Activation of ERK is regulated by Gas6.

<p>(<b>A</b>) rhGas6 induced signaling pathway in HRMECs. Cells were incubated with rhGas6 (400 ng/ml) at various times, and cell lysates were subjected to western blotting with specific antibodies, as described in Materials and Methods. (<b>B</b>) Immunofluorescence staining for pERK1/2 and Hoechst staining was performed, as described in Materials and Methods. Representative images of p-ERK1/2 staining in rhGas6-treated HRMECs. (<b>C, </b><b>D</b>) PD98059, SB203580, or U0126 was preincubated for 30 min, and HRMEC responses to rhGas6 or rhVEGF were determined by western blotting using specific antibodies. The experiment was repeated three times. (<b>E</b>) Phospho-ERK1/2 staining in the trunk of 26 hpf embryos [Ctrl-MO embryo (n = 7); <i>gas6</i>-MO injected embryo (n = 8); U0126-treated embryo (n = 8)] and quantitative analysis of zebrafish p-ERK1/2 expression by whole-mount immunostaining. The <i>gas6</i>-morphant and U0126 treated embryos showed reduction of p-ERK1/2 protein expression compared to controls in the zebrafish trunk. The experiment was repeated two times. **<i>p</i><0.01 vs. Ctrl-MO.</p

Knockdown of <i>gas6</i> induces the inhibition of angiogenesis in intersegmental vessels.

<p>(<b>A</b>) The red arrows (Ex5:In5 and Ex7:In7) indicate morpholino target sites for splicing blocks. Primers (blue arrows, exon 1, forward primer; exon 8, reverse primer) were designed for the testing of morpholino efficacy, as described in the Materials and Methods. (<b>B</b>, <b>C</b>) Testing and quantification of morpholino nucleotide efficacy by RT-PCR in standard control MO (Ctrl-MO) and <i>gas6</i> MO (EX5-MO and EX7-MO) treated embryos at 26 hpf. In control morphants, <i>gas6</i> mRNA (768 bp, black arrow) is detectable by RT-PCR (<b>B</b>, first line). In <i>gas6</i> EX5 morphants, the wild-type <i>gas6</i> mRNA is undetectable by RT-PCR at doses of 2 and 5 ng/embryo. The morphant mRNA encodes a truncated form of the Gas6 protein. (<b>C</b>) In <i>gas6</i> EX7 morphants, RT-PCR products reveal Ctrl-MO embryos expressing wild-type <i>gas6</i> transcript, while EX7-MO embryos express two transcript variants at doses of 1 or 2 ng/embryo. The black arrow shows reduced expression of wild-type <i>gas6</i> mRNA. The red arrow indicates results from aberrant splicing, resulting in a gain of ∼250 base pairs of intron 7, which encodes a premature stop codon that occurs in the <i>Gas6</i>. (<b>D</b>) Angiogenesis defects in <i>gas6</i> morphants. The flk:GFP transgenic zebrafish embryos were microinjected with Ctrl-MO (n = 30, 4 ng/embryo) and <i>gas6</i> MO (EX5-MO, n = 35, 4 ng/embryo; EX7-MO, n = 34, 2 ng/embryo), and their blood vessel formation was examined at a cellular level in living embryos at 30 hpf. Normal formation of intersegmental vessels, as shown by GFP-positive endothelial cells, is observed in Ctrl-MO embryos, but severe vascular defects is observed in <i>gas6</i> MO-injected embryos. The experiment was repeated two times.</p

Gas6 Stimulates Angiogenesis of Human Retinal Endothelial Cells and of Zebrafish Embryos via ERK1/2 Signaling

