Src Plays an Important Role in AGE-Induced Endothelial Cell Proliferation, Migration, and Tubulogenesis

Abstract

Advanced glycation end products (AGEs), produced by the non-enzymatic glycation of proteins and lipids under hyperglycemia or oxidative stress conditions, has been implicated to be pivotal in the development of diabetic vascular complications, including diabetic retinopathy. We previously demonstrated that Src kinase played a causative role in AGE-induced hyper-permeability and barrier dysfunction in human umbilical vein endothelial cells (HUVECs). While the increase of vascular permeability is the early event of angiogenesis, the effect of Src in AGE-induced angiogenesis and the mechanism has not been completely revealed. Here, we investigated the impact of Src on AGE-induced HUVECs proliferation, migration, and tubulogenesis. Inhibition of Src with inhibitor PP2 or siRNA decreased AGE-induced migration and tubulogenesis of HUVECs. The inactivation of Src with pcDNA3/flag-Src^K298M also restrained AGE-induced HUVECs proliferation, migration, and tube formation, while the activation of Src with pcDNA3/flag-Src^Y530F enhanced HUVECs angiogenesis alone and exacerbated AGE-induced angiogenesis. AGE-enhanced HUVECs angiogenesis in vitro was accompanied with the phosphorylation of ERK in HUVECs. The inhibition of ERK with its inhibitor PD98059 decreased AGE-induced HUVECs angiogenesis. Furthermore, the inhibition and silencing of Src suppressed the AGE-induced ERK activation. And the silencing of AGEs receptor (RAGE) inhibited the AGE-induced ERK activation and angiogenesis as well. In conclusions, this study demonstrated that Src plays a pivotal role in AGE-promoted HUVECs angiogenesis by phosphorylating ERK, and very likely through RAGE-Src-ERK pathway.

Keywords: ERK; Src; advanced glycation end products; angiogenesis; endothelial cells.

FIGURE 1

Src inhibitor PP2 attenuated AGE-enhanced HUVECs proliferation, migration, and tube formation. HUVECs were incubated with 15 μmol/L PP2 for 90 min and then exposed to 100 μg/mL AGEs for 24 h. CCK-8 was used to evaluate the proliferation of HUVECs (A). Scratch wound healing (B) and transwell migration assay (C) were used to measure the migration of HUVECs. The tube length, numbers of branch points, and vessel area were observed in Matrigel medium (D). n ≥ 4 independent experiments. ^∗P < 0.05 versus control, ^#P < 0.05 versus AGEs.

FIGURE 2

Src siRNA weakened AGE-induced endothelial angiogenesis. HUVECs were transfected with Src siRNA or control siRNA for 48 h and then treated with 100 μg/mL AGEs for 24 h. CCK-8 was used to evaluate the proliferation of HUVECs (A). Scratch wound healing (B) and transwell migration assay (C) were used to measure the migration of HUVECs. The tube length, numbers of branch points, and vessel area were observed in Matrigel medium (D). n ≥ 4 independent experiments. ^∗P < 0.05 versus control, ^#P < 0.05 versus AGEs, ^∗∗P < 0.05 versus Control siRNA.

FIGURE 3

Tyrosine kinase activity of Src was associated with AGE-induced endothelial angiogenesis. HUVECs were transfected with a kinase-deficient mutant at Lys298 (K298M) and an active mutant at Tyr530 (Y530F), also with a Mock as plasmid control. 48 h after transfection, HUVECs were incubated with 100 μg/mL AGEs for 24 h. CCK-8 was used to evaluate the proliferation of HUVECs (A). Scratch wound healing (B) and transwell migration assay (C) were used to measure the migration of HUVECs. The tube length, numbers of branch points, and vessel area were observed in Matrigel medium (D). n ≥ 4 independent experiments. ^∗P < 0.05 versus Mock, ^#P < 0.05 versus Mock + AGEs.

FIGURE 4

Advanced glycation end products induced ERK phosphorylation required Src and RAGE. HUVECs were treated with 100 μg/mL AGEs for 15, 30, 60, 180, 360 min (A) and the phosphorylation level of ERK was detected by Western Blot. To investigate the effect of AGEs concentration on ERK phosphorylation, HUVECs were incubated with 50, 100, 150, 200 μg/mL AGEs for 30 min (B). To study the role of Src in AGE-induced ERK phosphorylation, HUVECs were pretreated with 15 μmol/L PP2 for 90 min and then incubated with 100 μg/mL AGEs for 30 min (C). HUVECs were transfected with Src siRNA or control siRNA for 48 h and cultured with 100 μg/mL AGEs for 30 min to further investigate the effect of Src (D). Also, we transfected HUVECs with RAGE siRNA or control siRNA for 48 h and then incubated with 100 μg/mL AGEs for 30 min to further investigate the effect of RAGE (E). n ≥ 4 independent experiments. ^∗P < 0.05 versus Control, ^#P < 0.05 versus AGEs, ^∗∗P < 0.05 versus Control siRNA, ^##P < 0.05 versus Control siRNA + AGEs.

FIGURE 5

The role of ERK in AGE-induced angiogenesis. HUVECs were pretreated with 20 μmol/L PD98059 for 30 min and then incubated with 100 μg/mL AGEs for 24 h. CCK-8 was used to evaluate the proliferation of HUVECs (A), scratch wound healing (B) and transwell migration assay (C) were used to measure the migration of HUVECs. The tube length, numbers of branch points, and vessel area were observed in Matrigel medium (D). n ≥ 4 independent experiments. ^∗P < 0.05 versus control, #P < 0.05 versus AGEs.

FIGURE 6

The role of RAGE in AGE-induced angiogenesis. HUVECs were pre-transfected with RAGE siRNA or control siRNA for 48 h and then cultured with 100 μg/mL AGEs for 24 h. CCK-8 was used to evaluate the proliferation of HUVECs (A), scratch wound healing (B), and transwell migration assay (C) were used to measure the migration of HUVECs. The tube formation was observed in Matrigel medium (D). n ≥ 4 independent experiments. ^∗P < 0.05 versus control, ^#P < 0.05 versus AGEs.

FIGURE 7

The Role of Src/ERK pathway in AGE-induced aortic ring sprouting. The aortic rings were pretreated with 15 μmol/L PP2 for 90 min or 20 μmol/L PD98059 for 30 min respectively and then incubated with 100 μg/mL AGEs for 6 days. Fresh medium with or without AGEs was reintroduced every 2 days. An estimation of the capillary was performed by counting the branches from the aortic explants. Graph B displayed the statistical results of angiogenesis in mouse aortic ring. (A,B). n = 3 independent experiments. ^∗P < 0.05 versus control, #P < 0.05 versus AGEs. Scale bar: 200 μm. The aortic rings were incubated with isolectin B4 (green) for 1 h to stain endothelial cells for the indication of sprouts of neovessels (red arrows) (C).