Hyperoxia inhibits several critical aspects of vascular development

Abstract

Normal human retinal vascular development uses angiogenesis and vasculogenesis, both of which are interrupted in the vaso-obliteration phase of retinopathy of prematurity (ROP). Canine oxygen-induced retinopathy (OIR) closely resembles human ROP. Canine retinal endothelial cells (ECs) and angioblasts were used to model OIR and characterize the effects of hyperoxia on angiogenesis and vasculogenesis. Cell cycle analysis showed that hyperoxia reduced the number of G1 phase cells and showed increased arrest in S phase for both cell types. Migration of ECs was significantly inhibited in hyperoxia (P < 0.01). Hyperoxia disrupted the cytoskeleton of angioblasts but not ECs after 2 days. Differentiation of angioblasts into ECs (determined by acetylated low-density lipoprotein uptake) was evaluated after basic fibroblast growth factor treatment. Differentiation of angioblasts into pericytes was determined by smooth muscle actin expression after treatment with platelet-derived growth factor. Differentiation into ECs was significantly inhibited by hyperoxia (P < 0.0001). The percentage of CXCR4(+) cells (a marker for retinal vascular precursors) increased in both treatment groups after hyperoxia. These data show novel mechanisms of hyperoxia-induced disruption of vascular development.

Fig. 1

Cell counts, apoptosis, and necrosis after normoxia or hyperoxia exposure. A,B: Endothelial cells (A) and angioblasts (B) were counted after 24, 48, and 72 hr of normoxia (dashed lines) and hyperoxia (solid lines) exposure. Bars indicate SD. *P < 0.01 when normoxia is compared with hyperoxia; **P < 0.01 when hyperoxia values are compared with the 24-hr normoxia values. C: Induction of apoptosis and necrosis in endothelial cells by hyperoxia. Flow cytometry was used to determine the percentage of apoptotic (filled symbols) and necrotic endothelial cells (open symbols) with Annexin V and propidium iodide, respectively. The normoxic controls are shown with solid lines, and the hyperoxia-treated cells are represented by dashed lines. Bars indicate SD.

Fig. 2

Cell cycle alterations in hyperoxia. A,B: The DNA content histograms for normoxia and hyperoxia-treated endothelial cells and angioblasts are shown in A and B, respectively. The percentages of cells in the various phases of the cell cycle and apoptosis are shown, where the normoxic values are indicated by solid bars and hyperoxic values by open bars.

Fig. 3

Inhibition of endothelial cell migration by hyperoxia. The inset photos show cells that have migrated beyond the initial scrape line (bottom of the image) after 24 hr of normoxia (left photo) and hyperoxia (right photo) exposure. Mitomycin C was used to inhibit proliferation of endothelial cells before scraping. The graph depicts nuclei counts at various migration intervals. Bars indicate SEM values. *P < 0.01.

Fig. 4

Disruption of angioblast and endothelial cell cytoskeletons by hyperoxia. A–F: Photomicrographs were taken of angioblasts (A–C) and endothelial cells (D–F) exposed to 0 (A,D), 3 (B,E), and 5 (C,F) days of hyperoxia. These cells were then labeled with Phalloidin (green), and angioblasts were also labeled with anti-smooth muscle actin (red, SMA). The cells were counterstained with 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI, blue). G: The percentage of cells with an intact cytoskeleton, as shown in upper panels, was determined. The SEM is indicated by bars. *P < 0.05 to normoxic angioblasts, and **P < 0.05 when compared with either normoxic/hyperoxic quiescent or normoxic dividing/migrating endothelial cells. All angioblast counts were significantly less (P < 0.05) than endothelial counts after the first day.

Fig. 5

Effect of hyperoxia on basic fibroblast growth factor (bFGF) -treated angioblast differentiation and CXCR4 expression. A,B: Upper panels (A,B) show acetylated low-density lipoprotein (AcLDL) uptake by bFGF-treated angioblasts after normoxia (A) and hyperoxia (B) exposure. AcLDL uptake was observed as punctate staining in the cytoplasm (A,B). C: The graph shows the number of cells per 10 mm². D,E: Lower panels show CXCR4 immunostaining in bFGF-treated angioblasts after normoxia (D) and hyperoxia (E) exposure. F: The graph shows the number of cells per 10 mm². G: The percentages of AcLDL⁺ and CXCR4⁺ cells are shown. Bars in C, F, and G indicate SEM values. A,B,D,E: Nuclear counterstaining with DAPI is shown in blue, and AcLDL/CXCR4 is shown in red. NOX, normoxia; HOX, hyperoxia. *P < 0.05. Scale bar = 20 μm in A.

Fig. 6

Effect of hyperoxia on platelet-derived growth factor (PDGF) -treated angioblast differentiation. A,B: Upper panels show smooth muscle actin (SMA) immunostaining in PDGF-treated angioblasts after normoxia (A) and hyperoxia (B) exposure. C: The graph shows the number of cells per 10 mm². D,E: Lower panels show CXCR4 immunostaining in PDGF-treated angioblasts after normoxia (D) and hyperoxia (E) exposure. F: The number of nuclei and SMA⁺ or CXCR4⁺ cells per field of view (10 mm²) were also counted and are shown. G: The percentages of SMA⁺ and CXCR4⁺ cells are shown. Bars in C, F, and G indicate SEM values. A,B,D,E: Nuclear counterstaining with 4′,6-dia-midine-2-phenylidole-dihydrochloride (DAPI) is shown in blue. NOX, normoxia; HOX, hyperoxia. *P < 0.05. Scale bar = 20 μm in A.