Effect of nitric oxide on neointimal hyperplasia based on sex and hormone status

Abstract

Nitric oxide (NO)-based therapies decrease neointimal hyperplasia; however, studies have been performed only in male animal models. Thus, we sought to evaluate the effect of NO on vascular smooth muscle cells (VSMC) in vitro and neointimal hyperplasia in vivo based on sex and hormone status. In hormone-replete medium, male VSMC proliferated at greater rates than female VSMC. In hormone-depleted medium, female VSMC proliferated at greater rates than male VSMC. However, in both hormone environments, NO inhibited proliferation and migration to a greater extent in male compared to female VSMC. These findings correlated with greater G₀/G₁ cell cycle arrest and changes in cell cycle protein expression in male compared to female VSMC after exposure to NO. Next, the rat carotid artery injury model was used to assess the effect of NO on neointimal hyperplasia in vivo. Consistent with the in vitro data, NO was significantly more effective at inhibiting neointimal hyperplasia in hormonally intact males compared to females using weight-based dosing. An increased weight-based dose of NO in females was able to achieve efficacy equal to that in males. Surprisingly, NO was less effective at inhibiting neointimal hyperplasia in castrated animals of both sexes. In conclusion, these data suggest that NO inhibits neointimal hyperplasia more effectively in males compared to females and in hormonally intact compared to castrated rats, indicating that the effects of NO in the vasculature may be sex- and hormone-dependent.

Figure 1

Inhibition of proliferation by nitric oxide (NO) in male versus female vascular smooth muscle cells (VSMC) in hormone replete medium (HRM) and hormone deplete medium (HDM). VSMC proliferation was assessed using ³H-thymidine incorporation at 24 hours ± exposure (CPM=counts per minute) to the NO donor DETA/NO (NO, units are mol/L). A) Male VSMC proliferated faster than female VSMC in HRM (*P<0.05 vs. male control). NO caused a dose-dependent inhibition of VSMC proliferation in both sexes (^#P<0.05 vs. same-sex control). B) Dose response curve demonstrating the percent reduction of proliferation by a given concentration of NO in male and female VSMC (P<0.05 for male vs. female curve). C) Female VSMC proliferated faster than male VSMC in HDM (*P<0.05, compared to male control). NO caused a dose-dependent inhibition of VSMC cell proliferation in males and females (^#P<0.05, compared to same-sex control). D) Dose response curve demonstrating the percent reduction of proliferation by a given concentration of NO in male and female VSMC in HDM (P<0.05 for male vs. female curve). Data shown are representative of more than five separate experiments; n = 3/treatment group.

Figure 2

Inhibition of migration by nitric oxide (NO) in male versus female vascular smooth muscle cells (VSMC). Migration was assessed using the scrape assay at 24 hours ± exposure (HPF=high power field) to the NO donor DETA/NO (NO, units are mol/L). A) Male VSMC migrate faster than female VSMC (*P<0.05, vs. male control). NO caused a dose-dependent inhibition of VSMC migration in males and decreased VSMC migration in females at the highest concentration (^#P<0.05, vs. same-sex control). B) Graphical representation of the percent inhibition of migration by a given concentration of NO after adjusting the data to the control group (*P<0.05, vs. male NO counterpart). Data shown are representative of more than five separate experiments; n = 3/treatment group.

Figure 3

Effect of nitric oxide (NO) on cell cycle progression in male and female vascular smooth muscle cells (VSMC). NO=DETA/NO 500 μmol/L and C=control. A-C) NO induced G₀/G₁ cell cycle arrest and decreased G₂/M and S phase in male and female VSMC (^#P<0.05 vs. same sex control; *P<0.05 vs. male control; ⁺P<0.05 vs. male NO). However, the percent change is greater in males compared to females (^τP<0.05 vs. percent reduction in males). Data shown are representative of five separate experiments; n = 3/treatment group.

Figure 4

Nitric oxide (NO) differentially effects the expression of cell cycle proteins based on sex. A) NO differentially effects the expression of cell cycle proteins based on sex by Western blot analysis. B-E) Densitometries of Western blots normalized by beta-actin loading controls and graphed as a percent of the male control. Female vascular smooth muscle cells (VSMC) expressed more p21 at baseline than male VSMC and NO increased p21 expression in males and females. NO decreased cdc2 expression in males and females. Male VSMC had a greater expression of cdk2 at baseline than female VSMC and that NO decreased cdk2 in male VSMC but not in female VSMC. NO decreased the expression of cyclin B in male and female VSMC, but to a slightly greater extent in females. NO=DETA/NO in mol/L. Data are representative of at least five separate experiments.

Figure 5

A) Castration decreased estrogen levels in all female groups but did not affect estrogen levels in male rats (*P<0.05 compared to intact counterpart). B) Intact male injured rats had higher testosterone levels than any other rat groups (^#P<0.05 compared to intact male injury). Castration significantly reduced testosterone levels in every group (*P<0.05 compared to intact counterpart). n=6/group.

Figure 6

Hematoxylin-eosin stained cross-sections of A) intact and B) castrated rat carotid arteries at 400X magnification. C) Morphometric analysis of the effect of NO on neointimal hyperplasia in male and female hormonally intact and castrated rodents (*P<0.05). NO=PROLI/NO (n=6/group).

Figure 6

Hematoxylin-eosin stained cross-sections of A) intact and B) castrated rat carotid arteries at 400X magnification. C) Morphometric analysis of the effect of NO on neointimal hyperplasia in male and female hormonally intact and castrated rodents (*P<0.05). NO=PROLI/NO (n=6/group).