1. The κ opioid system regulates endothelial cell differentiation and pathfinding in vascular development
by Kohei Yamamizu, Sadayoshi Furuta, Shiori Katayama, Michiko Narita, Naoko Kuzumaki, Satoshi Imai, Hiroshi Nagase, Tsutomu Suzuki, Minoru Narita, and Jun K. Yamashita
Blood
Volume 118(3):
July 21, 2011
©2011 by American Society of Hematology

2. Inhibitory effects of KOR agonists U50,488H and TRK820 on EC induction from Flk1+ cells.
Inhibitory effects of KOR agonists U50,488H and TRK820 on EC induction from Flk1+ cells. Flk1+ cells after 3 days of culture (Flk-d3). (A) Flow cytometry. X-axis: CD31; y-axis: side scatter (SSC). Percentages of CD31+ ECs among total Flk1+ cell–derived cells are indicated. (B) Quantitative evaluation of the effect of VEGF on CD31+ EC induction from Flk1+ cells by FACS. Percentages of CD31+ cell population among total Flk1+ cell–derived cells. Control (n = 7) and VEGF (50 ng/mL; n = 7) treatments are shown (**P < .01 vs control). (C) Double fluorescent staining for CD31 and αSMA at Flk-d3. Left, CD31 (pan-ECs, green) and DAPI (blue); middle, αSMA (MCs, red) and DAPI (blue); and right, merged. Flk1+ cells were stimulated with VEGF (50 ng/mL), DAMGO (10μM), SNC80 (10μM), U50,488H (10μM), or TRK820 (10μM) as indicated. Scale bars, 200 μm. (D) Flow cytometry. X-axis: CD31; y-axis: SSC. Percentages of CD31+ ECs among total Flk1+ cell–derived cells are indicated. (E) Percentages of CD31+ cell population among total Flk1+ cell–derived cells. Treatments with VEGF alone (50 ng/mL; n = 7), and VEGF plus DAMGO (1, 3, 10μM; n = 4), SNC80 (1, 3, 10μM), U50,488H (1, 3, 10μM; n = 4), or TRK820 (1, 3, 10μM; n = 4) are shown (**P < .01, *P < .05 vs VEGF). (F) CD31+ cell number that appeared from 1.5 × 105 of Flk1+ cells. (**P < .01, *P < .05 vs VEGF).
Kohei Yamamizu et al. Blood 2011;118:
©2011 by American Society of Hematology

3. Inhibitory effects of KOR agonists U50,488H and TRK820 on 3D vascular formation from Flk1+ cells.
Inhibitory effects of KOR agonists U50,488H and TRK820 on 3D vascular formation from Flk1+ cells. Three-dimensional culture of Flk1+ cell aggregates in type I collagen gel. (A) Phase contrast images after 5 days of culture. Flk1+ cells were stimulated with control, VEGF (50 ng/mL), DAMGO (10μM), SNC80 (10μM), U50,488H (10μM), or TRK820 (10μM) as indicated. Scale bars, 200 μm. (B) In-gel immunostaining for CD31 (green). Scale bars, 200 μm. (C) Quantitative analysis of CD31+ area in 3D vascular formation (except for the aggregate area). Control (n = 3), VEGF alone (50 ng/mL; n = 3), and VEGF plus DAMGO (10μM; n = 3), SNC80 (10μM; n = 3), U50,488H (10μM; n = 3), or TRK820 (10μM; n = 3) are shown (**P < .01, *P < .05 vs control). (D) Quantitative analysis of length in CD31+ sprouting vessels (excluding the aggregate area; **P < .01 vs VEGF).
Kohei Yamamizu et al. Blood 2011;118:
©2011 by American Society of Hematology

4. KOR was highly expressed in Flk1+ vascular progenitors.
KOR was highly expressed in Flk1+ vascular progenitors. (A) RT-PCR showing mRNA expression of MOR, DOR, and KOR in ES cells, Flk1+ cells, cells after 1 or 3 days of Flk1+ cell culture (Flk-d1 or Flk-d3), CD31-positive cells (ECs) and CD31-negative cells (MCs) at Flk-d3. (B) Fluorescent staining for MOR, DOR, and KOR at Flk-d1. Nuclei are stained with DAPI (blue). Left, MOR; middle, DOR; right, KOR. Scale bars, 100 μm. (C) Double fluorescent staining for MOR, DOR, and KOR with CD31 (red) at Flk-d3. Nuclei are stained with DAPI (blue). Top, opioid (green) receptors (green); middle, CD31 (red); bottom, merged. Scale bars, 100 μm.
Kohei Yamamizu et al. Blood 2011;118:
©2011 by American Society of Hematology

