An in vivo chemical library screen in Xenopus tadpoles reveals novel pathways involved in angiogenesis and lymphangiogenesis by Roland E. Kälin, Nadja E. Bänziger-Tobler, Michael Detmar, and André W. Brändli Blood Volume 114(5):1110-1122 July 30, 2009 ©2009 by American Society of Hematology

A chemical library screening strategy to identify small-molecule modulators of vascular development in Xenopus embryos. A chemical library screening strategy to identify small-molecule modulators of vascular development in Xenopus embryos. Xenopus embryos were arrayed in multiwell plates, and single compounds from the LOPAC1280 chemical library were added to the water in each well. Embryos were screened visually for developmental defects (edema and/or late lethality). Positive hits are verified by repeating the phenotypic assay. Compounds interfering with vascular development are identified by whole-mount in situ hybridization of compound-treated Xenopus embryos. Roland E. Kälin et al. Blood 2009;114:1110-1122 ©2009 by American Society of Hematology

Compounds affecting distinct aspects of blood vessel development in vivo. Compounds affecting distinct aspects of blood vessel development in vivo. Compound-treated (A-N) and control (DMSO-treated) Xenopus embryos (O-P) were analyzed by whole-mount in situ hybridization for expression of the vascular marker gene apj. Stage 35/36 embryos are shown in lateral views with anterior to the left. Close-up views of the trunk illustrate the morphology of the blood vessels. Compound names are indicated. (A-D) Hypoplastic VVN (*) and PCV (arrowheads). (E-F) Lack of ISVs (arrowheads). Note that assembly of PCVs and VVN (*) is unaffected. (G-H) Ectopic ISVs (arrowheads) and dysplastic VVN (*). (I-J) Ectopic ISV (arrowheads) and hyperplastic VVN (*). (K-N) Hypoplastic, dispersed VVN (*), but normal ISV angiogenesis (arrowheads). (O-P) Control embryos with normal VVN (*), PCVs (arrow), and ISVs (arrowhead). (Q) Scheme of the blood vasculature (blue) of the stage 35/36 embryo. Calmidazolium indicates calmidazolium chloride; Indirubin, indirubin-3′-oxime; and PS, pronephric sinus. Roland E. Kälin et al. Blood 2009;114:1110-1122 ©2009 by American Society of Hematology

Compounds affecting blood and lymph vessel development in Xenopus tadpoles. Compounds affecting blood and lymph vessel development in Xenopus tadpoles. Control DMSO- (A-B) and compound-treated (C-J) Xenopus embryos were analyzed by whole-mount in situ hybridization for expression of the blood vascular marker gene apj at stage 35/36 and the lymphatic marker gene vegfr3 at stage 42. Panels of the embryonic blood vasculature (apj) are accompanied by close-up views illustrating ISV angiogenesis and VVN development in the embryonic trunk. The panels visualizing the developing lymphatic system (vegfr3) include close-ups of the head and midtrunk region (middle panels) for the ALSs and the ALHs, and enlargements (left panels) of the tail for PLVs. (A) Normal ISVs (arrowhead) and VVN (*). (B) Normal ALSs (arrow), ALHs (*), and PLVs (arrowheads). (C) Stunted ISVs (arrowhead), normal VVN (*). (D) Hypoplastic PLV (arrowheads), impaired ALS (arrow), and ALH (*) lymphatics. (E) Stunted ISVs (arrowhead), hypoplastic VVN (*). (F) Stunted ALS lymphatics (arrow), dysplastic ALH lymphatics (*), hypoplastic PLVs (arrowheads). (G) Normal ISVs (arrowhead), hyperplastic VVN. (H) Hypoplastic PLVs (arrowheads), impaired ALS (arrow), and ALH (*) lymphatics. (I) Stunted ISVs (arrowhead), hyperplastic VVN. (J) Complete lack of ALS (arrow), ALH (*) and tail lymphatics. Note that vegfr3 expression persists in the VVN (arrowhead). (K) Scheme of the blood vasculature (blue) of the stage 35/36 embryo. (L) Scheme of the lymphatic vasculature (orange) of the stage 42 embryo. 7-Cyclo indicates 7-cyclopentyl-5-(4-phenoxy)phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine; Naphthalimide, 4-amino-1,8-naphthalimide; Naphthyridine, 7-chloro-4-hydroxy-2-phenyl-1,8-naphthyridine; PS; pronephric sinus; DPLV, dorsal posterior lymph vessel; and VPLV, ventral posterior lymph vessel. Roland E. Kälin et al. Blood 2009;114:1110-1122 ©2009 by American Society of Hematology

