High-risk human papillomavirus-induced expression of endothelial and inducible nitric oxide synthase in human uterine cervix

Figures & data

Table I. Clinical characteristics of the women studied.

Variable	High-risk HPV present (n = 41)	High-risk HPV absent (n = 45)	P
Age (y ± SD)	34.1 ± 9.7	42.0 ± 7.7	< 0.0001
Parous	24 (59%)	29 (64%)	0.6
Phase of spontaneous menstrual cycle
Follicular	7 (17%)	15 (33%)	0.1
Luteal	9 (22%)	12 (27%)
Use of oral contraception	13 (32%)	6 (13%)	0.04
Use of intrauterine device
Levonorgestrel-releasing	8 (20%)	5 (11%)	0.2
Copper	2 (5%)	1 (2%)
Postmenopausal	3 (7%)	4 (9%)	0.8
Use of hormone therapy	3 (100%)	2 (50%)	0.7
Smoking	18 (44%)	13 (29%)	0.1
Histology
Normal	4 (11%)	25 (78%)	< 0.001
CIN 1	16 (44%)	7 (22%)	< 0.001
CIN 2	15 (42%)		< 0.001
Adenocarcinoma in situ	1 (3%)
No biopsy	5	13	< 0.001

Figure 1. A representative illustration of eNOS and iNOS protein expression (Western blotting) in cervical samples from women with (+) and without (–) hrHPV. Tissue homogenates (containing 25 µg of total protein) were loaded, separated by gel electrophoresis, and immunoblotted with specific monoclonal antibodies to eNOS and iNOS. Signal intensity was quantified by densitometry and normalized to that of β-actin (loading control).

Figure 2. Protein levels of endothelial and inducible nitric oxide synthase (eNOS, iNOS) determined by Western blotting in women with (+) and without (–) high-risk human papillomavirus (hrHPV). The NOS scales (left, eNOS; right, iNOS) represent density in arbitrary units relative to actin. The horizontal lines show the means of the values.

Table II. Expression of endothelial and inducible nitric oxide synthase (eNOS, iNOS) in smokers and non-smokers according to high-risk human papillomavirus (hrHPV) status. The values represent density in arbitrary units relative to actin (mean × 10² ± SE (95% CI)) as assessed by Western blotting.

	Smokers	Non-smokers	P
hrHPV + eNOS	17.4 ± 4.0^a (8.8–26.0) (n = 15)	48.3 ± 8.5^c (30.3–66.3) (n = 17)	0.001
hrHPV– eNOS	37.9 ± 22.1 ^a (13.1–89.0) (n = 9)	12.6 ± 2.3^c (7.8–17.4) (n = 21)	0.9
hrHPV + iNOS	5.6 ± 1.5^b (2.3–8.8) (n = 15)	17.6 ± 3.9^d (9.4–25.9) (n = 17)	0.008
hrHPV– iNOS	10.9 ± 4.9^b (0.4–22.2) (n = 9)	3.4 ± 1.2^d (0.925.9) (n = 21)	0.4

^aP = 0.93; ^bP = 1.0; ^cP < 0.001; ^dP < 0.001.

Figure 3. Immunohistochemical localization of endothelial nitric oxide synthase (eNOS) (left panels, a–c) and inducible nitric oxide synthase (iNOS) (right panels, d–f) in cervical specimens from women without (a, d) and with (b, e) high-risk human papillomavirus. Endothelial NOS was localized to the vascular endothelium and iNOS mainly to the basal layer of epithelial cells. Specimens incubated with irrelevant antibody (IgG) showed no immunoreactivity (c, f). Hematoxylin counterstain, × 200 magnification.

Table III. Expression of endothelial and inducible nitric oxide synthase (eNOS, iNOS) in women with different histological findings. The levels represent density in arbitrary units relative to actin (mean × 10² ± SE (95% CI)) as assessed by Western blotting. Comparisons are made to women with normal histology.

	Normal (n = 17)	CIN 1 (n = 18)	CIN ≥ 2 (n = 13)	P
eNOS	13.3 ± 4.3 (4.1–22.4)	50.3 ± 12.4^a (24.1276.6)	28.0 ± 6.1^b (14.6–41.4)	^a0.002, ^b0.07
iNOS	6.2 ± 2.6 (0.8–11.8)	13.8 ± 3.6^c (6.3–21.3)	11.6 ± 3.4^d (4.1–19.1)	^c0.01, ^d0.1