Inhibition of angiogenesis in endothelial cells by Human Lysyl oxidase propeptide

Figures

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  Figure 1. LOX-PP protein purification and antibody production: (a) SDS-PAGE of purified LOX-PP using Ni-NTA agarose (Lane-M: Mw Marker, lane-1: Crude, lane-2: Unbound, lane-3 to 8: washes 1 to 6, lane-9 to 13: Elution −1 to 5). (b) Western blot for His-tag and LOX-PP in purified protein (M - Mw marker, lane-1: purified protein stained with coomassie stain). The corresponding full length blots are represented in Supplementary Fig. 21. (c) Western blot of purified protein post His-tag cleavage using factor Xa protease (Lane-1: His-tag uncleaved, Lane-2: His-tag cleaved. The corresponding full length blots are represented in Supplementary Figs 22 and 23. (d) Mass spectrum of the purified LOX-PP and its protein coverage map. (e) Direct ELISA for LOX-PP with purified antibody showing the affinity of the raised antibody with purified LOX-PP protein. (f) SDS-PAGE of purified LOX-PP antibody using two different volumes (Lane-1: 2.5 μl, Lane-2: 5.0 μl) and stained with coomassie stain to show heavy chain (HC) at 55 kDa and light chain (LC) at 25 kDa.

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  Figure 2. Transient transfection of LOX-PP, confirmation by western blot and mass spectrometry: (a) mRNA expression of LOX-PP overexpressed in HUVECs after 48 h (b) After 48 h of transfection, cell extracts were collected and probed using LOX-PP antibody by western blot and was normalized with β- actin (EV- empty vector, LOX-PP-LOX-PP transfected cells). The corresponding full-length blots are represented in Supplementary Figs 3 and 4. (c) Conditioned medium was collected and it was immunoprecipitated using LOX-PP antibody, immunoprecipitated samples were subjected to western blot using antibody against LOX-PP and coomassie staining was considered as loading control, the corresponding full-length blots are represented in Supplementary Figs 5 and 6. (d) Mass spectrum of LOX-PP in cell lysate. (e) Mass spectrum of LOX-PP in conditioned medium. (f) Immunofluorescence for LOX-PP in HUVECs after overexpression (Magnification 63x).Cells were stained with Alexa 488 and counterstained with DAPI.

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  Figure 3. Effect of LOX-PP overexpression on HUVEC’s migration and tube formation: Overexpression of LOX-PP inhibits (a) cell migration and (b) Bar graph represents the quantification of scratch assay (c) tubule formation (d) Bar graph represents the quantification of tube formation assay. Values were expressed as mean ± SD, n = 3. ***p < 0.001, *p < 0.05 versus EV. (EV – Empty vector).

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  Figure 4. Proteomic analysis of LOX-PP overexpression in HUVECs: (a) Reactome and KEGG pathway analysis of the 372 uniquely expressed proteins in empty vector by ConsensusPathDB online tool (http://cpdb.molgen.mpg.de/) with p < 0.01, which showed components of VEGF signaling, MAPK signaling, RAF signaling and FGF signaling to be significantly increased. (b) Reactome and KEGG pathway analysis of the 75 uniquely expressed proteins in LOX-PP overexpressed cells by ConsensusPathDB online tool (http://cpdb.molgen.mpg.de/) with p < 0.01, which showed components of apoptosis, cell cycle and G2M check points to be increased. (c) Reactome and KEGG pathway analysis of proteins that are downregulated by LOX-PP overexpression. (d) Reactome and KEGG pathway analysis of proteins that are upregulated by LOX-PP overexpression.

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  Figure 5. Effect of LOX-PP on VEGF induced proliferation and cell cycle in HUVECs: (a) Bar graph represents the quantification of proliferation assay using BrdU ELISA kit. (b) Propidium iodide staining and flow cytometry analysis was performed to determine the percentage of cells in G1, S and G2 phase of cell cycle. (c) Bar graph represents the quantification of cell cycle analysis. (d) Bar graph represents the quantification of apoptosis assay using Cell death ELISA kit. Values were expressed as mean ± SD, n = 3. ***p < 0.001 versus VEGF + EV. #p < 0.05 versus CTRL. (CTRL – Control; EV – Empty vector).

