Thrombus weights in electrolytic IVC injury-induced DVT in wild-type and <i>Plg</i>^T/T mice.

<p>On day 2 or day 7 post-injury, thrombi formed in the IVC were weighed. No significant differences (<i>p</i> > 0.05) were observed in the thrombus weight between wild-type (WT) and <i>Plg</i>^T/T mice (n = 9 and n = 9 on day 2, and n = 5 and n = 5 on day 7, respectively). <i>Circles and squares</i> represent individual mouse data. <i>Bars</i> represent the mean values of groups.</p

Survival time and lung perfusion defect after tissue factor-induced PE.

<p>(<b><i>A</i></b>) After the tissue factor infusion via IVC, the survival time was recorded until 20 min. No significant difference was observed in the survival between wild-type (WT) (n = 28) and <i>Plg</i>^T/T (n = 29) mice (hazard ratio, 1.18; 95% confidence intervals, 0.40–3.50), or between <i>Pros1</i>^E/E (n = 10) and <i>Plg</i>^T/T<i>/Pros1</i>^E/E (n = 10) mice (hazard ratio, 0.81; 95% confidence intervals, 0.27–2.41). The survival rates of both WT and <i>Plg</i>^T/T mice were significantly longer than those of <i>Pros1</i>^E/E and <i>Plg</i>^T/T<i>/Pros1</i>^E/E mice (WT and <i>Pros1</i>^E/E mice: hazard ratio, 5.43; 95% confidence intervals, 1.81–16.28; WT and <i>Plg</i>^T/T<i>/Pros1</i>^E/E mice: hazard ratio, 4.38; 95% confidence intervals, 1.41–13.60; <i>Plg</i>^T/T and <i>Pros1</i>^E/E mice: hazard ratio, 4.62; 95% confidence intervals, 1.61–13.28; <i>Plg</i>^T/T and <i>Plg</i>^T/T<i>/Pros1</i>^E/E mice: hazard ratio, 3.73; 95% confidence intervals, 1.25–11.11). *Significant difference in comparison to WT mice and <i>Plg</i>^T/T mice. (<b><i>B</i></b>) The scale used to measure lung perfusion defect scores. A score of 0 indicates complete perfusion of Evans blue with no occlusion and a score of 4 indicates no Evans blue perfusion with complete occlusion. (<b><i>C</i></b>) Perfusion defect scores. No significant (ns) differences (<i>p</i> > 0.05) were observed among WT (n = 28), <i>Plg</i>^T/T (n = 29), <i>Pros1</i>^E/E (n = 10) and <i>Plg</i>^T/T<i>/Pros1</i>^E/E mice (n = 10). Perfusion defect scores were assessed by the Mann-Whitney test. <i>Circles</i> and <i>diamonds</i> represent individual mouse data. <i>Bars</i> represent the mean values of groups.</p

Effect of Plg-A622T mutation on fibrinolysis in plasma.

<p>Pooled plasma from 6 wild-type (<b><i>A</i></b>) or 6 <i>Plg</i>^T/T (<b><i>B</i></b>) mice was preincubated in the absence (○) or presence (◆) of human tPA, and clotting was induced with thrombin and CaCl₂. Human α₂-plasmin inhibitor (α₂-PI) was added before the induction of clotting (▽). The turbidity monitored by the absorbance at 405 nm was measured every 5 min as an index of fibrin formation and lysis. The time course of the turbidity was plotted to the maximum absorbance of each sample, which was taken as 100%.</p

No exacerbation of <i>Plg</i>^T/T mutation in the transient focal brain ischaemia model.

<p>(<b><i>A</i></b>) Representative images of coronal sections of wild-type (WT) and <i>Plg</i>^T/T mouse brains. Permanent occlusion of the distal M1 portion of the left middle cerebral artery and 15-min transient occlusion of the bilateral common carotid arteries were applied. After 24 hours, the brains were excised and stained with 2, 3, 5-triphenyl tetrazolium chloride. <i>White</i> areas represent brain infarction. (<b><i>B</i></b>) Infarct volumes. The infarct volume was adjusted for edema by dividing the volume by the edema index (left hemisphere volume / right hemisphere volume). No significant differences (<i>p</i> > 0.05) were observed between groups. <i>Circles</i> represent individual mouse data. <i>Bars</i> represent the mean values of groups.</p

No effects of <i>Plg</i>^T/T mutation in the skin-wound healing model.

<p>The time course of the wound area was measured every other day for two weeks. Data are the means ± SDs of wild-type (WT, n = 10) and <i>Plg</i>^T/T (n = 9) mice. No significant differences (<i>p</i> > 0.05) were observed between groups.</p

Plasma Plg antigen levels and plasmin activities of wild-type and <i>Plg</i>^T/T mice.

<p>(<b><i>A</i></b>) Plg antigen levels. Data are the means ± SDs of wild-type (WT, n = 10) and <i>Plg</i>^T/T (n = 10) mice. The levels measured in WT mice were defined as 1 U/ml. (<b><i>B</i></b>) Plasmin activities. Plasma from the indicated mice was preincubated with human uPA and reacted with a synthetic substrate, S-2403, for plasmin. Data are the means ± SDs of 10 mice for each genotype. The mean activity measured in WT mice was defined as 100%. (<b><i>C</i></b>) Western blot analysis of uPA-treated mouse plasma. Two mouse plasma samples of each genotype were incubated with human uPA and separated by SDS-PAGE under reducing conditions. Plg (<i>black triangle</i>) and heavy chains of plasmin (<i>white triangles</i>) were detected with anti-mouse Plg antibodies.</p

Generation of Plg-A622T mice.

