Atorvastatin inhibits calcification and enhances nitric oxide synthase production in the hypercholesterolaemic aortic valve.

OBJECTIVE: To study in a rabbit model the expression of endothelial nitric oxide synthase (eNOS) in association with the development of calcification of the aortic valve, and to assess the effects of atorvastatin on eNOS expression, nitrite concentration, and aortic valve calcification. METHODS: Rabbits (n = 48) were treated for three months: 16, forming a control group, were fed a normal diet; 16 were fed a 0.5% (wt/wt) high cholesterol diet; and 16 were fed a 0.5% (wt/wt) cholesterol diet plus atorvastatin (2.5 mg/kg/day). The aortic valves were examined with eNOS immunostains and western blotting. Cholesterol and high sensitivity C reactive protein (hsCRP) concentrations were determined by standard assays. Serum nitrite concentrations were measured with a nitric oxide analyser. eNOS was localised by electron microscopy and immunogold labelling. Calcification in the aortic valve was evaluated by micro-computed tomography (CT). RESULTS: Cholesterol, hsCRP, and aortic valve calcification were increased in the cholesterol fed compared with control animals. Atorvastatin inhibited calcification in the aortic valve as assessed by micro-CT. eNOS protein concentrations were unchanged in the control and cholesterol groups but increased in the atorvastatin treated group. Serum nitrite concentrations were decreased in the hypercholesterolaemic animals and increased in the group treated with atorvastatin. CONCLUSION: These data provide evidence that chronic experimental hypercholesterolaemia produces bone mineralisation in the aortic valve, which is inhibited by atorvastatin.

MicroCT quantification of in vitro bone resorption of neonatal murine calvaria exposed to IL-1 or PTH.

This study investigated how effectively a laboratory microCT (X-ray micro-computed tomography) system can quantify bone resorption in an in vitro calvarial model and how well this measure correlates with a conventional assay for calcium release (fluorometric titration). In vitro bone resorption in neonatal murine calvaria was quantified for 0.3 or 1.0 nM interleukin-1 (IL-1) or for 1.0 or 10.0 nM parathyroid hormone (PTH) treatment. Compared to control calvaria, a significantly greater fraction F of the calvarial "shell" (computed from the volumetric microCT data) was resorbed in treated calvaria of 5- to 7-day-old pups from the same litter. Excellent correlation (R2 = 0.8234) was observed between F and calcium release, and, unlike the calcium assay, the 3-D maps revealed where bone was resorbed. Mineral was preferentially lost near the sutures, and areas away from the suture were left relatively intact. MicroCT of calvaria before and after 96 h culture demonstrated that this X-irradiation neither increased control resorption nor prevented responses in the treated calvaria. Observations on calvaria from intact mice aged 1, 3, 5, 8, and 11 days showed uniformly distributed mineral (not a pronounced patchwork of "high" and "low" mineral regions) and increasing levels of mineral with age; this suggested that the spatial patterns of resorption were not related to inhomogeneities in the starting mineral distribution.

Three-dimensional microarchitecture of the plates (primary, secondary, and carinar process) in the developing tooth of Lytechinus variegatus revealed by synchrotron X-ray absorption microtomography (microCT).

This paper reports the first noninvasive, volumetric study of entire cross-sections of a sea urchin tooth in which the individual calcite structural elements could be resolved. Two cross-sectionally intact fragments of a Lytechinus variegatus tooth were studied with synchrotron microCT (microcomputed tomography) with 1.66 microm voxels (volume elements). These fragments were from the plumula, that is the tooth zone with rapidly increasing levels of mineral; one fragment was from a position aboral of where the keel developed and the second was from the zone where the keel was developing. The primary plates, secondary plates, carinar process plates, prisms, and elements of the lamellar-needle complex were resolved. Comparison of the microCT data with optical micrographs of stained thin sections confirmed the identifications and measured dimensions of the characteristic microarchitectural features. The interplay of reinforcing structures (plates and prisms) was more clearly revealed in the volumetric numerical data sets than in single or sequential slices. While it is well known that the primary plates and prisms in camarodont teeth are situated to improve resistance to bending (which can be termed primary bending), the data presented provide a new understanding of the mechanical role of the carinar process plates, that is, a geometry consistent with that required in the keel to resist lateral or transverse bending of the tooth about a second axis. The increase in robustness of teeth incorporating lateral keel reinforcement suggests that the relative development of carinar processes (toward a geometry similar to that of L. variegatus) is a character which can be used to infer which sea urchins among the stirodonts are most primitive and among the camarodonts which are more primitive.