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  Figure 6. Effect of LOX-PP on VEGF induced cell migration in HUVECs: (a) For scratch assay HUVEC cells were induced with 10 ng/mL of VEGF, 10 ng/mL of VEGF + EV and 10 ng/mL of VEGF + LOX-PP for 18 h and were imaged using Axio observer microscope at 5x magnification. (b) Bar graph represents the quantification of scratch assay. (c) For transwell migration assay HUVEC cells were induced with 10 ng/mL of VEGF, 10 ng/mL of VEGF + EV and 10 ng/mL of VEGF + LOX-PP for 8 h and were imaged using Axio observer microscope at 5x magnification. (d) Bar graph represents the quantification of transwell migration assay. Cells were counted using ImageJ software (expressed as % of controls). (e) HUVEC cells were induced with 10 ng/mL VEGF, 10 ng/mL VEGF + Vehicle ctrl and 10 ng/ml of VEGF + 2.5 μg/ml of rLOX-PP for 18 h and were imaged using Axio observer microscope at 5 x magnification. (f) Bar graph represents the quantification of scratch assay. Values were expressed as mean ± SD, n = 3. ***p < 0.001 versus VEGF + EV. ***p < 0.001 versus VEGF + Vehicle ctrl. #p < 0.05 versus CTRL. (CTRL – Control; EV – Empty vector; Vehicle ctrl – Vehicle control).

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  Figure 7. Effect of LOX-PP on VEGF induced cell attachment and reorganization of F-actin: (a) Bar graph represents the quantification of cell attachment after overexpression of LOX-PP, attached cells were counted using ImageJ software (expressed as % of controls). (b) Bar graph represents the quantification of cell attachment after addition of rLOX-PP, attached cells were counted using ImageJ software (expressed as % of controls). (c) Inhibitory effect of overexpressed LOX-PP on the reorganization of actin cytoskeleton induced by VEGF (10 ng/mL) under LSM 880 confocal laser scanning microscope. HUVECs were fixed and stained for filamentous actin with phalloidin FITC conjugate. Cells were also transfected with EV. Nucleus is counter-stained with DAPI. Values were expressed as mean ± SD, n = 3. **p < 0.01 versus VEGF + EV. **p < 0.01 versus VEGF + Vehicle ctrl. #p < 0.05 versus CTRL. (CTRL – Control; EV – Empty vector; Vehicle ctrl – Vehicle control).

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  Figure 8. Effect of LOX-PP on VEGF induced tube formation in HUVECs: (a) HUVEC cells were induced with 10 ng/mL VEGF, 10 ng/mL of VEGF + EV and 10 ng/mL of VEGF + LOX-PP for 4 h and were imaged using Axio observer microscope at 5x magnification. (b) Bar graph represents the quantification of tube length measured using ImageJ software (expressed as % of controls). (c) HUVEC cells were induced with 10 ng/mL VEGF, 10 ng/mL VEGF + Vehicle ctrl, 10 ng/mL of VEGF + 1.0 μg/ml of rLOX-PP and 10 ng/ml of VEGF + 2.5 μg/ml of rLOX-PP for 4 h and were imaged using Axio observer microscope at 5x magnification. (d) Bar graph represents the quantification of tube length measured using ImageJ software (expressed as % of controls). Values were expressed as mean ± SD, n = 3. ***p < 0.001 versus VEGF + EV. **p < 0.01 versus VEGF + Vehicle ctrl. #p < 0.05 versus CTRL. (CTRL – Control; EV – Empty vector; Vehicle ctrl – Vehicle control).