<div><p>Aim</p><p>To determine if growth arrest-specific 6 (Gas6) plays an important role in the regulation of angiogenesis in human retinal microvascular endothelial cells (HRMECs) and in vessel development of zebrafish.</p><p>Methods</p><p>Proliferation, wound-healing cell migration, and tube formation were measured in HRMECs treated with recombinant human Gas6 (rhGas6). Sprague-Dawley rat aortas in Matrigels were treated with rhGas6, and microvessel sprouting emanating from arterial rings was analyzed. Transgenic zebrafish embryos (flk:GFP) were microinjected with rhGas6 at 50 hours post-fertilization (hpf), and ectopic sprouting of subintestinal vessels (SIVs) was observed under a confocal microscope. Morpholino oligonucleotides (MOs) were microinjected to knockdown <i>gas6</i> in zebrafish embryos, and intersegmental vessel impairment was observed. The effect of the extracellular signal-regulated kinase (ERK1/2) inhibitor on the migration of HRMECs and on vessel development in zebrafish embryos was tested.</p><p>Results</p><p>rhGas6 stimulated proliferation, migration, and tube formation in HRMECs in a dose-dependent manner. In rat aortas, rhGas6 induced vessel outgrowth, and the sprouting length was longer than that of controls. The rhGas6-microinjected zebrafish embryos had significantly increased vessel outgrowth in the SIVs. Recombinant human vascular endothelial growth factor (rhVEGF) served as a positive control. Knockdown of <i>gas6</i> inhibited angiogenesis in the developing vessels of zebrafish. The ERK1/2 inhibitor inhibited HRMEC migration and intersegmental vessel formation in zebrafish embryos.</p><p>Conclusions/Interpretations</p><p>These data suggest that Gas6 plays a pivotal role in proliferation, migration, and sprouting of angiogenic endothelial cells in the retina and in zebrafish embryos. Furthermore, Gas6 induced angiogenic processes are induced via phosphorylation of ERK1/2.</p></div

Angiogenic responses are induced by rhGas6 in rat aortic rings and in zebrafish embryos.

<p>(<b>A</b>) Representative images after 5 day incubation with rhGas6 and rhVEGF. Sprouts from rat aortic rings are shown in the Matrigel control, rhGas6, and rhVEGF after treatment for 5 days, as described in the Materials and Methods. (<b>B</b>) Quantification of sprout length from aortic rings revealed increased sprout formation after a 5-day treatment with rhGas6 and rhVEGF in Matrigel. Data are expressed as mean ± SEM. (n = 4). ^***<i>p</i><0.001 vs. control. (<b>C</b>) Transgenic embryos (flk:GFP) at 50 hpf were injected into the perivitelline space with Texas Red dye (Ctrl), Texas Red dye and rhGas6 (300 ng/µl), or Texas Red dye and rhVEGF (5 ng/µl). After 30 h, embryos were photographed under a confocal microscope. In the controls, the formation of ectopic sprouts (arrow) is never observed, and the injected embryos of the rhGas6- and rhVEGF-treated embryos show ectopic sprouting of subintestinal vessels (SIVs). The experiment was repeated three times (Control, n = 28; rhGas6, n = 24; rhVEGF, n = 24).</p

rhGas6 induces proliferation and migration of HRMECs.

<p>HRMECs were incubated in the presence of the indicated concentrations of rhGas6 for 24(<b>A</b>). Proliferation was determined using the MTT assay, and absorption was analyzed at 550 nm using a microtiter plate reader. The results are presented as the mean ± SEM. (n = 4). ^***<i>p</i><0.001, ^**<i>p</i><0.01, and ^*<i>p</i><0.05 vs. control, # <i>p</i><0.01. (<b>B</b>) HRMEC responses to rhGas6 or rhVEGF were determined using a scratch-wound healing assay. Lines indicate the same width of the gap. Representative images are shown at 6 h after generating the scratch. (<b>C</b>) Warfarin (1 µM) was preincubated for 30 min prior to rhGas6 addition. Representative images are shown after generating the scratch.</p

rhGas6 induces tube formation in HRMECs.

