Suppression of Sproutys Has a Therapeutic Effect for a Mouse Model of Ischemia by Enhancing Angiogenesis

Sprouty proteins (Sproutys) inhibit receptor tyrosine kinase signaling and control various aspects of branching morphogenesis. In this study, we examined the physiological function of Sproutys in angiogenesis, using gene targeting and short-hairpin RNA (shRNA) knockdown strategies. Sprouty2 and Sprouty4 double knockout (KO) (DKO) mice were embryonic-lethal around E12.5 due to cardiovascular defects. The number of peripheral blood vessels, but not that of lymphatic vessels, was increased in Sprouty4 KO mice compared with wild-type (WT) mice. Sprouty4 KO mice were more resistant to hind limb ischemia and soft tissue ischemia than WT mice were, because Sprouty4 deficiency causes accelerated neovascularization. Moreover, suppression of Sprouty2 and Sprouty4 expression in vivo by shRNA targeting accelerated angiogenesis and has a therapeutic effect in a mouse model of hind limb ischemia. These data suggest that Sproutys are physiologically important negative regulators of angiogenesis in vivo and novel therapeutic targets for treating peripheral ischemic diseases

Preoperative estimation of distance between retinal break and limbus with wide-field fundus imaging: Potential clinical utility for conventional scleral buckling.

ObjectiveAccurate scleral marking of retinal breaks is essential for successful scleral buckling. This study aimed to investigate the use of wide-field fundus images obtained with an Optos for preoperative estimation of the distance from the limbus to the retinal breaks.Methods and analysisThis is a retrospective review of 29 eyes from 26 patients with rhegmatogenous retinal detachment who received scleral buckling with anatomically successful repair. They underwent wide-field fundus photography with Optos California. In the pre- and postoperative fundus images, we measured distances from the macula to the retinal tears (TM), to the center of the vortex veins (VM), to the optic disc (DM), and to the posterior edge of the scleral buckle (BM).Results(BM-VM) / DM was significantly correlated with the distance from the limbus to the posterior edge of the scleral buckle that had been determined intraoperatively. (r = 0.705; pConclusionPreoperative analysis of Optos images may be useful for estimating the distance from the limbus to retinal breaks, which might aid scleral marking during scleral buckling surgery

Spreds Are Essential for Embryonic Lymphangiogenesis by Regulating Vascular Endothelial Growth Factor Receptor 3 Signaling▿

Spred/Sprouty family proteins negatively regulate growth factor-induced ERK activation. Although the individual physiological roles of Spred-1 and Spred-2 have been investigated using gene-disrupted mice, the overlapping functions of Spred-1 and Spred-2 have not been clarified. Here, we demonstrate that the deletion of both Spred-1 and Spred-2 resulted in embryonic lethality at embryonic days 12.5 to 15.5 with marked subcutaneous hemorrhage, edema, and dilated lymphatic vessels filled with erythrocytes. This phenotype resembled that of Syk−/− and SLP-76−/− mice with defects in the separation of lymphatic vessels from blood vessels. The number of LYVE-1-positive lymphatic vessels and lymphatic endothelial cells increased markedly in Spred-1/2-deficient embryos compared with WT embryos, while the number of blood vessels was not different. Ex vivo colony assay revealed that Spred-1/2 suppressed lymphatic endothelial cell proliferation and/or differentiation. In cultured cells, the overexpression of Spred-1 or Spred-2 strongly suppressed vascular endothelial growth factor-C (VEGF-C)/VEGF receptor (VEGFR)-3-mediated ERK activation, while Spred-1/2-deficient cells were extremely sensitive to VEGFR-3 signaling. These data suggest that Spreds play an important role in lymphatic vessel development by negatively regulating VEGF-C/VEGFR-3 signaling

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

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

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