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Figures
- Fig. 1. Endothelial colony-forming cells (ECFCs) accumulate the anandamide (AEA) analog SKM4-45-1 in a time and concentration dependent manner.
(A) Representative images of phase contrast (upper images) and SKM4-45-1-derived fluorescence in ECFCs (green, lower images) at incubation time-points 0, 10, 20 and 30 min (at 20× magnification). (B) Kinetics of SKM-45-1 uptake in ECFCs. Increase in fluorescence was documented every 10 sec throughout 30 min. (n = 8; ± SEM). (C) Concentration response of SKM4-45-1 uptake in ECFCs. Increase in fluorescence intensity with various concentrations of SKM4-45-1 (0.1, 0.3, 1, 3, 10, 30 µM) after 30 min (± SEM) of incubation in % of the maximal fluorescence intensity is blotted (n = 3).
- Fig. 2. ECFCs express various (endo)cannabinoid receptors (CBR) of those the pharmacological inhibition exhibits different effects on the uptake of the AEA analog SKM4-45-1.
(A) Western blots representing protein levels of CB1R, CB2R, G-protein coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and β-Actin in ECFCs and EAhy.926 (EA) cells. (B) Columns represent SKM 4-45-1 (1 µM) uptake in ECFCs with vehicle or antagonists against CB1R (AM251, 0.1 µM; n = 3), CB2R (SR144528, 1 µM; n = 4), GPR55 (CID16020046, 20 µM; n = 4) and TRPV1 (SB366791, 10 µM; n = 4) after 30 minutes of incubation. Values show % fluorescence as compared to vehicle control (± SEM); *** indicates p ≤ 0.001 significance as compared to vehicle control, # indicates p ≤ 0.01 significance as compared to other treatments.
- Fig. 3. SKM4-45-1 uptake in ECFCs is diminished by either the inhibition of TRPV1, the TRPV1 agonist capsaicin, and a siRNA-mediated down-regulation of TRPV1.
(A) Comparison of the inhibitory effects of the TRPV1 antagonist SB366791 and the TRPV1 agonist capsaicin. SKM4-45-1 (1 µM) was added to ECFCs treated with vehicle, antagonist (SB366791, 10 µM; n = 4) or agonist (Capsaicin, 0.1 µM; n = 3) of TRPV1. Values show % fluorescence as compared to vehicle control (± SEM); *** indicates p ≤ 0.001 significance as compared to vehicle control. (B) Genetic knock-down of TRPV1 mRNA levels. EA.hy926 cells were treated with siRNA against TRPV1. Effectiveness of knock-down was verified by RT-PCR (left panel) and quantitative real time (q) PCR (right panel). Values show % TRPV1 corrected for GAPDH as compared to nonspecific siRNA controls (n = 3; ± SEM); *** indicates p ≤ 0.001 significance as compared to siRNA control. (C) Genetic knock-down of TRPV1 protein level. EA.hy926 cells were treated with non-specific siRNA or siRNA against TRPV1. Effectiveness of knock-down was verified by western blot using antibodies against TRPV1 and β-Actin (left panel), as indicated, and quantified by ImageJ® (right panel). Values show % TRPV1 corrected for β-Actin as compared to untreated and non-specific siRNA controls (n = 3; ± SEM); *** indicates p ≤ 0.001 significance as compared to control. (D) Columns represent SKM4-45-1 uptake in untreated control cells and EA.hy926 cells treated with non-specific siRNA or siRNA against TRPV1, as indicated. SKM4-45-1 fluorescence was measured 30 minutes after incubation (n = 3; ± SEM); *** indicates p ≤ 0.001 significance as compared to control.
- Fig. 4. Overexpression of TRPV1 leads to an increase in SKM4-45-1 uptake in human endothelial cells.
