of mature cells by blood stem cells is very well understood at the cellular level and we know many of the key transcription factors that control fate decisions. However, many upstream signalling and downstream effector processes are only partially understood. Genome wide association studies (GWAS) have been particularly useful in providing new directions to dissect these pathways. A GWAS meta-analysis identified 68 genetic loci controlling platelet size and number. Only a quarter of those genes, however, are known regulators of hematopoiesis. To determine function of the remaining genes we performed a medium-throughput genetic screen in zebrafish using antisense morpholino oligonucleotides (MOs) to knock down protein expression, followed by histological analysis of selected genes using a wide panel of different hematopoietic markers. The information generated by the initial knockdown was used to profile phenotypes and to position candidate genes hierarchically in hematopoiesis. Further analysis of brd3a revealed its essential role in differentiation but not maintenance and survival of thrombocytes. Using the from-GWAS-to-function strategy we have not only identified a series of genes that represent novel regulators of thrombopoiesis and hematopoiesis, but this work also represents, to our knowledge, the first example of a functional genetic screening strategy that is a critical step toward obtaining biologically relevant functional data from GWA study for blood cell traits

<i>brd3a</i> is an important regulator of thrombopoiesis.

<p>(A) Live confocal imaging of zebrafish embryos injected with h<i>BRD3-</i>GFP mRNA revealed the nuclear localization of h<i>BRD3</i> and that it binds to mitotic chromosomes (white arrows). Expression of h<i>BRD3</i> in <i>brd3a</i> MO injected embryos resulted in a partial rescue of the number of thrombocytes as shown in (B). (C) A graph to illustrate the number of thrombocytes in control, splice <i>brd3a</i> MO and splice <i>brd3a</i> MO plus h<i>BRD3</i> mRNA injected embryos. Each dot represents the number of thrombocytes in the individual MO-injected embryos with respect to control. A blue horizontal line represents the mean value of the number of thrombocytes for each group of embryos. Student t test, * p = 0.016; n = 17. All embryos are oriented with anterior to the left and dorsal to the top.</p

Heat map summarising hematopoietic phenotypes of knock-down of 19 candidate genes.

<p>Data obtained from the initial knock-down were used to generate a heat map of phenotype profiles. Each colored cell in the heat map shows the severity of the observed phenotype relative to control. The most severe decrease in the number of cells is displayed in red, a moderate reduction is displayed in orange and green denotes a cell number comparable to control. A moderate increase in the cell number is displayed in blue and grey indicates that the test was not performed.</p

<i>In vivo</i> morpholino screen in zebrafish identifies 15 new regulators of thrombopoiesis.

<p>MOs were injected into one-cell stage transgenic <i>Tg(cd41:EGFP)</i> zebrafish embryos and assayed for their effect on the number of thrombocytes (<i>cd41^high</i>) at 3 dpf. Representative confocal images were taken of the CHT. For <i>akap10</i>, <i>brd3a</i>, <i>brf1b</i>, <i>kalrn 1</i>, <i>kalrn 2</i>, <i>kif1b</i>, <i>mfn2</i>, <i>pdia5</i>, <i>psmd13</i> and <i>satb1</i> a severe decrease in the number of <i>cd41^high</i> positive cells was observed. <i>brf1a</i>, <i>rcor1</i>, <i>waspla</i>, <i>wasplb</i> and <i>wdr66</i> depletion resulted in a mild phenotype, and <i>fen1</i>, <i>grtp1b</i> and <i>tmcc2</i> MO injected embryos showed no phenotype. All embryos are oriented with anterior to the left and dorsal to the top. White arrow – thrombocytes; white arrowhead – HSCs.</p

Characterization of HSCs in candidate gene depleted embryos.

<p>To assess the stage at which hematopoiesis of each MO injected embryo was defective, we performed whole mount <i>in situ</i> hybridization using a <i>c-myb</i> probe at 3 dpf. Although more than half of MOs had no effect on the number of HSCs, depletion of <i>rcor1</i> resulted in increased numbers of HSCs and depletion of <i>kalrn1</i>, <i>kalrn2</i>, <i>mfn2</i>, <i>pdia5</i>, <i>psmd13</i> and <i>wasplb</i> resulted in decreased numbers of HSCs in CHT at 3 dpf. Representative images of CHT region are shown. All embryos are oriented with anterior to the left and dorsal to the top.</p