cell culture medium mv2  (PromoCell)

Journal: Cardiovascular Research

Article Title: Role of R-spondin 2 in arterial lymphangiogenesis and atherosclerosis

doi: 10.1093/cvr/cvaa244

Figure Lengend Snippet: RSPO2 inhibits VEGF-C-stimulated AKT and eNOS activation in LEC. (A–G) LEC were serum-starved in 0.5% FBS containing basal media MV2 for 16 h. Then, LEC were pretreated with RSPO2 (6 h), stimulated with VEGF-C for 15 min, and subjected to western blot analysis. (A and E) Representative western blot images are shown. (B–D and F) Bar diagrams represent averaged protein levels expressed as a ratio of phospho to total proteins, eNOS (B), AKT (C), ERK1/2 (D), and PKCδ (F) (n = 6–8). (G) Bar diagram represents the mean expression of VEGFR3 normalized with GAPDH (n = 6). (H–J) Control and LGR4-silenced LEC were used for this experiment. The details of treatment are same as in (A). (H) Representative western blot images are shown. (I and J) Bar diagrams represent averaged protein levels expressed as a ratio of phospho to total proteins, eNOS (I) (n = 4) and AKT (J) (n = 5). Statistical analyses were performed using one-way ANOVA (B–D, F, and G) and two-way ANOVA (I and J). Data represent mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. eNOS, endothelial nitric oxide synthase; LEC, lymphatic endothelial cells; RSPO2, R-spondin 2; VEGF, vascular endothelial growth factor.

Article Snippet: Cell culture Primary human dermal LEC (PromoCell GmbH, Heidelberg, Germany) were cultured in endothelial cell growth medium MV 2 (PromoCell) containing 5% heat-inactivated foetal bovine serum (FBS), 100 IU/mL of penicillin, 100 μg/mL streptomycin, and growth factors bullet kit provided by PromoCell.

Techniques: Activation Assay, Western Blot, Expressing, Control

Journal: Cardiovascular Research

Article Title: Role of R-spondin 2 in arterial lymphangiogenesis and atherosclerosis

doi: 10.1093/cvr/cvaa244

Figure Lengend Snippet: Supplementation of nitric oxide by SNP abrogates RSPO2’s anti-lymphangiogenic activity. (A) Vehicle or RSPO2-pretreated LEC (6 h) were stimulated with VEGF-C for 1 h, washed, incubated with DAF-FM diacetate, and fluorescence determined (excitation/emission 495/515 nm). L-NAME pretreated cells were used as negative controls. Data are representative of three independent experiments performed in triplicate. (B) LEC were treated with vehicle or RSPO2 for 30 min, incubated with H2DCFDA and fluorescence analysed using flow cytometry. Representative histograms showing H2DCFDA fluorescence are shown. The X-axis is logarithmic. Bar diagram indicates mean fluorescence intensity in different groups (n = 4). (C) Human LEC were used to extract RNA and qRT-PCR was performed to determine levels of Nox1, Nox2, Nox4, and Nox5 expression. GAPDH was used as an internal control. Bar graph represents mRNA levels of different Nox isoforms in comparison to Nox2 (gene with the lowest expression). Data are representative of three independent experiments performed in triplicate. (D and E) LEC were pretreated with vehicle, SNP (1 µM, D) or EUK-134 (100 nM, E) for 30 min, then incubated with RSPO2 (6 h), stimulated with VEGF-C for 48 h and proliferation investigated using MTT assay. Data are representative of three to four independent experiments performed at least in triplicate. (F) LEC pretreated with vehicle or SNP (30 min) were incubated with RSPO2 for 6 h, trypsinized, and seeded in wells of Matrigel-coated plate in basal medium containing VEGF-C, VEGF-C + RSPO2, and VEGF-C + RSPO2 + SNP, and tube formation determined (6 h). Representative photomicrographs are shown. Scale bar 200 µm. Bar diagrams indicate tube length and number of branching points (n = 6–7). (G) Matrigel plugs mixed with VEGF-C, VEGF-C + RSPO2, or VEGF-C + RSPO2 + SNP were implanted subcutaneously in wild-type mice. Plugs were isolated after 2 weeks, sectioned and immunostained for LYVE-1. Bar diagram shows quantitative analysis of LYVE-1 positive area in implanted Matrigel plugs (n = 5–8). Statistical analyses were performed using one-way ANOVA (A and C–G) and two-tailed unpaired t-test (B). Data represent mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. L-NAME, L-NG-Nitro arginine methyl ester; LEC, lymphatic endothelial cells; LYVE-1, lymphatic vessel endothelial hyaluronan receptor-1; RSPO2, R-spondin 2; SNP, sodium nitroprusside; VEGF, vascular endothelial growth factor.

Article Snippet: Cell culture Primary human dermal LEC (PromoCell GmbH, Heidelberg, Germany) were cultured in endothelial cell growth medium MV 2 (PromoCell) containing 5% heat-inactivated foetal bovine serum (FBS), 100 IU/mL of penicillin, 100 μg/mL streptomycin, and growth factors bullet kit provided by PromoCell.