Compounds specifically affecting lymph vessel development in Xenopus tadpoles. Compounds specifically affecting lymph vessel development in Xenopus tadpoles. Control DMSO- (A-B) and compound-treated (C-N) Xenopus embryos were analyzed by whole-mount in situ hybridization as described in the legend to Figure 3. Close-up views of apj-stained embryos illustrate normal ISV angiogenesis and VVN development in the embryonic trunk. ALSs, ALHs, and PLVs are highlighted by arrows, asterisks, and arrowheads, respectively. (A-B) Normal ALS and ALH lymphatics, and PLVs. (C-D) Dysplastic ALH lymphatics. (E-F) Dysplastic ALS and ALH lymphatics, and hypoplastic PLVs. Note persistent vegfr3 expression in the VVN (black arrowhead). (G-H) Stunted ALS lymphatics, dysplastic ALH lymphatics, and hypoplastic PLVs. (I-J) Impaired ALS lymphangiogenesis, stunted ALH lymphatics, and hypoplastic PLVs. (K-N) Impaired ALS and ALH lymphangiogenesis, and hypoplastic PLVs. (O) Scheme of the lymphatic vasculature (orange) of the stage 42 embryo. DPLV indicates dorsal posterior lymph vessel; and VPLV, ventral posterior lymph vessel. Roland E. Kälin et al. Blood 2009;114:1110-1122 ©2009 by American Society of Hematology

Selective and cell-type specific in vitro responses of endothelial cells to treatment with small-molecule compounds. Selective and cell-type specific in vitro responses of endothelial cells to treatment with small-molecule compounds. Twenty-four compounds were tested in vitro on human lymphatic (LECs; black bars) and blood vascular (HUVECs; open bars) endothelial cell cultures for effects on cell proliferation and tube formation. (A) Results of the compound screens using cell proliferation assays. Compounds were screened at a dose of 10 μM in 0.1% DMSO. Control cultures were treated with 0.1% DMSO. (B) Results of the compound screens using tube formation assays. Compounds were screened at a dose of 1 μM in 0.1% DMSO. Control cultures were treated with 0.1% DMSO. 7-Cyclo indicates 7-cyclopentyl-5-(4-phenoxy)phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine; L-687,384, L-687,384 hydrochloride; Naphthalimide, 4-amino-1,8-naphthalimide; Naphthyridine, 7-chloro-4-hydroxy-2-phenyl-1,8-naphthyridine; Nicardipine, nicardipine hydrochloride; Phenylxanthine, 1,3-diethyl-8-phenylxanthine. Bars represent mean values and SD of 3 independently performed assays. Roland E. Kälin et al. Blood 2009;114:1110-1122 ©2009 by American Society of Hematology

Differential, cell-type specific effects of selected small-molecule compounds on endothelial tube formation. Differential, cell-type specific effects of selected small-molecule compounds on endothelial tube formation. Confluent monolayers of HUVEC or LECs were overlaid with collagen type I gels containing the indicated compounds at a dose of 1 μM in 0.1% DMSO. Scale bars represent 100 μm. Roland E. Kälin et al. Blood 2009;114:1110-1122 ©2009 by American Society of Hematology

The adenosine A1 receptor antagonist naphthyridine inhibits VEGFA-induced angiogenesis and lymphatic vessel enlargement in mice. The adenosine A1 receptor antagonist naphthyridine inhibits VEGFA-induced angiogenesis and lymphatic vessel enlargement in mice. VEGFA-containing Matrigel plugs were implanted subcutaneously into adult mice, and the mice were subsequently treated systemically with naphthyridine or with vehicle control for 6 days. (A-C) Differential immunofluorescence analysis for the lymphatic-specific marker LYVE1 (green, examples highlighted by arrows) and the pan-vascular marker CD31 (red) demonstrated lymphatic vessel enlargement and enhanced numbers of blood vessels in the skin surrounding VEGFA-containing Matrigels (B), compared with Matrigels containing PBS (A). Treatment with naphthyridine resulted in a reduction of blood vessel numbers and lymphatic vessel enlargement (C). (D-G) Quantitative image analyses confirmed that the density of lymphatic vessels was unchanged by VEGFA alone or by combined VEGFA and naphthyridine treatment (D). In contrast, the tissue area covered by lymphatic vessels surrounding VEGFA-containing Matrigels was significantly reduced by treatment with naphthyridine (E). Treatment with naphthyridine also reduced the number of VEGFA-induced blood vessels (F) and the tissue area covered by blood vessels (G). Scale bars represent 100 μm. Roland E. Kälin et al. Blood 2009;114:1110-1122 ©2009 by American Society of Hematology