<資料>土耳古和議法と瑞西和議法

MicroRNAs (miRNAs, miRs) emerged as key regulators of gene expression. Germline hemizygous deletion of the gene that encodes the miR-17∼92 miRNA cluster was associated with microcephaly, short stature and digital abnormalities in humans. Mice deficient for the miR-17∼92 cluster phenocopy several features such as growth and skeletal development defects and exhibit impaired B cell development. However, the individual contribution of miR-17∼92 cluster members to this phenotype is unknown. Here we show that germline deletion of miR-92a in mice is not affecting heart development and does not reduce circulating or bone marrow-derived hematopoietic cells, but induces skeletal defects. MiR-92a-/- mice are born at a reduced Mendelian ratio, but surviving mice are viable and fertile. However, body weight of miR-92a-/- mice was reduced during embryonic and postnatal development and adulthood. A significantly reduced body and skull length was observed in miR-92a-/- mice compared to wild type littermates. µCT analysis revealed that the length of the 5th mesophalanx to 5th metacarpal bone of the forelimbs was significantly reduced, but bones of the hindlimbs were not altered. Bone density was not affected. These findings demonstrate that deletion of miR-92a is sufficient to induce a developmental skeletal defect

The small fibrinopeptide bβ15-42 as renoprotective agent preserving the endothelial and vascular integrity in early ischemia reperfusion injury in the mouse kidney

Disruption of the renal endothelial integrity is pivotal for the development of a vascular leak, tissue edema and consequently acute kidney injury. Kidney ischemia amplifies endothelial activation and up-regulation of pro-inflammatory mechanisms. After restoring a sufficient blood flow, the kidney is damaged through complex pathomechanisms that are classically referred to as ischemia and reperfusion injury, where the disruption of the inter-endothelial connections seems to be a crucial step in this pathomechanism. Focusing on the molecular cell-cell interaction, the fibrinopeptide Bβ15–42 prevents vascular leakage by stabilizing these inter-endothelial junctions. The peptide associates with vascular endothelial-cadherin, thus preventing early kidney dysfunction by preserving blood perfusion efficacy, edema formation and thus organ dysfunction. We intended to demonstrate the early therapeutic benefit of intravenously administered Bβ15–42 in a mouse model of renal ischemia and reperfusion. After 30 minutes of ischemia, the fibrinopeptide Bβ15–42 was administered intravenously before reperfusion was commenced for 1 and 3 hours. We show that Bβ15–42 alleviates early functional and morphological kidney damage as soon as 1 h and 3 h after ischemia and reperfusion. Mice treated with Bβ15–42 displayed a significantly reduced loss of VE-cadherin, indicating a conserved endothelial barrier leading to less neutrophil infiltration which in turn resulted in significantly reduced structural renal damage. The significant reduction in tissue and serum neutrophil gelatinase-associated lipocalin levels reinforced our findings. Moreover, renal perfusion analysis by color duplex sonography revealed that Bβ15–42 treatment preserved resistive indices and even improved blood velocity. Our data demonstrate the efficacy of early therapeutic intervention using the fibrinopeptide Bβ15–42 in the treatment of acute kidney injury resulting from ischemia and reperfusion. In this context Bβ15–42 may act as a potent renoprotective agent by preserving the endothelial and vascular integrity

The early activation of toll-like receptor (TLR)-3 initiates kidney injury after ischemia and reperfusion

