Skip to main content
. Author manuscript; available in PMC: 2017 Feb 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2015 Dec 3;36(2):350–360. doi: 10.1161/ATVBAHA.115.306713

Figure 3. LRP1 knockdown in HRECs promotes angiogenesis, endothelial proliferation and cell cycle progression.

Figure 3

A, LRP1 protein level decreases in HRECs that were transfected with LRP1 siRNA, compared to control siRNA. Lysates of LRP1-knockdown or control HRECs treated were analyzed by Western blotting to detect LRP1 β chain at 85 kDa. B–C, LRP1 knockdown in HRECs increases tube formation. HRECs were incubated in normoxia (21% O2) or hypoxia (2% O2) condition for 24 hours on Matrigel coated plates. Phase contrast images were used for quantitative measurements of tube numbers per sample and shown in B. *, P<0.05 compared to the same HRECs at normoxia condition, #, P<0.05 compared to the control siRNA-treated HRECs at hypoxia condition, n=3. Analysis was two-way ANOVA followed by Bonferroni multiple comparison test. D–E, LRP1 knockdown in HRECs increases sprouting angiogenesis. Spheroid angiogenesis assays were performed with HRECs that were transfected with LRP1 or control siRNAs. Hypoxia (2% O2) was used to induce sprout formation. Images of HRECs spheroids demonstrate the formation of sprouts following 72 hours of hypoxia (2% O2) incubation. The number of sprouts per spheroid were counted and quantified in E. *, P<0.05 via unpaired Student’s t-test compared to control cells, n=6. F, LRP1 knockdown in HRECs increases hypoxia-induced cell growth. Cell numbers were counted daily in HRECs that were transfected with LRP1 or control siRNAs, and subsequently cultured in normoxia or hypoxia (2% O2) for 4 days. *, P<0.05, compared to same HRECs at day 0. #, P<0.05, compared to control siRNA-transfected HRECs that were incubated at hypoxia condition. n= 4. Analysis was two-way ANOVA followed by Bonferroni multiple comparison test. G–H, LRP1 knockdown increases cell cycle progression to S phase from G1/G0 stage. HRECs were transfected with LRP1 or control siRNA. Two days later, cells were incubated under normoxia (N, 21% O2) or hypoxia (H, 2% O2) for 24 hours. Cells were then stained with propidium iodide and analyzed by flow cytometry. The representative flow cytometry images are shown in G, and the percentages of cells at different cell cycle stages were quantified and present in H. *, P<0.05, compared to same cells under normoxia. #, P<0.05, compared to control siRNA-transfected HRECs under hypoxia. n=3. Analysis was two-way ANOVA followed Fisher’s LSD multiple comparison test.