(A) Verification of TRPV1 mRNA overexpression in EA.hy926 cells. Cells were transfected with a TRPV1 encoding plasmid conjugated to a red fluorescence protein (TRPV1-RFP) or an empty vector control. Effectiveness of overexpression was verified by RT-PCR (left panel) and quantitative real time (q) PCR (right panel). Values show % TRPV1 corrected for GAPDH as compared to empty vector (vector control) (n = 3; ± SEM); *** indicates p ≤ 0.001 significance as compared to control. (B) Verification of TRPV1 protein overexpression in EA.hy926 cells. Effectiveness of TRPV1-RFP overexpression was verified by western blot using antibodies against TRPV1 and β-Actin, as indicated, and quantified by ImageJ® (right panel). Values show % TRPV1 corrected for β-Actin as compared to untreated and empty vector controls (n = 3; ± SEM); *** indicates p ≤ 0.001 significance as compared to control. (C) Single cell fluorescence images of SKM4-45-1 accumulation in control EA.hy926 cells and cells overexpressing TRPV1-RFP (at 100× magnification). RFP expression (left panel; red signal) and SKM4-45-1 uptake (middle panel; green signal) was measured after 30 minutes incubation and single channel pictures where overlaid (right panel). (D) Columns represent SKM4-45-1 accumulation in untransfected EA.hy926 cells, vector control cells and cells overexpressing TRPV1-RFP. SKM4-45-1 uptake was measured after 30 minutes incubation. Values show % fluorescence as compared to untransfected controls (n = 3; ± SEM); *** indicates p ≤ 0.001 significance as compared to untransfected control. (E) Columns represent SKM4-45-1 accumulation in control EA.hy926 cells, cells overexpressing TRPV1 in the presence of SB366791. SKM4-45-1 uptake in untransfected, empty vector control transfected and TRPV1-RFP transfected EA.hy926 was measured after 30 minutes simultaneous incubation of 1 µM SKM4-45-1 with 10 µM SB366791. Values show % fluorescence as compared to untransfected controls (n = 3; ± SEM). There is no significant difference to untransfected controls. # indicates p ≤ 0.01 significant decrease as compared to TRPV1-RFP transfected cells without SB366791 treatment.
- Fig. 5. ECFC proliferation is promoted equally by SKM4-45-1 and AEA which is strongly reduced by inhibition of TRPV1.
(A) Quantification of cell proliferation in response to anandamide (AEA) and SKM4-45-1. ECFCs where treated with or without 1 µM AEA (n = 10), 1 µM SKM4-45-1 (n = 3) (fluorescent AEA analogue) or same amounts vehicle. ECFC cell numbers were counted after 48 h treated with agonists. Values represent cell numbers (×103; ± SEM); *** indicates p ≤ 0.001 significance as compared to vehicle control. Representative respective pictures of ECFC cell culture after 48 h treatment with vehicle (left picture), 1 µM AEA (middle picture) or 1 µM SKM4-45-1 (right picture) (at 10× magnification). (B) Concentration response curve of AEA on ECFC proliferation. ECFCs where treated with different concentrations of AEA (0.001; 0.01; 0.1 or 1 µM). Proliferation increase was compared to vehicle control (n = 3; ± SEM). (C) Columns represent cell proliferation increase. ECFCs were treated with vehicle (light grey bar) or anandamide (AEA, 1 µM; n = 10) (dark grey bar), with or without an antagonist against TRPV1 (SB366791, 10 µM; n = 3), as indicated. Effects are presented in % as compared to vehicle control (± SEM); *** indicates p ≤ 0.001 significance as compared to vehicle control. # indicates p ≤ 0.001 significance as compared to AEA treated cells without SB366791 treatment. (D) Columns represent cell proliferation increase of knock-down cells. ECFCs were treated with vehicle (light grey bar) or anandamide (AEA, 1 µM; n = 10) (dark grey bar), with or without transfection with non-specific siRNA or siRNA against TRPV1, as indicated. Effects are presented in % as compared to vehicle control (± SEM); *** indicates p ≤ 0.001 significance as compared to individual vehicle control. # indicates p ≤ 0.001 significance as compared to AEA treated cells without siRNA against TRPV1.
- Fig. 6. AEA-induced ECFC network-formation is efficiently reduced by the TRPV1 antagonist SB366791.
(A) Representative pictures of ECFC network-formations after 12 h treatment with vehicle, anandamide (AEA, 1 µM; n = 10) or AEA + TRPV1-inhibitor (SB366791, 10 µM; n = 3), as depicted (at 10× magnification). (B) Columns represent number of branch-points formed by ECFCs. ECFCs were treated with vehicle (light grey bar) or anandamide (AEA, 1 µM; n = 10) (dark grey bar), with or without TRPV1 inhibitor (SB366791, 10 µM; n = 3), as indicated. Branch-point numbers were counted by ImageJ® (± SEM); *** indicates p ≤ 0.001 significance as compared to vehicle control. # indicates p ≤ 0.001 significance as compared to AEA only treatment.
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