Real Time PCR primers.

<p>Real Time PCR primers.</p

Targeted disruption of the heparanase gene and generation of heparanase deficient mice.

<p>A. Structure of the 5′ end of the heparanase (<i>Hpse)</i> gene (top-normal allele), the targeting vector (middle- K/O allele), and the expected size of products obtained by digestion with Sca1 or EcoRv (bottom). The orientation of the <i>neo</i> cassette and the southern blot probes are indicated. B. Southern blot analysis. Genomic DNA was extracted from embryos of the intercross of <i>Hpse</i> +/− heterozygous mice and subjected to Southern blot analysis after digestion with ERv. Wild type (<i>wt</i>) embryos exhibited only the normal allele, heterozygous embryos exhibited both the normal and the mutated allele, while <i>Hpse-</i>KO mice exhibited only the shorter, KO allele. Samples were hybridized with the external probe 3, shown in A. C. Heparanase mRNA expression. RNA was extracted from lungs and spleens of <i>wt</i> and <i>Hpse-</i>KO mice and subjected to PCR analysis of heparanase expression using 3 different PCR primer pairs designed to amplify different regions of the <i>Hpse</i> gene, as indicated. Heparanase expression was identified in samples derived from <i>wt</i> but not from <i>Hpse-</i>KO mice. D. Heparanase activity. Blood samples derived from 4 <i>wt</i> and 4 <i>Hpse-</i>KO mice, as well as total cell lysate derived from JAR cells, were incubated (16 h, 37°C, pH 6.2) with sulfate labeled ECM. Labeled degradation products released into the incubation medium were subjected to gel filtration analysis on Sepharose 6B, as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005181#s2" target="_blank">Materials and Methods</a>”. High heparanase activity was noted only in samples derived from <i>wt</i> mice; no heparanase activity was detected in <i>Hpse-</i>KO samples or JAR cell lysate. E. HS degradation. Liver, kidney and spleen tissue extracts derived from <i>wt</i> and <i>Hpse-</i>KO mice were prepared as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005181#s2" target="_blank">Materials and Methods</a>” and incubated (18 h, 37°C, pH 5.8) with ³H-acetyl labeled HS. The reaction mixture was then subjected to gel chromatography on Superose-12. The upper panel shows blank (peak I; control ³H-acetyl labeled HS substrate) and positive control (control substrate treated with recombinant heparanase; peak II). Incubations of <i>Hpse</i>-KO tissue extracts (black) resulted in the same elution profile as the blank incubation (upper panel), indicating no detectable heparanase activity. In contrast, incubations with <i>wt</i> tissue extracts (purple) resulted in substantial cleavage of the HS substrate, similar to incubation with recombinant heparanase (upper panel).</p

MMP expression and ChIP analysis for β-catenin in MEF cells derived from <i>Hpse-</i>KO and <i>wt</i> mice.

<p>A. Real-time PCR. RNA was extracted from MEF derived from <i>wt</i> and <i>Hpse-</i>KO mice and subjected to quantitative real time PCR analysis to evaluate the expression of MMP-2, MMP-9, and MMP-14. The expression level determined for each MMP in the <i>wt</i> tissue (blue) was regarded as 100% and the corresponding levels determined in the <i>Hpse-</i>KO cells (purple) are presented as percentage relative to it. Each reaction was repeated 6 times and the mean±SD is indicated. B. Schematic representation of regions along the murine MMP-14 promoter with ≥95% homology to the consensus Lef/Tcf motif. Numbers show the location of putative Lef/Tcf motifs relative to the transcription initiation site (<i>bent arrow</i>). C. ChIP analysis. Following cross-linking of proteins to DNA, chromatin derived from <i>Hpse-</i>KO and <i>wt</i> MEF was sonicated into fragments of average length ≤500 bp; the β-catenin protein was immunoprecipitated with anti β-catenin antibody, and PCR analysis was performed on the immunoprecipitated DNA samples using primer sets MMP14-Pro1 & Pro2, as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005181#s2" target="_blank">Materials and Methods</a>’. Samples were equilibrated for DNA loading amounts using primers specific to actin. The results are representative of three independent experiments.</p

MMP expression in <i>Hpse-</i>KO mice.

<p>A. Real-time PCR. RNA was extracted from liver, kidney and mammary gland of <i>wt</i> and <i>Hpse-</i>KO mice and subjected to quantitative real time PCR analysis to evaluate the expression of MMP-2, MMP-9, MMP-14 and MMP-25. The expression level determined for each MMP in the <i>wt</i> tissue (blue) was regarded as 100% and the corresponding expression determined in the <i>Hpse-</i>KO tissue (Purple) are presented as percentage relative to it. Each reaction was repeated 6 times and the mean±SD is indicated. B. Western blot analysis. Liver, kidney and mammary gland tissue extracts, prepared as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005181#s2" target="_blank">Materials and Methods</a>”, were subjected to Western blot analysis using anti-mouse MMP-2 monoclonal antibodies (mA801B; upper panels), anti mouse β-catenin (mAb610154; middle panels), or anti mouse α-tubulin (B-5-1-2; lower panels). Higher protein levels of MMP-2 and β-catenin were detected in samples derived from <i>Hpse-</i>KO vs. <i>wt</i> tissues. C. MMP2 zymography. Serum samples derived from <i>Hpse-</i>KO and <i>wt</i> mice were evaluated for MMP-2 activity. MMP-2 activity was approximately 3 fold higher in plasma samples derived from <i>Hpse-</i>KO vs. <i>wt</i> mice. D. β-catenin immunostaining. Parafin embedded kidney tissue sections were subjected to immunostaining with antibody directed against β-catenin. Increased staining was observed in kidney derived from <i>Hpse-</i>KO vs. <i>wt</i> mice. C. MMP2 zymography. Serum samples derived from <i>Hpse-</i>KO and <i>wt</i> mice were evaluated for MMP-2 activity. MMP-2 activity was approximately 3 fold higher in plasma samples derived from <i>Hpse-</i>KO vs. <i>wt</i> mice.</p