<p>(<b>A</b>) HRMECs were incubated with rhGas6 or rhVEGF. Representative images at 9 h after treatment with rhGas6 or rhVEGF are shown. (<b>B</b>) Tube formation by HRMECs on Matrigels was observed by fluorescence microscopy. Relative density was measured using ImageJ software. Data are expressed as mean ± SEM. (n = 4). ^**<i>p</i><0.01 and ^*<i>p</i><0.05 vs. control. (<b>C</b>) Cumulative sprout length was quantified as described in the Materials and Methods. Data are expressed as mean ± SEM. (n = 4). ^**<i>p</i><0.01 vs. control.</p

Phenolic Compounds from the Leaves and Twigs of <i>Osteomeles schwerinae</i> That Inhibit Rat Lens Aldose Reductase and Vessel Dilation in Zebrafish Larvae

Three new phenolic biphenyl derivatives (<b>1</b>–<b>3</b>) and one new lignan glycoside (<b>4</b>) were isolated from the leaves and twigs of <i>Osteomeles schwerinae</i>. The structures of the new compounds were established by spectroscopic data interpretation. The inhibitory effects of <b>1</b>–<b>4</b> on rat lens aldose reductase in vitro were examined, and compounds <b>1</b>–<b>3</b> markedly inhibited the enzyme with IC₅₀ values of 3.8 to 13.8 μM. In addition, the effects of these isolates on the dilation of hyaloid-retinal vessels induced by high glucose (HG) in zebrafish larvae were investigated. Compound <b>1</b> was the most effective in reducing HG-induced dilation of hyaloid-retinal vessels

New Variants Including <i>ARG1</i> Polymorphisms Associated with C-Reactive Protein Levels Identified by Genome-Wide Association and Pathway Analysis

<div><p>C-reactive protein (CRP) is a general marker of systemic inflammation and cardiovascular disease (CVD). The genetic contribution to differences in CRP levels remains to be explained, especially in non-European populations. Thus, the aim of this study was to identify genetic loci associated with CRP levels in Korean population. We performed genome-wide association studies (GWAS) using SNPs from 8,529 Korean individuals (7,626 for stage 1 and 903 for stage 2). We also performed pathway analysis. We identified a new genetic locus associated with CRP levels upstream of <i>ARG1</i> gene (top significant SNP: rs9375813, <i>P_meta</i> = 2.85×10⁻⁸), which encodes a key enzyme of the urea cycle counteract the effects of nitric oxide, in addition to known <i>CRP</i> (rs7553007, <i>P_meta</i> = 1.72×10⁻¹⁶) and <i>HNF1A</i> loci (rs2259816, <i>P_meta</i> = 2.90×10⁻¹⁰). When we evaluated the associations between the CRP-related SNPs with cardiovascular disease phenotypes, rs9375813 (<i>ARG1</i>) showed a marginal association with hypertension (<i>P</i> = 0.0440). To identify more variants and pathways, we performed pathway analysis and identified six candidate pathways comprised of genes related to inflammatory processes and CVDs (<i>CRP, HNF1A</i>, <i>PCSK6, CD36</i>, and <i>ABCA1</i>). In addition to the previously reported loci (<i>CRP, HNF1A</i>, and <i>IL6</i>) in diverse ethnic groups, we identified novel variants in the <i>ARG1</i> locus associated with CRP levels in Korean population and a number of interesting genes related to inflammatory processes and CVD through pathway analysis.</p></div

Candidate CRP-associated SNPs identified by ICSNPathway analysis.

<p>*The number indicates the index of pathways that are ranked by their statistical significance (FDR) (details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095866#pone-0095866-t004" target="_blank">Table 4</a>).</p>†<p>-log₁₀(<i>P</i>) in stage 1 phase of the GWAS. The ‘-’ denotes that the SNP was not present in the stage 1 phase of the GWAS.</p>‡<p>-log₁₀(<i>P</i>) for the SNP in the stage 1 phase of the GWAS, which is in LD with the SNP identified by pathway analysis.</p

Regional plot of the SNPs in the <i>ARG1</i> locus (up) and the LD relationship among these SNPs (down).

<p>Data are shown for the <i>ARG1</i> locus around rs9375813. Diamond-shaped dots represent -log₁₀ (<i>P</i>-values) of SNPs, and green diamond in the LD plot indicates the most significant SNP. The strength of LD relationship (<i>r</i>²) between the most strongly associated SNP and the other SNPs is presented with red color intensities based on JPT+CHB HapMap data. The light blue curve shows recombination rates drawn based on JPT+CHB HapMap data. Green bars represent the coding genes in this region.</p