Effect of standing frames used in real life on bone remodeling in non-walking children with cerebral palsy.

Children with severe cerebral palsy are prone to low bone mineral density. No clear recommendation exists for an optimal use of standing frame to enhance bone health in this context. Used in real life, this study suggests for the first time that standing practice improved bone mineralization by limiting bone resorption. INTRODUCTION: To compare the bone health of children with severe cerebral palsy who use a static standing frame in real life to that of children who do not. METHODS: A total of 24 children with severe cerebral palsy GMFCS IV & V were included in the study and were divided into two groups: 13 were using a passive standing frame and 11 were not. We performed a single center retrospective cross-sectional study comparing the two groups using dual X-ray absorptiometry data and tests on biological samples, including bone remodeling factors. RESULTS: Total body (less head) bone mineral content was significantly higher in children who used a standing frame for an average of 30 min/day. This was confirmed in the lumbar spine. Although the total body bone mineral density (less head and proximal femur) densitometric data were not significantly higher, a positive trend favored the use of a standing frame in the children. Bone resorptive factors (CTX) were higher in the non-standing-frame group, whereas there was no difference among osteoformation factors. No difference in fracture history was found. CONCLUSIONS: We show that non-ambulant children with cerebral palsy who use a static standing frame in real life have better bone health, with lower bone resorption, than children who do not. Further studies are needed to determine how standing practice could impact bone mineralization over time in real life and to explore more bone remodeling factors.

The onset of left ventricular diastolic dysfunction in SHR rats is not related to hypertrophy or hypertension.

Left ventricular (LV) diastolic dysfunction, particularly relaxation abnormalities, are known to be associated with the development of LV hypertrophy (LVH). Preliminary human and animal studies suggested that early LV diastolic dysfunction may be revealed independently of LVH. However, whether LV diastolic dysfunction is compromised before the onset of hypertension and LVH remains unknown. We therefore evaluated LV diastolic function in spontaneously hypertensive rats (SHR) at different ages and tested whether LV diastolic dysfunction is associated with abnormal intracellular calcium homeostasis. LV systolic and diastolic functions were evaluated by invasive and echocardiographic methods in 3-week-old (without hypertension) and 5-week-old (with hypertension) SHR and Wistar-Kyoto control rats. Basal intracytoplasmic calcium and sarcoplasmic reticulum (SR) Ca(2+) contents were measured in cardiomyocytes using fura-2 AM. Sarco(endo)plasmic Ca(2+)-ATPase isoform 2a (SERCA 2a) and phospholamban (PLB) expressions were quantified by Western blot and quantitative RT-PCR techniques. LV relaxation dysfunction was observed in 3-week-old SHR rats before onset of hypertension and LVH. An increase in basal intracytoplasmic Ca(2+) and a decrease in SR Ca(2+) release were demonstrated in SHR. Decreased expression of SERCA 2a and Ser16 PLB (p16-PLB) protein levels was also observed in SHR rats, whereas mRNA expression was not decreased. For the first time, we have shown that LV myocardial dysfunction precedes hypertension in 3-week-old SHR rats. This LV myocardial dysfunction was associated with high diastolic [Ca(2+)](i) possibly due to decreased SERCA 2a and p16-PLB protein levels. Diastolic dysfunction may be a potential predictive marker of arterial hypertension in genetic hypertension syndromes.

Is vascular calcification associated with bone mineral density and osteoporotic fractures in ambulatory, elderly women?

UNLABELLED: We analyzed the relationship between aortic calcification and two osteoporotic parameters (bone mineral density (BMD) and incident osteoporotic fractures) in 667 ambulatory, elderly women from the Epidemiology of Osteoporosis (EPIDOS) cohort (mean age, 80 years; range, 72-94 years). We did not find any correlation between the aortic calcification score and BMD or osteoporotic fractures. INTRODUCTION: The aging process is associated with osteoporosis and aortic calcification; conditions which may have similar disease mechanisms. However, the relationship between these two settings remains to be elucidated. We analyzed the relationship between aortic calcification and osteoporotic parameters (BMD and incident osteoporotic fractures) in a cohort of ambulatory, elderly women. METHODS: The study included 667 ambulatory women from the EPIDOS cohort (mean age, 80 years; age range, 72-94 years). The baseline examination included bone investigations, a clinical and functional examination, and a comprehensive questionnaire on health status and lifestyle. Semiquantitative methods were used to determine the abdominal aortic calcification score on baseline radiographs. Incident fractures were recorded via postal questionnaires issued every 4 months for about 4 years. RESULTS: Five hundred three women (75%) had aortic calcification. The mean aortic calcification score was 5.5 (median, 4). During the follow-up period, 186 (28%) women reported one or more incident osteoporotic fractures. We did not find any correlation between the aortic calcification score on one hand and the BMD or the occurrence of incident osteoporotic fractures on the other. Only age and systolic blood pressure were found to be independently associated with the aortic calcification score. Osteoporotic fractures were independently associated with age and BMD. CONCLUSIONS: Osteoporosis and aortic calcification appear to be independent processes in a cohort of ambulatory, elderly women. However, potential confounding factors may be present and prospective studies are needed to investigate this situation further.

