Increased ischemia-induced angiogenesis by <i>in vivo</i> shRNA targeting <i>Sprouty2 and Sprouty4</i>.

<p>(A) Representative laser Doppler images for each group are depicted. Arrowheads indicate ischemic limbs. The interval of low perfusion is displayed as dark blue; the highest perfusion interval is displayed as red. (B) Recovery of limb perfusion in C57BL/6J mice (8 weeks old) injected with the control shRNA (n = 10) or <i>Sprouty2/Sprouty4</i> shRNA vectors (n = 12) after hind limb ischemia as assessed by laser Doppler blood flow analysis on day 14. Data shown are means±SD. *: <i>P</i><0.05. (C) Blood vessels (green) in the non-ischemic or ischemic adductor muscle injected with the control shRNA or <i>Sprouty2/Sprouty4</i> shRNA vectors stained with anti-PECAM-1/CD31Ab. Nuclei were stained with Hoechst 33342 dye (blue). The CD31-positive vessel area was quantified. Data shown are means±SEM. *: <i>P</i><0.05. Scale bars (C): 100 µm.</p

<i>In vivo</i> effects of shRNA targeting <i>Sprouty2</i> and <i>Sprouty4</i> in corneal micropocket assay.

<p>(A) Corneal neovascularization was induced by mouse VEGF-A (200 ng) on day 12 after hydron pellets had been implanted into male BALB/c mouse corneas. After implantation, 10 µg shRNA plasmids per eye were delivered by subconjunctival injection. Representative photos are shown. (B) Quantitative analysis of neovascularization on day 12. Areas are expressed in mm². Bars show the mean±SEM (n = 5). *: <i>P</i><0.05. (C) Sections of corneas implanted with VEGF-A stained by anti-PECAM-1/CD31Ab on day 12. Scale bars (C): 100 µm.</p

Characterization of <i>Sprouty2/Sprouty4</i> DKO mice.

<p>(A, B) Gross appearance of wild-type (WT) (A) and <i>Sprouty2/Sprouty4</i> DKO (B) embryos at embryonic day 12.5. The arrow and arrowheads indicate hemorrhage and edema, respectively. (C, D) Hematoxylin-eosin (H&E) staining of sections of WT (C) and <i>Sprouty2/Sprouty4</i> DKO (D) skin. (E, F) H&E staining and immunohistochemical staining with von Willebrand factor (vWF) of sections of hepatic hemangiomas in <i>Sprouty2/Sprouty4</i> DKO liver. vWF was used as a blood vessel marker. (G) Expression of <i>Sproutys</i> in endothelial cells. About 5.0×10⁴ BECs and LECs were FACS-sorted at embryonic day 14.5, and were used for RT-PCR analysis. <i>GAPDH</i> served as a loading control. Good separation of BECs and LECs was confirmed by BEC markers (<i>Nrp1</i>, <i>CD44</i>) and LEC markers (<i>LYVE1</i>, <i>Prox1</i>). Scale bars (C–F): 100 µm.</p

Blood and lymphatic vessels of <i>Sprouty4</i> single KO mice.

<p>(A) Blood vessels (green) and lymphatic vessels (red) in the ears of WT and <i>Sprouty4</i> KO mice (8 weeks old) were analyzed by whole-mount immunohistochemical staining with anti-PECAM-1/CD31Ab and anti-LYVE-1 Ab, respectively. (B) CD31-positive vessel area or LYVE1-positive area was quantified. Data shown are means±SEM. *: <i>P</i><0.05. (C) Blood vessels (green) and lymphatic vessels (red) in the dorsal skin of WT and <i>Sprouty4</i> KO mice (8 weeks old) were analyzed by immunohistochemical staining with anti-PECAM-1/CD31Ab and anti-LYVE-1 Ab, respectively. Nuclei were stained with Hoechst 33342 dye (Blue). (D) CD31-positive vessel area or LYVE1-positive area was quantified. Data shown are means±SEM. *: <i>P</i><0.05. (E) FITC-dextran-perfused flat-mounted retinal samples of WT and <i>Sprouty4</i> KO mice at postnatal day 3. Scale bars (A, C): 100 µm.</p

<i>In vivo</i> effects of shRNA targeting <i>Sprouty2</i> and <i>Sprouty4</i>.