Acute kidney injury (AKI) is one of the most important complications in hospitalized patients and its pathomechanisms are not completely elucidated. We hypothesize that signaling via toll-like receptor (TLR)-3, a receptor that is activated upon binding of double-stranded nucleotides, might play a crucial role in the pathogenesis of AKI following ischemia and reperfusion (IR). Male adult C57Bl6 wild-type (wt) mice and TLR-3 knock-out (-/-) mice were subjected to 30 minutes bilateral selective clamping of the renal artery followed by reperfusion for 30 min 2.5h and 23.5 hours or subjected to sham procedures. TLR-3 down-stream signaling was activated already within 3 h of ischemia and reperfusion in post-ischemic kidneys of wt mice lead to impaired blood perfusion followed by a strong pro-inflammatory response with significant neutrophil invasion. In contrast, this effect was absent in TLR-3-/- mice. Moreover, the quick TLR-3 activation resulted in kidney damage that was histomorphologically associated with significantly increased apoptosis and necrosis rates in renal tubules of wt mice. This finding was confirmed by increased kidney injury marker NGAL in wt mice and a better preserved renal perfusion after IR in TLR-3-/- mice than wt mice. Overall, the absence of TLR-3 is associated with lower cumulative kidney damage and maintained renal blood perfusion within the first 24 hours of reperfusion. Thus, we conclude that TLR-3 seems to participate in the pathogenesis of early acute kidney injury

Inhibition of microRNA-92a protects against ischemia/reperfusion injury in a large-animal model

BACKGROUND: MicroRNAs (miRs) are small noncoding RNAs that posttranscriptionally control gene expression. Small-animal studies suggest that miRs might offer novel therapeutic targets in cardiovascular diseases such as cardioprotection of murine hearts after myocardial infarction via miR-92a inhibitors. Because the functional benefits of miR-92a inhibitors in larger preclinical models are not known, we assessed the therapeutic efficacy of miR-92a inhibition in a porcine model of ischemia and reperfusion. METHODS AND RESULTS: Pigs (n=5 per group) underwent percutaneous ischemia/reperfusion (60 min/72 h or 7 days, respectively). Locked nucleic acid-modified antisense miR-92a (LNA-92a) was applied either regionally (antegrade or retrograde) with a catheter or systemically (intravenously). LNA-92a significantly (P<0.01) reduced miR-92a expression in the infarct zone regardless of the application venue. However, catheter-based delivery, but not intravenous infusion, of LNA-92a significantly (P<0.05) reduced the infarct size compared with control LNA-treated pigs, which correlated with an improved ejection fraction and left ventricular end-diastolic pressure (P<0.05). Histochemistry revealed that LNA-92a increased capillary density but decreased leukocyte influx and cardiac cell death. Complete loss of miR-92a in mice attenuated the infarct-related myocardial dysfunction to a larger extent than cardiomyocyte-specific miR-92a deletion. In vitro, LNA-92a protected against hypoxia/reoxygenation-induced cardiomyocyte cell death. CONCLUSIONS: Regional LNA-92a delivery reduces miR-92a levels and infarct size and postischemic loss of function. LNA-92a exerts cell-protective, proangiogenic, and anti-inflammatory effects. miR-92a inhibition might be a novel therapeutic tool to preserve cardiac function after ischemia

MiR-92a^−/− mice survive at a reduced Mendelian ratio.

<p>(<b>A</b>) Representative agarose gel picture of PCR products (WT allele: 1718 bp; miR-92a knockout (KO) allele: 422 bp) for genotyping of WT, miR-92a^+/− and miR-92a^−/− mice. (<b>B</b>) MiR-92a expression in the heart of WT, miR-92a^+/− and miR-92a^−/− mice. (<b>C</b>) Expression of the miR-17∼92 cluster members miR-17, miR-18a, miR-19a, miR-20a and miR-19b in the heart of WT, miR-92a^+/− and miR-92a^−/− mice. (<b>D</b>) Observed as well as by Mendelian ratios predicted percentage of weaned WT, miR-92a^+/− and miR-92a^−/− mice derived from mating of miR-92a^+/− mice. Data are represented as mean ± SEM, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 by one-way ANOVA; ^#<i>P</i><0.05 by Clopper-Pearson interval, ^$<i>P</i><0.01 by chi-square test.</p

Inhibition of miR-92a improves re-endothelialization and prevents neointima formation following vascular injury