The pathophysiology of vascular calcification: are osteoclast-like cells the missing link?

There is increasing evidence to suggest that the initiation of vascular calcification is an active process involving vascular smooth muscle cell (VSMC) apoptosis and trans-differentiation into calcifying cells. This active process results in the deposition of an osteogenic extracellular matrix and may be exacerbated by a reduction in the levels of one or more native calcification inhibitors (such as fetuin A and pyrophosphate). Here, we present data which strongly suggest that the regression of vascular calcification might also be an active cellular process involving osteoclast-like cells. However, the presence of osteoclast like cells in the vascular wall is rather limited. To explain this rarity of osteoclast-like cells, we recently observed that the same factors, which promote the trans-differentiation of VSMCs into osteoblast-like cells are also capable of inhibiting the in vitro differentiation of monocytes/macrophages into osteoclast-like cells. An imbalance between osteoblast-like and osteoclast-like cell activities would therefore favour the occurrence of a pathological calcification process in vessel walls. Our new data are strongly evocative of a vascular remodelling process similar to that observed in bone tissue. To confirm this hypothesis, strategies for activating osteoclasts in the vascular wall (with a view to preventing or reversing vascular calcifications) are required.

The calcium sensing receptor is directly involved in both osteoclast differentiation and apoptosis.

Intracellular transduction pathways that are dependent on activation of the CaR by Ca(o)2+ have been studied extensively in parathyroid and other cell types, and include cytosolic calcium, phospholipases C, A2, and D, protein kinase C isoforms and the cAMP/protein kinase A system. In this study, using bone marrow cells isolated from CaR-/- mice as well as DN-CaR-transfected RAW 264.7 cells, we provide evidence that expression of the CaR plays an important role in osteoclast differentiation. We also establish that activation of the CaR and resultant stimulation of PLC are involved in high Ca(o)2+-induced apoptosis of mature rabbit osteoclasts. Similar to RANKL, Ca(o)2+ (20 mM) appeared to trigger rapid and significant nuclear translocation of NF-kappaB in a CaR- and PLC-dependent manner. In summary, our data suggest that stimulation of the CaR may play a pivotal role in the control of both osteoclast differentiation and apoptosis in the systems studied here through a signaling pathway involving activation of the CaR, phospholipase C, and NF-kappaB.

Involvement of capacitive calcium entry and calcium store refilling in osteoclastic survival and bone resorption process.

Bone resorption is closely dependent on osteoclastic survival and osteoclast apoptotic cell death could represent a key step at the end of this process. In order to precise the possible role of calcium movement in osteoclastic cell death, we investigated whether intracellular calcium store replenishment and capacitive calcium entry (CCE) are involved in osteoclastic survival and bone resorption. We demonstrate that (i). thapsigargin, a sarco-endoplasmic reticulum calcium ATPase pump (SERCA) blocker, decreases both osteoclastic survival and bone resorption process, (ii). 2-aminoethoxydiphenyl borate (2-APB) and SKF-96365, two store-operated channel (SOC) blockers, dramatically decrease osteoclastic survival and bone resorption and (iii). culture in calcium-free medium and thapsigargin exposure synergically inhibit osteoclastic survival which falls dramatically to a value close to 0% (P<0.001). Inversely, osteoclastic survival increases significantly when thapsigargin-treated cells are cultured in the presence of 20mM calcium, suggesting that increasing extracellular calcium concentration stimulates osteoclasts survival when the filling of intracellular stores is prevented. Taken together, our data strongly suggest that in osteoclasts, calcium movements between cellular compartments involved in the regulation of calcium signalling, such as calcium stores refilling and CCE, are closely associated to the regulation of osteoclast survival and bone resorption.

The relationships between serum sclerostin, bone mineral density, and vascular calcification in rheumatoid arthritis.

CONTEXT: Recent data indicate that the secreted glycoprotein sclerostin may be involved in vascular calcification (VC). OBJECTIVE: The objective of the study was to establish whether serum sclerostin levels are associated with VC in patients with rheumatoid arthritis (RA). DESIGN: This was a cross-sectional study. SETTING: The study was conducted with ambulatory care. PATIENTS: We compared 75 RA patients with 75 age- and gender-matched control participants. INTERVENTION: Coronary artery calcification (CAC) and abdominal aortic calcification (AAC) scores were evaluated by computed tomography. MAIN OUTCOME MEASURE: Serum sclerostin levels (determined with an ELISA) were assessed. A statistical analysis was performed to identify the determinants of serum sclerostin and VC. RESULTS: AAC and CAC were more prevalent and more severe in patients with RA than in controls. Higher levels of AAC (P = .02) and a higher lumbar bone mineral density (BMD; P = .03) were identified as independent determinants of higher serum sclerostin levels in RA patients, whereas male gender (P = .03), higher lumbar BMD (P < .0001), and low estimated glomerular rate (P < .001) were identified as determinants in controls. In RA patients, a multivariate logistic regression analysis indicated that older age [P < .01, with an odds ratio (OR) per year 1.10] and male gender (P = .02, OR 6.79) were independent determinants of CAC and that older age (P < .001, OR 1.16) were independent determinants of AAC. In controls, the independent determinants were older age (P < .01, OR 1.19), hypertension (P < .01, OR 7.31), and lumbar BMD (P = .03, OR per 30 mg/cm(2) increment of 1.14) for CAC and older age (P = .01, OR 1.11) for AAC. CONCLUSIONS: Serum sclerostin levels were significantly and independently associated with AAC in RA patients.

