title: Osteocytic expression of constitutive NO synthase isoforms in the femoral neck cortex: a case-control study of intracapsular hip fracture.
abstract: 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.
authors.affiliation: Bone Research Group (MRC), Department of Medicine, University of Cambridge, Addenbrookes Hospital, Cambridge, United Kingdom. amc59@medschl.cam.ac.uk
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Caballero-Alías
authors.forename: Nigel
authors.initials: N
authors.lastname: Loveridge
authors.forename: Andrew
authors.initials: A
authors.lastname: Pitsillides
authors.forename: Martyn
authors.initials: M
authors.lastname: Parker
authors.forename: Stephen
authors.initials: S
authors.lastname: Kaptoge
authors.forename: Alan
authors.initials: A
authors.lastname: Lyon
authors.forename: Jonathan
authors.initials: J
authors.lastname: Reeve
mesh_terms: 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
chemical_list: 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