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 17␤-estradiol and had an attenuated chemotactic response to transforming growth factor-␤. Bone is a vital dynamic connective tissue that has evolved to maintain a balance between its two major functions: provision of mechanical integrity for locomotion and modulation and control of mineral homeostasis. 1 Mineralized bone is continuously resorbed by osteoclasts and new bone is formed by osteoblasts. This process, known as bone remodeling, is highly regulated with maintenance of normal integrity and structure. 2 Systemic hormones including calcitonin, parathyroid hormone, and sex steroids, particularly estrogen, are known to be important regulators of bone cell function. Their effects on bone turnover are in general exerted by activation of local mediators and second messengers present within bone cells. 3 Recent investigations have focused on the role of nitric oxide (NO) as one of these possible local regulators of bone metabolism and bone cell activity. NO is a shortlived radical gas generated from L-arginine by nitric oxide synthase (NOS) isoenzymes. 4 Three distinct isoforms of NOS have been identified: a neuronal form (type I; nNOS) originally isolated from brain, 5 an endothelial form (type III; eNOS) originally isolated from bovine aortic endothelial cells, 6 and an inducible form (type II; iNOS) originally isolated from murine macrophages. 7 Both eNOS and nNOS are expressed constitutively and are characterized by highly regulated rapid but low-output NO production. 4 In contrast the iNOS pathway is generally only activated after stimulation by certain pro-inflammatory cytokines such as interferon-␥, interleukin-1␤, and tumor necrosis factor-␣. The inducible NOS isoform is characterized by production of persistent and high concentrations of NO. 8 There is now am...

Increasing dietary nitrate has no effect on cancellous bone loss or fecal microbiome in ovariectomized rats

“…High levels of NO stimulate OPG, which binds to RANKL and prevents the binding of RANKL to the receptor activator of NF‐kappaB (RANK), decreasing osteoclast activity 48. eNOS global knockout animals exhibit lower BMD, bone formation, and osteoblast activity; with little to no effect on bone resorption, suggesting NO may be important for osteoblast function 49, 50. Previous research shows NO synthesis is induced in osteoblasts and osteocytes by mechanical strain and shear stress 51, 52, 53, 54.…”

Mobilizing Endothelial Progenitor Cells

“…30 Because statins profoundly augment eNOS expression and activity, 31 one may speculate that statins increase the concentration of eNOS-derived NO in the bone marrow. 30,32,33 Similar to statins, the antidiabetic and anti-inflammatory peroxisome proliferator-activated receptor-␥ agonists promote differentiation and mobilization of angiogenic progenitor cells and improve re-endothelialization after vascular intervention. 34 Recently, physical exercise, an important atheroprotective factor, was shown to enhance circulating EPC levels.…”

Pulsed electromagnetic fields rapidly modulate intracellular signaling events in osteoblastic cells: Comparison to parathyroid hormone and insulin

“…8 The second of these three molecules to be examined is endothelial nitric oxide synthase (eNOS/NOS III), which is thought to be the most highly expressed NO synthase in osteoblasts. 9 This choice is based on observations that mice deficient in eNOS expression exhibit juvenile osteopenia and skeletal dysplasias. 9 In addition, the anabolic effect of IGF-I in marrow-derived osteoprogenitor cells obtained from eNOS deficient mice is reported to be attenuated.…”

“…9 This choice is based on observations that mice deficient in eNOS expression exhibit juvenile osteopenia and skeletal dysplasias. 9 In addition, the anabolic effect of IGF-I in marrow-derived osteoprogenitor cells obtained from eNOS deficient mice is reported to be attenuated. 10 Finally, the third of these three molecules to be examined is the S6 ribosomal subunit.…”