Osteocytic expression of constitutive NO synthase isoforms in the femoral neck cortex: a case-control study of intracapsular hip fracture.
NO is an osteocytic signaling molecule that can inhibit osteoclasts. The NO synthases eNOS and nNOS were expressed by >50% of osteonal osteocytes in controls. Hip fracture cases showed +NOS osteocytes only in deep osteonal bone, and 25-35% reduced expression overall. These data are consistent with increased osteonal vulnerability to deep osteoclastic attack.


BACKGROUND
Osteocytes may regulate the response to mechanical stimuli in bone through the production of local signaling molecules such as NO derived from the NO synthase eNOS. Because NO is inhibitory to osteoclastic resorption, it has been suggested that osteocytes expressing eNOS act as sentinels, confining resorption within single osteons. Recently, nNOS has been shown to be present in osteocytes of adult human bone.


METHODS
Cross-sections of the femoral neck (eight female cases of intracapsular hip fracture and seven postmortem controls; age, 68-91 years) were analyzed by immunohistochemistry. The percentages of osteocytes expressing each of these two isoforms were calculated, and their distances to the nearest canal surface were measured.


RESULTS
The percentage of +nNOS osteocytes was lower in the fracture cases than in the controls (cases: 43.12 +/- 1.49, controls: 56.68 +/- 1.45; p < 0.0001). Compared with nNOS, eNOS expression was further reduced (p = 0.009) in the cases but was not different in the controls (cases: 36.41 +/- 1.53, controls: 56.47 +/- 2.41; p < 0.0001). The minimum distance of +eNOS or +nNOS osteocytes to a canal surface was higher in the cases compared with controls (eNOS: controls; 44.4 +/- 2.2 microm, cases: 61.7 +/- 2.0 microm; p < 0.0001; nNOS: controls: 52.4 +/- 1.7 microm, cases: 60.2 +/- 2.1 microm; p = 0.0039). +eNOS osteocytes were closer to the canal surfaces than +nNOS osteocytes in the controls by 8.00 +/- 4.0 microm (p = 0.0012).


CONCLUSIONS
The proportions of osteocytes expressing nNOS and eNOS were both reduced in the fracture cases, suggesting that the capacity to generate NO might be reduced. Furthermore, the reduction in NOS expression occurs in those osteocytes closest to the canal surface, suggesting that the ability of NO to minimize resorption depth might be impaired. Further studies are needed on the regulation of the expression and activity of these distinct NOS isoforms.
Caballero-Alías
Ana Maria
AM
Bone Research Group (MRC), Department of Medicine, University of Cambridge, Addenbrookes Hospital, Cambridge, United Kingdom. amc59@medschl.cam.ac.uk
Loveridge
Nigel
N
Pitsillides
Andrew
A
Parker
Martyn
M
Kaptoge
Stephen
S
Lyon
Alan
A
Reeve
Jonathan
J
D000368:Aged; D000369:Aged, 80 and over; D001862:Bone Resorption; D016022:Case-Control Studies; D005260:Female; D005265:Femoral Neck Fractures / Q000378:metabolism*; D006621:Hip Joint / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D009419:Nerve Tissue Proteins / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D010010:Osteoclasts / Q000378:metabolism*; D010011:Osteocytes / Q000378:metabolism*; D020033:Protein Isoforms; D013997:Time Factors
D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; C496320:NOS1 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III