<p>(A) The <i>in vivo</i> effects of shRNA plasmids targeting <i>Sproutys</i> in the hind limb model were evaluated by RT-PCR analysis. (B, C) Real-time PCR analysis of <i>Sprouty2</i> (B) or <i>Sprouty4</i> (C) mRNA expression in MEFs stably infected with control retroviruses and retroviruses expressing either <i>Sprouty2</i> shRNA (B) or <i>Sprouty4</i> shRNA (C). (D, E) Western blot analysis of protein extracts from MEFs stably infected with control retroviruses and retroviruses expressing either <i>Sprouty2</i> shRNA (D) or <i>Sprouty4</i> shRNA (E). The relative intensities of Sprouty2 and Sprouty4 bands normalized by STAT5 expression levels are shown above. (F) Effect of both <i>Sprouty2</i> and <i>Sprouty4</i> knockdown on ERK and Akt activities. MEFs stably expressing VEGFR-2 were infected with control retroviruses and retroviruses expressing <i>Sprouty2/Sprouty4</i> shRNA, and stimulated with 100 ng/mL VEGF-A. Cell extracts were immunoblotted with the indicated antibodies.</p

<i>Sprouty4</i> KO mice are also more resistant in a soft tissue ischemia model.

<p>(A) Representative photos of ischemic dorsal skin of male WT and <i>Sprouty4</i> KO mice (8–10 weeks old). Arrows indicate necrotic skin. (B) Left: Blood vessels (green) in the ischemic dorsal skin of male WT and <i>Sprouty4</i> KO mice were analyzed by immunohistochemical staining with anti-PECAM-1/CD31Ab. Nuclei were stained with Hoechst 33342 dye (blue). Right: The CD31-positive vessel area was quantified. Data shown are means±SEM. *: <i>P</i><0.05. Scale bars (B): 100 µm.</p

<i>Sprouty4</i> KO mice are more resistant in a hind-limb ischemia model.

<p>(A) Representative photos of ischemic limbs, indicated by arrows. (B) Representative laser Doppler images for each group are depicted. Arrowheads indicate ischemic limbs. The interval of low perfusion is displayed as dark blue; the highest perfusion interval is displayed as red. (C) Recovery of limb perfusion in WT (n = 10) and <i>Sprouty4</i> KO (n = 7) mice after hind limb ischemia as assessed by laser Doppler blood flow analysis on day 14. Data shown are means±SD. *: <i>P</i><0.001. (D) Blood vessels (green) in the non-ischemic or ischemic adductor muscles of male WT and <i>Sprouty4</i> KO mice (8–10 weeks old) were analyzed by immunohistochemical staining with anti-PECAM-1/CD31Ab. Nuclei were stained with Hoechst 33342 dye (blue). The CD31-positive vessel area was quantified. Data shown are means±SEM. *: <i>P</i><0.05. Scale bars: (D) 100 µm.</p

pDLCs expressed mDC specific antigens.

<p>pDLCs were cultured with Flt3-Ligand GM-CSF for six days. (A) CD123, CD11c and CD56 expressions were analyzed at day 0 and 6. CD123^highCD11c^dim and CD123^dimCD11c^high populations were observed at day 6. Results of one representative experiment are presented (left). The average frequencies of CD123^highCD11c^dim and CD123^dimCD11c^high populations are indicated (n = 5, middle). The average MFI of CD56 at day 0 and 6 are indicated (n = 5, right). * P < 0.05 compared to day0. (B) BDCA1 expression was compared by flow cytometry. pDLCs did not express BDCA1 at day 0, but they did express BDCA1 at day 6 especially CD123^dimCD11c^high population. * P < 0.05 compared to day0 (n = 5). (C) Sorted cells were CFSE-labeled and incubated for six days with Flt3-L and GM-CSF. The filled histograms represent PHA-stimulated CFSE-labeled CD4⁺ T cells that were included as a control for cell division (left). The average frequencies of divided cells are indicated (n = 5, right).</p

Immunophenotypic characteristics of pDLCs.

<p>Expression of cell-surface and intracellular markers in pDLCs, mDLCs, pDCs, and mDCs were measured by flow cytometry. Upper graph showed the results of one representative experiment. pDLCs (solid line), pDCs (dotted line), mDCs (dashed line) and isotype control (filled histogram). Lower graph showed mean fluorescence intensity of pDLC, mDLC, pDC and mDC (n=5). * P < 0.05 compared to pDLC.</p

pDLCs are a rare DC population that are morphologically similar to pDCs.

<p>(A) PBMNCs were analyzed using flow cytometry. The gated Lin^-HLA−DR⁺ population (left) was divided according to CD56 expression (middle), and these two subpopulations were analyzed according to CD123 and CD11c expression to define pDLCs, mDLCs, pDCs, and mDCs (right). The results of one representative experiment are presented. (B) The frequency of pDLC, mDLCs, pDCs, and mDCs in PBMCs (n = 5). pDLCs comprised a very small (0.03%) proportion in PBMCs. (C) The pDLC to pDC ratio was compared between peripheral blood (PB) and bone marrow (BM) cells (n=5). *P < 0.05 compared to PB. (D) MFI of CD123 (left panel) and CD11c (right panel) expression was measured on pDLCs, pDCs, and mDCs in PBMCs (n = 5). *P < 0.05 compared to pDLC. (E) Sorted cells were stained with May–Giemsa solution and observed by light microscopy at 1000× magnification.</p