HIF-1α-dependent upregulation of angiogenic factors by mechanical stimulation in retinal pigment epithelial cells

Abstract

Mechanical stimulation as a mimic of drusen formation in the eye increases the expression of angiogenic factors in retinal pigment epithelial (RPE) cells, but the underlying molecular mechanisms remain unclear. We investigated and characterized the effects of mechanical stimulation on the expression of angiogenic factors in RPE cells both in vitro and in a mouse model. Mechanical stimulation increased the expression of vascular endothelial growth factor (VEGF, encoded by VEGFA) and other angiogenesis-related genes in cultured RPE1 cells. The presence of hypoxia-inducible factor 1α (HIF-1α, encoded by HIF1A) was also increased, and both knockdown of HIF-1α and treatment with the HIF-1α inhibitor CAY10585 attenuated the effect of mechanical stimulation on angiogenesis factor gene expression. Signaling by the tyrosine kinase SRC and p38 mitogen-activated protein kinase was involved in HIF-1α activation and consequent angiogenesis-related gene expression induced by mechanical stimulation. Our results suggest that SRC-p38 and HIF-1α signaling are involved in the upregulation of angiogenic factors in RPE cells by mechanical stimulation. Such in vivo suppression of upregulated expression of angiogenesis-related genes by pharmacological inhibitors of HIF-1α suggests a new potential approach to the treatment of age-related macular degeneration.

Keywords: Age-related macular degeneration; Angiogenesis; HIF-1α; Mechanotransduction; Retinal pigment epithelial cells.

Fig. 1.

Mechanical stimulation-induced expression of angiogenesis-related factor genes in RPE1 cells. (A) qPCR analysis of VEGFA, ANG2 and IL6 mRNA abundance in RPE1 cells subjected to mechanical stimulation at 30 counts/min for the indicated times. Data were normalized to the amount of 18S rRNA and expressed relative to the corresponding value for non-stimulated cells. (B) qPCR analysis of the amounts of VEGFA, ANG2 and IL6 mRNAs in RPE1 cells subjected to mechanical stimulation at the indicated frequencies for 3 h. (C) qPCR analysis of VEGFA, ANG2, IL6, ANG1, IL8 and COL1A1 mRNA levels in RPE1 cells subjected (or not) to mechanical stimulation at 30 counts/min for 3 h. (D) ELISA analysis of the concentration of VEGF in culture supernatants of RPE1 cells subjected (or not) to mechanical stimulation at 30 counts/min for 3 h. All data are mean±s.e.m. for four independent experiments. NS, not significant; *P<0.05; **P<0.01; versus non-stretched control (Student's two-tailed paired t-test).

Fig. 2.

Role of HIF-1α in the upregulation of angiogenesis-related gene expression by mechanical stimulation in RPE1 cells. (A) Immunoblot analysis of HIF-1α in RPE1 cells subjected (or not) to mechanical stimulation for 3 h in the absence or presence of the HIF-1α inhibitor CAY10585 (left panel). Band intensity for HIF-1α in the three replicates shown was normalized to that for β-tubulin and presented as the mean±s.e.m. (right panel). (B,C) qPCR analysis of HIF1A mRNA (B) and VEGFA, ANG1, ANG2, IL6 and IL8 mRNAs (C) for cells treated as in A. Data were normalized to the amount of 18S rRNA and expressed relative to the corresponding value for non-stimulated cells not exposed to the inhibitor. (D) ELISA analysis of VEGF concentration in culture supernatants of cells treated as in A. (E,F) qPCR analysis of HIF1A mRNA (E) and VEGFA, ANG1, ANG2, IL6, IL8 and COL1A1 mRNAs (F) in RPE1 cells transfected with HIF-1α or control siRNAs and then subjected (or not) to mechanical stimulation for 3 h. Quantitative data are mean±s.e.m. NS, not significant; *P<0.05; **P<0.01; ***P<0.001 (Student's two-tailed paired t-test).

Fig. 3.

Roles of SRC and p38 MAPK in the HIF-1α-dependent upregulation of angiogenesis-related gene expression by mechanical stimulation in RPE1 cells. (A) qPCR analysis of VEGFA, ANG1, ANG2, IL6 and IL8 mRNA levels in RPE1 cells subjected (or not) to mechanical stimulation for 3 h in the absence or presence of the p38 inhibitor SB203580 or the SRC inhibitor dasatinib. Data were normalized to the amount of 18S rRNA and expressed relative to the corresponding value for non-stimulated cells not exposed to inhibitor. Data are means±s.e.m. for four independent experiments. NS, not significant; *P<0.05; **P<0.01 (Student's two-tailed paired t-test). (B,C) Immunoblot analysis of SRC (B) and phosphorylated (P-) and total p38 MAPK (C) in RPE1 cells subjected (or not) to mechanical stimulation for 3 h in the absence or presence of the HIF-1α inhibitor CAY10585. Data are representative of three independent experiments.

Fig. 4.

Angiogenesis induced by factors released from RPE1 cells in response to mechanical stimulation. (A,B) Light microscopy images of tube formation by vascular endothelial cells (top) as well as quantitation of tube length and number (bottom). Assays were performed in the presence of culture supernatants of RPE1 cells subjected (or not) to mechanical stimulation for 3 h, and without the inhibitor (A) or with CAY10585 (B). Scale bars: 300 µm. The quantitative data are means±s.e.m. for five independent experiments. NS, not significant; *P<0.05 (Student's two-tailed paired t-test).

Fig. 5.

Role of HIF-1α in regulation of angiogenesis-related gene expression in RPE cells by mechanical stimulation in a mouse model. (A) In vivo physical stimulation model. Two incisions were made in the conjunctiva and two beads were inserted into each incision and sutured in place. (B) Hematoxylin and Eosin staining of a control eye (left) and an eye at 2 days after bead insertion (right). Images are representative of ten eyes. Scale bars: 100 µm. (C) qPCR analysis of Vegfa and Ang2 mRNA levels in RPE cells isolated from control eyes or eyes at 2 days after bead insertion. (D) qPCR analysis of Vegfa and Ang2 mRNA levels in RPE cells isolated from control eyes or mechanically stimulated eyes that had been injected with the HIF-1α inhibitor CAY10585 at doses of 1 µg (low) or 10 µg (high) at the same time as bead insertion. All quantitative data were normalized to the amount of 18S rRNA and expressed relative to the corresponding control value, and are mean±s.e.m. for five eyes. NS, not significant; *P<0.05; **P<0.01 (Student's two-tailed paired t-test).