Endothelial nitric oxide synthase gene-deficient mice demonstrate marked retardation in postnatal bone formation, reduced bone volume, and defects in osteoblast maturation and activity

Abstract

Nitric oxide (NO) has been implicated in the local regulation of bone metabolism. However, the contribution made by specific NO synthase (NOS) enzymes is unclear. Here we show that endothelial NOS gene knockout mice (eNOS-/-) have marked abnormalities in bone formation. Histomorphometric analysis of eNOS-/- femurs showed bone volume and bone formation rate was reduced by up to 45% (P: < 0.01) and 52% (P: < 0.01), respectively. These abnormalities were prevalent in young (6 to 9 weeks old) adults but by 12 to 18 weeks bone phenotype was restored toward wild-type. Dual energy X-ray absorptiometry analysis confirmed the age-related bone abnormalities revealing significant reductions in femoral (P: < 0.05) and spinal bone mineral densities (P: < 0.01) at 8 weeks that were normalized at 12 weeks. Reduction in bone formation and volume was not related to increased osteoclast numbers or activity but rather to dysfunctional osteoblasts. Osteoblast numbers and mineralizing activity were reduced in eNOS-/- mice. In vitro, osteoblasts from calvarial explants showed retarded proliferation and differentiation (alkaline phosphatase activity and mineral deposition) that could be restored by exogenous administration of a NO donor. These cells were also unresponsive to 17ss-estradiol and had an attenuated chemotactic response to transforming growth factor-beta. In conclusion, eNOS is involved in the postnatal regulation of bone mass and lack of eNOS gene results in reduced bone formation and volume and this is related to impaired osteoblast function.

Figure 1.

Genotyping of eNOS+/+ and eNOS−/− mice. The set of primers (A) that amplified a 900-bp PCR product identified wild-type. The set of primers (B) that amplified a 603-bp PCR product identified the eNOS gene-deficient phenotype.

Figure 2.

Sections (3 μm) of distal femur from 6-week-old eNOS+/+ (a) and eNOS−/− (b) mice stained with von Kossa and toluidine blue. Fewer, thinner trabeculae are seen in the femurs of eNOS−/− mice compared to age-matched eNOS+/+ mice. Original magnification, ×3.

Figure 3.

Sections (3 μm) of distal femur from 9-week-old eNOS+/+ (a and c) and eNOS−/− (b and d) mice stained with von Kossa and toluidine blue. Fewer, thinner trabeculae are seen in the femurs of eNOS−/− mice compared to age-matched eNOS+/+ mice. Original magnifications: ×3 (a and b); ×10 (c and d).

Figure 4.

Sections (3 μm) from 18-week-old eNOS +/+ (a) and eNOS−/− (b) mice stained with von Kossa and toluidine blue. There are no obvious differences between wild-type and knockout in bone architecture. Original magnification, ×3.

Figure 5.

Bone mineral density measurements of femur/pelvis (a) and spine (b) from eNOS+/+ and eNOS−/− mice. *, P < 0.05; **, P < 0.01.

Figure 6.

Assessment of cell proliferation (a) and alkaline phosphatase activity (b) of primary calvarial osteoblasts from eNOS+/+ and eNOS−/− mice. It can been seen that retarded growth and differentiation of eNOS−/− osteoblasts is restored toward wild-type levels by SNAP. a: *, P < 0.0001 eNOS+/+ versus eNOS−/−; **, P < 0.001 eNOS−/− versus eNOS−/− plus SNAP; ^#, P < 0.05 eNOS+/+ versus eNOS+/+ plus SNAP. b: *, P < 0.05 eNOS−/− versus eNOS−/− plus SNAP.

Figure 7.

Assessment of formation of mineralized bone nodules (A and B) of primary calvarial osteoblasts from eNOS+/+ and eNOS−/− mice. It can be seen that there are fewer bone nodules formed in the eNOS−/− cultures but this is restored toward wild-type by SNAP. **, P <0.001 eNOS−/− versus eNOS +/+; *, P < 0.01 eNOS +/+ versus eNOS+/+ plus SNAP.

Figure 8.

Addition of 17-β estradiol (10⁻⁷ mol/L) induced an increase in cell number in eNOS+/+ osteoblasts but had no effect on eNOS−/− cells. *, P < 0.05 eNOS +/+ versus eNOS+/+ plus 17-β estradiol.

Figure 9.

Chemotaxis response of primary calavarial explant osteoblasts to transforming growth factor-β. It can be seen that although eNOS+/+ migrated toward the cytokine this effect was attenuated in the eNOS−/− cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001.