Hpa2 expression.

<p>RNA samples derived from liver, kidney and mammary glands of <i>wt</i> and <i>Hpse-</i>KO mice were subjected to quantitative real time PCR analysis to evaluate the expression of Hpa2. The expression level determined for each MMP in the <i>wt</i> tissue (blue) was regarded as 100% and the corresponding expression level determined in the <i>Hpse-</i>KO tissue (purple) is presented as percentage relative to the 100% value. Each reaction was repeated 6 times and the mean±SD is indicated. No significant difference was detected in Hpa2 expression between <i>wt</i> and <i>Hpse-</i>KO mice.</p

Morphological appearance of mammary glands from <i>wt</i> vs. <i>Hpse</i>-KO mice.

<p>Whole-mount preparations of mammary glands from 3-month-old virgin mice were stained with hematoxylin. <i>Hpse-</i>KO derived mammary glands (right panel) showed abundant side branches and alveolar structures compared with glands from age-matched <i>wt</i> animals (left panel).</p

PCR primers.

<p>PCR primers.</p

Molecular structure of HS from <i>wt vs. Hpse-</i>KO mice.

<p> Total metabolically ³⁵S-labeled HSPGs was isolated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005181#s2" target="_blank">Materials and Methods</a>”. The samples were analyzed on a Superose 12 column as shown for liver (A) and kidney (C). The HSPGs were treated with alkali and the released free HS chains were analyzed on the same column as show for liver (B) and kidney (D). (Blue- <i>wt</i>; red- <i>Hpse</i>-KO). Standard heparin is eluted at a volume of 14 ml.</p

Endothelial sprouting and angiogenesis.

<p>A. FGF-2 induced vascular sprouting in the aortic ring model. <i>Hpse-</i>KO and <i>wt</i> derived aortic rings were subjected to FGF-2 induced vascular sprouting for 6 days. The rings were then fixed, stained with 0.02% crystal violet and evaluated for vascular sprouting. A more extensive endothelial sprouting was noted in <i>Hpse-</i>KO derived rings (right panel) as compared to <i>wt</i> derived rings (left panel). B. Matrigel plug assay. <i>Hpse-</i>KO (lower panels) and <i>wt</i> (upper panels) mice were injected subcutaneously with 200 µl of growth factor depleted Matrigel supplemented with FGF-2 (80 ng/ml). Seven days later, the Matrigel plugs were excised and photographed, followed by homogenization and determination of hemoglobin content using Drabkin's reagent (right). A pronounced angiogenic response was noted in <i>Hpse-</i>KO vs. <i>wt</i> mice (55±7.18 mg/dl vs. 21±6.2 mg/dl; p≤0.0002, respectively).</p

Stereotactic Aspiration versus Craniotomy for Primary Intracerebral Hemorrhage: A Meta-Analysis of Randomized Controlled Trials

<div><p>Background</p><p>A wealth of evidence based on the randomized controlled trials (RCTs) has indicated that surgery may be a better choice in the management of primary intracerebral hemorrhage (ICH) compared to conservative treatment. However, there is considerable controversy over selecting appropriate surgical procedures for ICH. Thus, this meta-analysis was performed to assess the effects of stereotactic aspiration compared to craniotomy in patients with ICH.</p><p>Methods</p><p>According to the study strategy, we searched PUBMED, EMBASE and Cochrane Central Register of Controlled Trials. Other sources such as the internet-based clinical trial registries, relevant journals and the lists of references were also searched. After literature searching, two investigators independently performed literature screening, assessment of quality of the included trials and data extraction. The outcome measures included death or dependence, total risk of complication, and the risk of rebleeding, gastrointestinal hemorrhage and systematic infection.</p><p>Results</p><p>Four RCTs with 2996 participants were included. The quality of the included trials was acceptable. Stereotactic aspiration significantly decreased the odds of death or dependence at the final follow-up (odds ratio (OR): 0.80, 95% confidence interval (CI): 0.69–0.93; P = 0.004) and the risk of intracerebral rebleeding (OR: 0.44, 95% CI: 0.26–0.74; P = 0.002) compared to craniotomy with no significant heterogeneity among the study results.</p><p>Conclusions</p><p>The present meta-analysis provides evidence that the stereotactic aspiration may be associated with a reduction in the odds of being dead or dependent in primary ICH, which should be interpreted with caution. Further trials are needed to identify those patients most likely to benefit from the stereotactic aspiration.</p></div