Cytokine-based expansion of bone marrow-derived murine DCs.

<p>Bone marrow (from female C3H/He)-derived lineage-negative cells (CD45R⁻, CD5⁻, CD11b⁻, Gr-1⁻, TER119⁻, and 7/4⁻) were enriched using a SpinSep mouse hematopoietic progenitor enrichment kit. These cells were subjected to progenitor expansion by various cytokines under a floating condition in an MPC treated flask. At each time point, the culture medium was replaced with new DC-differentiation medium (RPMI 1640 containing GM-CSF/IL-4) for 1 week. The data are expressed as the means<u>+</u>SEM. a. Growth curve of hematopoietic progenitor cells (HPs). Neither FLT3-L, SCF, nor IL-6 could stimulate the growth of HPs over 10 days. Only the use of IL-3 was associated with long-term growth, and this growth was greatly accelerated when a mixture of cytokines was used (FS36). Note the log scale on the HP cell number. b. FACS analyses indicating the time course of the scatter plot of HPs (left row), in populations of CD11c⁺CD11b⁺ cells at pre- and post-treatment with GM-CSF/IL-4 (middle two 2-center rows), and of the expression of c-Kit/CD131 (a receptor of GM-CSF/IL-3) and CD11c/CD11b in the R1-gated increasing population indicated in SSC/FSC (right two rows at right). These experiments were performed in triplicate, and showed similar results. c. Summary of triplicate FACS experiments representing the time course of positive cell ratios for CD11b, c-Kit, and CD131 of expanded HPs. Note the culture duration-dependent increase of c-Kit⁺ and CD131⁺ HPs. d. Bar graph indicating the relative increase of CD11b⁺CD11c⁺ DC-like cells produced after 1-week cultivation in GM-CSF/IL-4 using expanded HPs at various time points. The data was gathered from three independent experiments. Note that a period of 3 weeks was [optimal][most efficient], yielding a more than 3-log increase in the production of CD11b⁺CD11c⁺ DC-like cells.</p

Assessment of functions that are typically seen in DCs.

<p>a. FITC-dextran uptake assay assessing endo-/phagocytotic activity, a typical feature of antigen-presenting cells such as DCs. This experiment was performed in triplicate, and showed similar results. b. A graph showing MLR activity for allo-antigen (C57BL6) by iDCs or activated DCs (C3H/He) by LPS derived from the conventional technique or expansion of HPs. c. Antitumor effect of subcutaneous vaccination with rSeV/dF-activated DCs that were derived from conventional or expansion techniques. Female C3H/He mice (7-week-old) were subcutaneously vaccinated via the left flank three times each weeks with 1×10⁶ conventional/expanded DCs pulsed with tumor lysate. Two days after the final vaccination, 1×10⁶ LM8 cells were inoculated intradermally into the left flank of mice. Note that 3-weeks expanded HPs treated with rSeV/dF did not show any effect on tumor growth. d. Antimetastatic activity via bolus intravenous injection of various DCs. Female C3H/He mice (7-week-old) were intravenously vaccinated with 1×10⁶ conventional/expanded DCs once via the tail vein, and 2 days later, 1×10⁶ of LM8 cells were inoculated intravenously. Seventeen days later, mice were sacrificed and the macroscopically recognized nodules on the surface of the bilateral lungs were counted.</p

<i>In vitro</i> characterization of expanded murine DCs.

<p>a. Schematic diagram of expansion/differentiation/maturation/activation sequences and microscopic morphology of conventional and expanded DCs that were stimulated by LPS. Note that typical dendrites were found in both DCs. b. FACS analyses assessing the expression of typical surface markers. Conventional and expanded DCs after treatment with GM-CSF/IL-4 without further stimulus were subjected to FACS analyses. c. Expression of typical murine inflammatory cytokines/chemokines of conventional (open bars) and expanded (black bars) DCs in response to various stimuli for RIG-I helicase (rSeV) or Toll-like receptors (LPS for TLR-4, poly I:C for TLR-3, CpG-DNA for TLR-9, and R848 for TLR-7). The upper three panels (mIL-6, mIFN-β, and mIL-12/p70) were assessed by ELISA, and contain data from three independent experiments, and the bottom three panels (mIL-6, mMCP-1/JE, and mTNF-α) were performed using a Cytometric Bead Array (CBA) system and show one typical result taken from three independent experiments.</p

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

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

<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

<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

<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

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