miR-29b protects fibrotic responses in NIH/3T3 cells induced with TGF-β without activating TLRs.

<p>(A) <i>Col1a1</i> mRNA expression in NIH/3T3 cells was reduced after transfection with miR-29b Psh-match compared with cells treated only with TGF-β. The degree of reduction in <i>COL1A1</i> expression induced by miR-29b Psh-match transfection was greater than that induced by either miR-29b scrambled control or miR-29b mimic (Fig 2A) (*P<0.05). (B) miR-29b Psh-match does not induce TLR signaling activity. TLR signaling activity was measured using an NF-κB reporter in which luciferase cDNA was placed under the control of the κB response element. The dsRNA and ssRNA induce the activation of NF-κB through TLR3 and TLR7, respectively.</p

Bleomycin-induced pulmonary fibrosis in mouse lungs.

<p>(A) Images of lungs from mice that were either subject to nasal administration of methylene blue (right) or not (left). (B) Fold changes in <i>Col1a1</i> mRNA showed a peak one week after bleomycin administration. (*P<0.05) (C) Histological staining by HE and Masson’s trichrome showed pronounced diffuse fibrosis in the lungs three weeks after bleomycin administration.</p

miR-29b Psh-match suppresses pulmonary fibrosis in mice to a greater degree than miR-29b mimic.

<p>(A) In bleomycin-induced pulmonary fibroblast model mice, quantitative real-time PCR data indicates an increase in <i>Col1a1</i> mRNA expression. Expression of <i>Col1a1</i> decreased in the lungs of mice administered with miR-29b Psh-match relative to mice administered with either PBS, miR-29b scrambled control, or miR-29b mimic (*P<0.05). (B) HE and Masson’s trichrome staining showed the pronounced diffuse fibrosis on the lungs in the bleomycin-treated mice. Administration of miR-29b Psh-match suppressed pulmonary fibrosis in an established mouse model of bleomycin-induced pulmonary fibrosis (*P<0.05). High magnification images of the lungs of mice administered with either miR-29b mimic or Psh-match mice are shown in the frames. (C) Hydroxyproline content was measured as micrograms of hydroxyproline per weight in grams of the left lung’s tissues in mice.</p

Summary of clinical detalis of acute leukemia patients used for analysis.

<p>Summary of clinical detalis of acute leukemia patients used for analysis.</p

MicroRNA expression in leukemic cells.

<p><i>In situ</i> hybridization was performed using LNA probes for miR-92a and negative control. Blue signals represent positive for the microRNAs. Bars indicate 50 µm.</p

Expression profiling of microRNAs in normal plasmas.

<p>A. MicroRNA expression profiles in seven normal samples by microRNA microarray analysis. Y axis represents relative intensity of hybridization signals. B. Comparison of the signal intensities of various microRNAs among normal plasmas. The signal intensity of each microRNA by microarray analysis is evaluated as a rank order among the detected microRNAs. Y axis represents log₁₀ (Rank of signal intensity in each sample/average rank of that in all samples).</p

Comparison of microRNA expressions in the plasmas of normal and acute leukemia.

<p>A. Comparison of signal intensity ranks of various microRNAs in normal (n = 7) and leukemia (n = 2) plasmas by microarray analysis. Y axis represents mean ratio of the signal intensity rank of leukemia to the rank of normal of each microRNA. B. Comparison of the ratio of miR92a signal intensity to miR-638 signal intensity by <i>Taq</i>Man qRT-PCR among the plasmas of normal and leukemia. Mann-Whitney's U test was used to determine statistical significance.</p

Angiopoietin-Like 7 Is an Anti-Angiogenic Protein Required to Prevent Vascularization of the Cornea

<div><p>Purpose</p><p>We sought to identify the anti-angiogenic molecule expressed in corneal keratocytes that is responsible for maintaining the avascularity of the cornea.</p><p>Methods</p><p>Human umbilical vein endothelial cells (HUVECs) were cultured with either human dermal fibroblasts or with human corneal keratocytes under serum-free conditions. The areas that exhibited blood vessel formation were estimated by immunostaining the cultures with an antitibody against CD31, a blood vessel marker. We also performed microarray gene-expression analysis and selected one molecule, angiopoietin-like 7 (ANGPTL7) for further functional studies conducted with the keratocytes and <i>in vivo</i> in mice.</p><p>Results</p><p>Areas showing blood vessel formation in normal serum-free medium were conditions were markedly smaller when HUVECs were co-cultured with corneal keratocytes than when they were co-cultured with the dermal fibroblasts under the same conditions. Microarray analysis revealed that <i>ANGPTL7</i> expression was higher in keratocytes than in dermal fibroblasts. <i>In vitro</i>, inhibiting <i>ANGPTL7</i> expression by using a specific siRNA led to greater tube formation than did the transfection of cells with a control siRNA, and this increase in tube formation was abolished when recombinant <i>ANGPTL7</i> protein was added to the cultures. <i>In vivo</i>, intrastromal injections of an <i>ANGPTL7</i> PshRNA into the avascular corneal stroma of mice resulted in the growth of blood vessels.</p><p>Conclusions</p><p><i>ANGPTL7</i>, which is abundantly expressed in keratocytes, plays a major role in maintaining corneal avascularity and transparency.</p></div

PshRNA platform.

<p>PshRNA was synthesized in solid phase as single-stranded RNAs that, following synthesis, self-anneal into a unique helical structure containing a single stem loop.</p

<i>ANGPTL7</i> siRNA tranfection increased tube formation in HCK-HUVEC co-culture.

<p><i>ANGPTL7</i> mRNA expression was drastically inhibited following the administration of <i>ANGPTL7</i> siRNA for up to 240 h after transfection (A). The tube-formation areas under the normal serum-free medium (normal) and control siRNA treatment were 0.13 ± 0.15% and 0.31 ± 0.09%, respectively (B). In the cells transfected with the <i>ANGPTL7</i> siRNA the tube formation areas increased significantly (1.41 ± 0.54%, <i>p</i> < 0.001, B). This siRNA effect was potently suppressed when human recombinant ANGPTL7 (A7 siRNA+rhANGPTL7) was added to the cells (0.69 ± 0.30%, <i>p</i> < 0.001, B). N = 12 per condition. In the cells in which <i>ANGPTL7</i> expression was suppressed, the mRNA expression levels of <i>ANGPTL1</i> and <i>ANGPTL4</i> were also decreased (by 48.4% and 56.1%, respectively), but the expression of <i>ANGPTL2</i> was increased (119%). N = 2 per each condition.</p