Techniques: Activity Assay, Incubation, Fluorescence, Flow Cytometry, Quantitative RT-PCR, Expressing, Control, Comparison, MTT Assay, Isolation, Two Tailed Test

Journal: Cardiovascular Research

Article Title: Role of R-spondin 2 in arterial lymphangiogenesis and atherosclerosis

doi: 10.1093/cvr/cvaa244

Figure Lengend Snippet: RSPO2 suppresses lymphangiogenesis in vitro and in vivo. (A) Human LEC were pretreated with vehicle or RSPO2 (100 ng/mL) in basal media MV2 containing 0.5% FBS for 6 h, stimulated with VEGF-C (100 ng/mL) and proliferation investigated after 48 h using MTT assay. Data are representative of six independent experiments performed at least in triplicate. (B) LEC grown on coverslips were pretreated with vehicle or RSPO2 for 6 h and stimulated with VEGF-C for 24 h. Cells were fixed and immunostained for Ki67 (red). Nuclei and actin filaments were counterstained with To-Pro 3 (blue) and phalloidin (green), respectively. Images were captured from five to nine random fields. Representative images are shown. Scale bar 50 µm. Bar graph represents the mean number of Ki67 positive cells/field (n = 4). (C) Vehicle or RSPO2-pretreated LEC were seeded in upper chambers of transwell plate in basal medium containing VEGF-C ± RSPO2 and migration investigated after 12 h. Images of seven to nine randomly selected fields were acquired and number of migrated cells counted. Representative photomicrographs are shown. Scale bar 500 µm. (D) Bar graph represents the number of migrated cells (n = 5). (E–G) Vehicle or RSPO2-pretreated LEC were seeded in wells of a Matrigel-coated plate in basal medium containing VEGF-C ± RSPO2 and tube formation determined (6 h). Representative photomicrographs are shown. Scale bar 500 µm (E). Images of random fields were taken, and tube length (F) and number of branching points (G) quantified (n = 8). (H) Matrigel plugs mixed with either VEGF-C or VEGF-C + RSPO2 were implanted subcutaneously in wild-type mice. Plugs were isolated after 2 weeks, sectioned and immunostained for LYVE-1. (I) Quantitative analysis of LYVE-1 in implanted Matrigel plugs. Bar diagram represents mean LYVE-1 positive area (n = 6–8). Statistical analyses were performed using a two-tailed unpaired t-test. Data represent mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. LYVE-1, lymphatic vessel endothelial hyaluronan receptor-1; RSPO2, R-spondin 2; VEGF, vascular endothelial growth factor.

Article Snippet: Cell culture Primary human dermal LEC (PromoCell GmbH, Heidelberg, Germany) were cultured in endothelial cell growth medium MV 2 (PromoCell) containing 5% heat-inactivated foetal bovine serum (FBS), 100 IU/mL of penicillin, 100 μg/mL streptomycin, and growth factors bullet kit provided by PromoCell.

Techniques: In Vitro, In Vivo, MTT Assay, Migration, Isolation, Two Tailed Test

Journal: Cardiovascular Research

Article Title: Role of R-spondin 2 in arterial lymphangiogenesis and atherosclerosis

doi: 10.1093/cvr/cvaa244

Figure Lengend Snippet: RSPO2 impairs Wnt-β-catenin signalling in VEGF-C-stimulated LEC. (A) HEK293 were treated with RSPO2 for 6 h and subjected to western blot analysis for LRP6 phosphorylation (left panel). LEC were serum-starved in 0.5% FBS containing basal media MV2 for 16 h. Then, LEC were pretreated with RSPO2 (6 h), stimulated with VEGF-C for 15 min, and subjected to western blot (right panel). (B) Bar diagram represents mean protein levels expressed as a ratio of phospho to total protein (n = 5). (C) LEC grown on coverslips were pretreated with vehicle or RSPO2 (6 h) and stimulated with VEGF-C for 15 min. Cells were fixed and immunostained for β-catenin (green). Nuclei were counterstained with Hoechst 33342 (red). At least five images of randomly chosen microscopic fields were captured. Representative images are shown. Scale bar 20 µm. Enlarged images; scale bar 10 µm. (D) Bar graph represents the mean co-localization coefficient for Hoechst 33342 and β-catenin (n = 14–17). (E) LEC were treated as indicated, nuclear-cytoplasmic fractionation conducted, and western blot experiments performed using nuclear and cytoplasmic fractions of LEC. GAPDH was used as cytoplasmic control. TBP and histone 3 were used to determine the purity of nuclear preparations. Bar diagram represents mean β-catenin levels in cytoplasmic and nuclear fractions (n = 4). Statistical analyses were performed using one-way ANOVA (B, D, and E). Data are presented as mean ± SEM. *P < 0.05 and **P < 0.01. L-NAME, L-NG-Nitro arginine methyl ester; LEC, lymphatic endothelial cells; RSPO2, R-spondin 2; TBP, TATA-binding protein; VEGF, vascular endothelial growth factor.

Article Snippet: Cell culture Primary human dermal LEC (PromoCell GmbH, Heidelberg, Germany) were cultured in endothelial cell growth medium MV 2 (PromoCell) containing 5% heat-inactivated foetal bovine serum (FBS), 100 IU/mL of penicillin, 100 μg/mL streptomycin, and growth factors bullet kit provided by PromoCell.

Techniques: Western Blot, Phospho-proteomics, Fractionation, Control, Binding Assay