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  Figure 9. Inhibitory effect of LOX-PP on FAK (Tyr397) and ERK (Thr202/Tyr204) phosphorylation induced by VEGF in HUVECs: (a) HUVECs were transfected with LOX-PP and EV. After transfection, cells were treated with 10 ng/mL of VEGF for 15 min. Whole cell extract were prepared and the samples were subjected to western blotting using antibodies against pFAK (Tyr397), total FAK and β–actin was used as loading control. The full-length blots are represented in Supplementary Figs 7, 8 and 11. (b) HUVECs were transfected with LOX-PP and EV. After transfection, cells were treated with 10 ng/mL of VEGF for 15 min. Whole cell extract were prepared and the samples were subjected to western blotting using antibodies against pERK (Thr202/Tyr204), total ERK and β–actin was used as loading control. The full-length blots are represented in Supplementary Figs 9, 10 and 11. (c) Bar graph represents quantification of the western blot using ImageJ software for FAK (Tyr397). (d) Bar graph represents quantification of the western blot using ImageJ software for ERK (Thr202/Tyr204). Values were expressed as mean ± SD, n = 3. **p < 0.01, *p < 0.05 versus VEGF + EV. #p < 0.05 versus CTRL. (CTRL – Control; EV – Empty…

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  Figure 10. Effect of rLOX-PP on FAK (Tyr397) and ERK (Thr202/Tyr204) phosphorylation induced by VEGF in HUVECs: (a) HUVECs were treated with rLOX-PP and vehicle ctrl. After treatment, cells were treated with 10 ng/mL of VEGF for 15 min. Whole cell extract were prepared and the samples were subjected to western blotting using antibodies against pFAK (Tyr397), total FAK, pERK (Thr202/Tyr204), total ERK and β–actin was used as loading control. The full-length blots are represented in Supplementary Figs 12, 13, 14, 15 and 16. (b) Bar graph represents the quantification of western blot using ImageJ software for pFAK (Tyr397) and (c) pERK (Thr202/Tyr204). (d) HUVECs were treated with rLOX-PP without stimulation of VEGF. Cell lysate were subjected to western blotting using pFAK (Tyr397), total FAK, pERK (Thr202/Tyr204), total ERK and β–actin. The full-length blots are represented in Supplementary Figs 12, 13, 14, 15 and 16. (e) Densitogram of the western blot shows the ratio of pFAK to total FAK, normalized with β-actin. (f) Densitogram of the western blot shows the ratio of pERK to total ERK, normalized with β-actin. Values were expressed as mean ± SD, n = 3. **p < 0.01, *p < 0.05 versus VEGF +…

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  Figure 11. Effect of rLOX-PP in in vivo angiogenesis CAM assay: (a) Representative photographs showing the rLOX-PP (2.5 μg/ mL) treated CAM, vehicle ctrl treated CAM and 10 ng/ml of VEGF treated CAM (positive control) at 0 h, 2 h and 4 h. (b) Quantification of the blood vessels in terms of measuring tubule length and (c) tubule junction. Vessel length and junction were noted (indicated in arrow) in each CAM and changes are shown in percentage in relation to the vehicle ctrl. Values are expressed as mean ± SD, n = 3. **p < 0.01 and *p < 0.05 versus Vehicle ctrl. *p < 0.05 versus 10 ng/mL of VEGF. (Vehicle ctrl – Vehicle control).

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  Figure 12. Uptake and localization of rLOX-PP in HUVECs. (a) LOX-PP protein expression was increased upon addition of rLOX-PP in HUVECs when compared with vehicle ctrl. The full-length blots are represented in Supplementary Figs 17 and 18. (b) Densitogram of the western blot shows LOX-PP protein normalized with β-actin. (c) Western blot for His-tag indicates rLOX-PP with His-tag which was added exogenously to HUVECs was found in the cell lysate indicating its uptake. The full-length blots are represented in Supplementary Figs 19 and 20. (d) Densitogram of the western blot shows His-tagged LOX-PP protein expression normalized with β-actin. Values were expressed as mean ± SD, n = 3. **p < 0.01 versus vehicle ctrl. (Vehicle ctrl – Vehicle control). (e) Increased LOX-PP expression is observed upon addition of rLOX-PP to HUVECs under LSM 880 confocal laser scanning microscope. Cells were stained with Alexa 488 and counterstained with DAPI.