5. Activation of cAMP/PKA signaling rescues the inhibitory effects of KOR on EC induction from Flk1+ vascular progenitors.
Activation of cAMP/PKA signaling rescues the inhibitory effects of KOR on EC induction from Flk1+ vascular progenitors. (A) Double fluorescent staining for CD31 and αSMA at Flk-d3. Left, CD31 (pan-ECs, green) and DAPI (blue); middle, αSMA (MCs, red) and DAPI (blue); and right, merged. Flk1+ cells stimulated with VEGF (50 ng/mL), U50,488H (10μM), TRK820 (10μM), 8br-cAMP (0.5mM), and 6-Bnz-cAMP (0.1mM) as indicated. Scale bars, 200 μm. (B) Flow cytometry. X-axis: CD31; y-axis: SSC. Percentages of CD31+ ECs among total Flk1+ cell–derived cells are indicated. (C) Three-dimensional culture of Flk1+ cell aggregates in type I collagen gel. In-gel immunostaining for CD31 (green). Scale bars, 200 μm.
Kohei Yamamizu et al. Blood 2011;118:
©2011 by American Society of Hematology

6. VEGF receptors Flk1 and NRP1 were decreased by KOR activation in Flk1+ vascular progenitors.
VEGF receptors Flk1 and NRP1 were decreased by KOR activation in Flk1+ vascular progenitors. (A) Quantitative RT-PCR showing mRNA expression at Flk-d1. Control mRNA expression was set as 1.0. Flk1+ cells were stimulated with control, VEGF (50 ng/mL), DAMGO (10μM), SNC80 (10μM), U50,488H (10μM), TRK820 (10μM), 8br-cAMP (0.5mM), and 6-Bnz-cAMP (0.1mM) as indicated (**P < .01 vs VEGF). (B) Fluorescent staining for Flk1 (green) at Flk-d1. Nuclei are stained with DAPI (blue). Scale bars, 200 μm. (C) Flow cytometry at Flk-d1. X-axis: Flk1; y-axis: SSC. Percentages of Flk1+ cells among vascular progenitor-derived cells are indicated. (D) Fluorescent staining for NRP1 (green) at Flk-d1. Nuclei are stained with DAPI (blue). Scale bars, 200 μm.
Kohei Yamamizu et al. Blood 2011;118:
©2011 by American Society of Hematology

7. KOR or Dynorphin KO mice show increased vascular formation in brain and intersomitic vessels.
KOR or Dynorphin KO mice show increased vascular formation in brain and intersomitic vessels. (A) Representative results of WT, PDYN KO, and KOR KO mouse embryo at E10.5. Whole-mount CD31 (red) staining. Left, WT mice. Pn, perineural vascular plexus; Isv, intersomitic vessels. Middle, PDYN KO mice. Right, KOR KO mice. Scale bars, 2 mm. (B) High-magnification views of CD31-stained Pn region. Scale bars, 200 μm. (C) Greater magnification views corresponding to boxed regions in panel B. Scale bars, 40 μm. (D) Quantitative evaluation of CD31+ area in Pn. CD31 staining of WT mice was set as 1.0. (n = 3, **P < .01 vs WT). (E) Flow cytometry. X-axis: VE-cadherin; y-axis: CD31. Percentages of CD31+/VE-cadherin+/CD45− ECs in the embryo are indicated. (F) Quantitative evaluation of CD31+/VE−cadherin+/CD45− ECs in the embryo. (n = 3, *P < .05 vs WT). (G) Quantitative RT-PCR showing mRNA expression in purified CD31+/VE-cadherin+/CD45− ECs in the embryo. WT mRNA expression was set as 1.0. (n = 3, **P < .01 vs WT). (H) High-magnification views of CD31-stained Isv region in E10.5 embryo. Ectopic invasion of Isv into somite was observed in both KO mice. Scale bars, 100 μm. (I) Quantitative RT-PCR showing mRNA expression in purified CD31+/VE−cadherin+/CD45− ECs in the embryo. WT mRNA expression was set as 1.0. (n = 3, **P < .01 vs WT).
Kohei Yamamizu et al. Blood 2011;118:
©2011 by American Society of Hematology

8. Molecular mechanisms of inhibition of vascular formation and pathfinding by KOR signaling activation.
Molecular mechanisms of inhibition of vascular formation and pathfinding by KOR signaling activation. KOR signaling induced by dynorphin activates cAMP/PKA pathway. This pathway reciprocally regulates expression of VEGF receptors, Flk1 and Neuropilin1, and plexinD1 in vascular progenitors. Expression balance of these receptors controls vascular formation and pathfinding during vascular development.
Kohei Yamamizu et al. Blood 2011;118:
©2011 by American Society of Hematology