Endogenously produced nitric oxide inhibits endothelial cell growth as demonstrated using novel antisense cell lines

Abstract

Proliferation of endothelial cells is a vital component of vascular repair and angiogenesis. The endothelial cell mediator, nitric oxide (NO) has been reported both to inhibit and to promote endothelial cell proliferation. In this study we have generated cell lines which constitutively express antisense RNA to a region of inducible nitric oxide synthase (iNOS) from a murine endothelial cell line, sEnd-1. In response to stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) these antisense cells had no detectable RNA for endogenous iNOS, barely detectable iNOS protein and produced 82% less NO than did the control transfected line. Stimulation of the control transfected line caused significant NO production and inhibition of cell growth whereas for the antisense line, producing little NO in response to stimulation, proliferation remained the same as for unstimulated cells. No differences in cell death were observed between unstimulated and LPS/IFN-gamma stimulated cells. The data presented in this study directly demonstrate that NO derived endogenously from iNOS inhibits proliferation of endothelial cells. This approach overcomes problems in other studies where NO donors or non-isoform specific inhibitors of NO synthase have been used.

Figure 1

Northern blot analysis of iNOS expression in cell lines transfected with pSG5/iNOS. RNA was electrophoresed through a 1.5% agarose gel, transferred to Hybond N+ membranes and hybridized with ³²P-labelled double-stranded probe to murine iNOS (includes the region used for sense/antisense). Cells were either unstimulated or stimulated for 4 h with LPS/IFN-γ (1 μg ml⁻¹ LPS, 50 u ml⁻¹ IFN-γ, 25 ml⁻¹ 175 cm³ flask 2 × 10⁷ cells⁻¹). The bands of antisense or sense sequence plus β-globin from pSG5 are approximately 1.2 kb. The band for endogenous iNOS mRNA (in the stimulated sEnd-1 and S7 but not visible in A1) is approximately 3.6 kb. Lane 1: stimulated S7; lane 2: unstimulated S7; lane 3: stimulated A1; lane 4: unstimulated A1; lane 5: stimulated untransfected sEnd-1; lane 6: unstimulated untransfected sEnd-1.

Figure 2

Western blot analysis of iNOS expression in cell lines transfected with pSG5/iNOS. The control sense transfected line S7 and antisense transfected line A1 (2.5 × 10⁶ cells 9 cm⁻¹ plate) were stimulated with LPS/IFN-γ (1 μg ml⁻¹ LPS, 50 u ml⁻¹ IFN-γ) for 24 h. J774.2 cells (2.5 × 10⁶ cells 9 cm⁻¹ plate) were stimulated with LPS (10 μg ml⁻¹) for 24 h and were included as a positive control for iNOS production. Cells were harvested, lysed and separated by SDS polyacrylamide electrophoresis (200 μg protein per track) and transferred to a nitrocellulose membrane. Following incubation with rabbit polyclonal anti-mouse iNOS and anti-rabbit IgG peroxidase, bound antibodies were detected by chemiluminescence. Lane 1 shows the positive control of stimulated J774.2 cells. Lane 2 shows stimulated S7 cells. Lane 3 shows stimulated A1 cells.

Figure 3

Nitrite accumulation from cell lines transfected with pSG5/iNOS. Transfected lines were seeded at 3.5 × 10⁵ cells ml⁻¹ (24-well plate, 500 μl well⁻¹) and incubated for 24 h. LPS (1 μg ml⁻¹) and IFN-γ (50 u ml⁻¹) were added in a volume of 500 μl and the incubation continued for 24 h. Nitrite accumulation was measured by applying the Greiss reaction to 75 μl medium. The remainder of the medium was removed, the cells washed with PBS and lysed with 1 M NaOH. Protein content of each well was measured and the Greiss reaction results adjusted to a standard protein amount. Results shown are mean+s.e.mean of combined results from four separate experiments, each in duplicate, with triplicate measurements of each replicate in the Greiss reaction. P<0.0001 for LPS/IFN-γ stimulated S7 cells compared to unstimulated S7 cells. P=0.0112 for LPS/IFN-γ stimulated A1 cells compared to unstimulated A1 cells.

Figure 4

Growth of resting and LPS/IFN-γ stimulated cell lines transfected with pSG5/iNOS determined by protein analysis. Transfected lines were seeded at 5 × 10⁴ cells ml⁻¹ (24-well plates, 500 μl well⁻¹) and incubated for 7 h to allow them to adhere. Cells were stimulated with LPS (1 μg ml⁻¹) and IFN-γ (50 u ml⁻¹) for 24–72 h. The medium was removed after the stated times (and kept for nitrite determination by the Greiss reaction, Table 1) and the cells washed with PBS, lysed with 1 M NaOH and the protein content determined. The results presented are mean±s.e.mean of combined results from three separate experiments, each in quadruplicate, with duplicate measurements of each replicate in the protein assay.

Figure 5

Growth studies of resting and LPS/IFN-γ stimulated cell lines transfected with pSG5/iNOS determined by cell number analysis. Transfected lines were seeded at 5 × 10⁴ cells ml⁻¹ (24 well plates, 500 μl well⁻¹) and incubated for 7 h to allow them to adhere. Cells were stimulated with LPS (1 μg ml⁻¹) and IFN-γ (50 u ml⁻¹) for 72 h and then washed with PBS and detached using trypsin/EDTA. Cells were centrifuged at 150 × g for 10 min, resuspended in 200 μl medium and the cell number determined by counting using a haemocytometer. The results presented are mean+s.e.mean of combined results from three separate experiments, each in quadruplicate, with duplicate cell counts of each replicate. P<0.0001 for LPS/IFN-γ stimulated S7 cells compared to unstimulated S7 cells.