<p>(<b><i>A</i></b>) Structure of the targeted locus in the mouse <i>Plg</i> gene. Exons are represented by <i>filled boxes</i>. A <i>lox</i>P-flanked (<i>filled triangles</i>) neomycin-resistance cassette (<i>NEO</i>) and a diphtheria toxin A fragment expression cassette (<i>DT-A</i>) are indicated by <i>open boxes</i> with <i>arrows</i> that represent the transcriptional orientation. The A622T mutant allele was produced by homologous recombination and <i>NEO</i> deletion mediated by Cre recombinase. The c.1864G>A (p.A622T) mutation and three translationally silent mutations (c.1857T>G, c.1858C>T, c.1860G>A) creating a new <i>Hpa</i>I site (<i>GTTAAC</i>) were introduced into exon 15. Homologous fragments are indicated by <i>dotted lines</i>, while the <i>Mfe</i>I-<i>Hpa</i>I fragments detected by Southern blot analysis of the wild-type (WT) and Plg-A622T alleles are indicated by <i>double-headed arrows</i>. (<b><i>B</i></b>) Southern blot analysis. Genomic DNA from targeted ES cells was digested with <i>Mfe</i>I/<i>Hpa</i>I and detected with the specific probe (WT allele: 9.5 kb; Plg-A622T allele: 6.6 kb). (<b><i>C</i></b>) Quantitative RT-PCR analysis. Total RNA was extracted from mouse liver and subjected to real-time RT-PCR with dual-labeled probes for mouse <i>Plg</i> and <i>Rn18s</i>. Expression levels of <i>Plg</i> mRNA were normalized to <i>Rn18s</i> mRNA. Data are the means ± SDs of WT (n = 8) and <i>Plg</i>^T/T (n = 7) mice. The levels measured in WT mice were defined as 100%.</p

Dilution plots of FL-PS-E in the PS K196E ELISA system.

<p>FL-PS-E was serially diluted from 0 μg mL⁻¹ to 2.5 μg mL⁻¹ in TBS (▲), 20-fold diluted PS-deficient plasma (+), or a 20-fold diluted KK plasma sample (■).</p

Effects of ILK mutants with defects in either PINCH or parvin binding.

<p>The activation indexes of transfected cells (A, C, E). ILK-deficient mutant cells were transiently transfected with GFP cDNA, GFP-fused wild-type ILK (GFPILK-WT) cDNA, GFP-fused ILK mutant with defective PINCH binding (GFPILK-H99D/F109A/W110A) cDNA (A, E), or GFP-fused ILK mutant with defective parvin binding (GFPILK-M402A/K403A) cDNA (C, E). After transfection, the binding of either PAC-1 (A, C) or fibrinogen (E) to the cells was analyzed by flow cytometry. The activation index was determined by the formula shown in Materials and Methods. A value of 100% represents the maximal binding of PAC-1 or fibrinogen to the cells treated with dithiothreitol. Data represent means ± SD of three independent experiments. ** indicates <i>P</i> < 0.01. Immunoblotting showing protein expression of GFP (B, D), GFP-fused wild-type ILK (GFPILK-WT) (B, D), GFP-fused ILK mutant with defective PINCH binding (GFPILK-H99D/F109A/W110A) (B), and GFP-fused ILK mutant with defective parvin binding (GFPILK-M402A/K403A) (D) in ILK-deficient mutant cells. Cell lysates were electrophoresed and immunoblotted with indicated Abs. </p

A Deficiency of Herp, an Endoplasmic Reticulum Stress Protein, Suppresses Atherosclerosis in ApoE Knockout Mice by Attenuating Inflammatory Responses

<div><p>Herp was originally identified as an endoplasmic reticulum (ER) stress protein in vascular endothelial cells. ER stress is induced in atherosclerotic lesions, but it is not known whether Herp plays any role in the development of atherosclerosis. To address this question, we generated Herp- and apolipoprotein E (apoE)-deficient mice (Herp^−/−; apoE^−/− mice) by crossbreeding Herp^−/− mice and apoE^−/− mice. Herp was expressed in the endothelial cells and medial smooth muscle cells of the aorta, as well as in a subset of macrophages in the atherosclerotic lesions in apoE^−/− mice, while there was no expression of Herp in the Herp^−/−; apoE^−/− mice. The doubly deficient mice developed significantly fewer atherosclerotic lesions than the apoE^−/− mice at 36 and 72 weeks of age, whereas the plasma levels of cholesterol and triglycerides were not significantly different between the strains. The plasma levels of non-esterified fatty acids were significantly lower in the Herp^−/−; apoE^−/− mice when they were eight and 16 weeks old. The gene expression levels of ER stress response proteins (GRP78 and CHOP) and inflammatory cytokines (IL-1β, IL-6, TNF-α and MCP-1) in the aorta were significantly lower in Herp^−/−; apoE^−/− mice than in apoE^−/− mice, suggesting that Herp mediated ER stress-induced inflammation. In fact, peritoneal macrophages isolated from Herp-deficient mice and RAW264.7 macrophages in which Herp was eliminated with a siRNA expressed lower levels of mRNA for inflammatory cytokines when they were treated with tunicamycin. Herp deficiency affected the major mediators of the unfolded protein response, including IRE1 and PERK, but not ATF6. These findings suggest that a deficiency of Herp suppressed the development of atherosclerosis by attenuating the ER stress-induced inflammatory reactions.</p></div