AIMS: MicroRNA (miR)-92a is an important regulator of endothelial proliferation and angiogenesis after ischaemia, but the effects of miR-92a on re-endothelialization and neointimal lesion formation after vascular injury remain elusive. We tested the effects of lowering miR-92a levels using specific locked nucleic acid (LNA)-based antimiRs as well as endothelial-specific knock out of miR-92a on re-endothelialization and neointimal formation after wire-induced injury of the femoral artery in mice. METHODS AND RESULTS: MiR-92a was significantly up-regulated in neointimal lesions following wire-induced injury. Pre-miR-92a overexpression resulted in repression of the direct miR-92a target genes integrin α5 and sirtuin1, and reduced eNOS expression in vitro. MiR-92a impaired proliferation and migration of endothelial cells but not smooth muscle cells. In vivo, systemic inhibition of miR-92a expression with LNA-modified antisense molecules resulted in a significant acceleration of re-endothelialization of the denuded vessel area. Genetic deletion of miR-92a in Tie2-expressing cells, representing mainly endothelial cells, enhanced re-endothelialization, whereas no phenotype was observed in mice lacking miR-92a expression in haematopoietic cells. The enhanced endothelial recovery was associated with reduced accumulation of leucocytes and inhibition of neointimal formation 21 days after injury and led to the de-repression of the miR-92a targets integrin α5 and sirtuin1. CONCLUSION: Our data indicate that inhibition of endothelial miR-92a attenuates neointimal lesion formation by accelerating re-endothelialization and thus represents a putative novel mechanism to enhance the functional recovery following vascular injury

Bβ_15–42 attenuates cellular damage and apoptosis after early renal ischemia and reperfusion.

<p>(<b>A</b>) Quantification of intact cells remaining within the groups (left graph) and quantification of the apoptosis rates (right graphs). (<b>B</b>) Representative micrographs of H&E (left micrographs) and TUNEL (right micrographs) stained kidney sections of Bβ_15–42 and untreated mice at 1 h and 3 h after IR.10 fields within the cortex and outer medulla in each group (n = 8) have been evaluated at 400fold magnification. Quantification was performed using an automatized Matlab script measuring proportional colored pixels in a defined relative area. For H&E staining (left graph) quantification was done by setting blue to the total number of colored pixels. For TUNEL staining (right graph) brown was set into relationship to blue colored pixels. Error bars represent means ± SEM.*P<0.05, ***P<0.001; one-way ANOVA followed by Bonferroni’s multiple comparison test.</p

Better preserved vascular function in Bβ_15–42 treated mice after IR.

<p>(<b>A</b>) Kidney perfusion was evaluated by determining the resistive index in %. (left graph) and blood velocity in mm/s (right graph). Dotted lines indicate upper and lower normal limits (left graph). Systolic (upper lines) and diastolic (lower lines) values are plotted (right graph). Measurements were performed on 10 s video sequences by evaluating at least 15flow signatures per animal. (<b>B</b>) Representative pictures of duplex sonography measurements performed pre-operatively, 1 h and 3 h after completed IR in NaCl (upper micrographs) and Bβ_15–42 (lower micrographs) treated mice.*Significantly different from mice treated with saline; ∫ significantly different from pre-operative measurements. Time points and error bars represent means ± SEM. ∫∫∫ and ***P<0.001; one-way ANOVA followed by Bonferroni’s multiple comparison test.</p

Significant preservation of VE-Cadherin in mice kidneys treated with Bβ_15–42.

<p>(<b>A</b>) Western blot immunoassays of renal homogenates on VE-Cadherin (left graph) and immunofluorescence double-staining of the area occupied by VE-Cadherin and CD13 (right graphs), respectively.10 fields within the cortex and outer medulla in each group (n = 8) have been evaluated at 400fold magnification. Data are expressed as the ratios of VE-Cadherin (green) positive vs. VE-Cadherin negative areas per field and CD13 (green) positive vs. CD13 negative areas per field. (<b>B</b>) Representative micrographs of selective immunofluorescence staining on VE-Cadherin (upper micrographs) and CD13 (lower micrographs) of all groups (sham, NaCl 1 h, Bβ 1 h, NaCl 3 h, and Bβ 3 h). Error bars represent means ± SEM. *P<0.05, **P<0.01, ***P<0.001. Values are expressed in % expression. One-way ANOVA followed by Bonferroni’s multiple comparison test.</p