Vascular calcification in rheumatoid arthritis: prevalence, pathophysiological aspects and potential targets.

Individuals with rheumatoid arthritis (RA) are at increased risk for morbidity and mortality from cardiovascular disease. Excess cardiovascular mortality in RA patients cannot be fully explained by conventional cardiovascular risk factors. The purpose of this review is to discuss recent progress concerning the prevalence and pathophysiological aspects of vascular calcification in RA. RA patients have early-onset diffuse calcification involving multiple vascular beds compared to age and sex-matched controls. Pathogenesis of vascular calcification in RA patients is not fully understood, but specific mediators such as proinflammatory cytokines and not global inflammation could be involved. The possible link between osteoporosis and vascular calcification in RA will not be discussed. Finally, potential targets to reduce vascular calcification in RA will be discussed.

[Osteoblastic regulation of osteoclast survival: effect of calcitriol]. / Régulation ostéoblastique de la survie des ostéoclastes: effets du calcitriol.

Throughout life, bone is remodelled in a dynamic process which results in a balance between bone formation by osteoblasts and bone resorption by osteoclasts. It is now clearly established that osteoblasts/stromal cells are crucial for differentiation of osteoclasts, through a mechanism involving cell-to-cell contact. However, the possible involvement of osteoblasts and stromal cells in the survival of osteoclasts has not yet been clearly demonstrated. In this study, we assessed the influence of cellular microenvironment, especially osteoblasts, on the osteoclast survival. Our results have shown significant differences in osteoclastic survival between unfractionated bone cells and pure osteoclasts. Furthermore, we have shown that addition of 1.25(OH)2D3 to unfractionated bone cells resulted in a dose-dependent increase in osteoclast survival. Finally, we have shown that a conditioned medium obtained from rat osteoblastic cells cultured with calcitriol was able to increase significantly survival of pure osteoclasts. Taken together, these results strongly suggest that osteoblastic cells present in the bone microenvironment might play a role in the osteoclastic survival by producing soluble factor which modulate osteoclast apoptosis.

High extracellular calcium concentrations directly stimulate osteoclast apoptosis.

Although the inhibitory effects of high extracellular calcium concentrations ([Ca](e)) on osteoclastic bone resorption have been known for several years, the exact mechanism remains poorly understood. The present study was performed to investigate the possible effect of [Ca](e) on osteoclast apoptosis. Using highly purified rabbit osteoclasts, we have shown that calcium directly promotes apoptosis in a dose-dependent manner which correlates with the dose range of calcium for the inhibition of bone resorption. A time-course experiment of apoptotic changes of osteoclasts cultured in presence of 1.8 or 20 mM calcium showed a significant difference after as early as 8 h of culture. After 72 h of culture, we observed that 80% of the cells cultured in the presence of 20 mM calcium displayed the typical features of apoptosis compared to only 20% in the medium containing 1.8 mM calcium. Calcium channel blockers and ryanodine abrogated the effects of [Ca](e) on apoptosis while neomycin, a calcium-sensing receptor agonist, did not alter cell viability. Taken together, these results suggest that calcium influx is involved in calcium-induced osteoclast apoptosis. Our results are consistent with the concept that in the presence of high [Ca](e) generated during bone demineralization, osteoclasts are subjected to negative-feedback regulation due, at least in part, to the induction of apoptosis.

Regulation of bone resorption and osteoclast survival by nitric oxide: possible involvement of NMDA-receptor.

Nitric oxide has been shown to play an important role in regulation of bone resorption. However, the role of endogenous nitric oxide on osteoclast activity remains still controversial. In this work, using RT-PCR amplification, we demonstrated that rabbit mature osteoclasts express mRNA encoding for neuronal nitric oxide synthase suggesting that this enzyme could be involved in basal nitric oxide production in these cells. Then we assessed the effect of carboxy-PTIO, a nitric oxide scavenger, on in vitro bone resorption and osteoclast survival. Carboxy-PTIO (10-100 microM) inhibited osteoclastic bone resorption in a dose dependent manner and induced osteoclast apoptosis by a mechanism involving caspase 3 activation. These results suggest that basal concentration of endogenous nitric oxide may be essential for normal bone resorption by supporting osteoclast survival. Because osteoclasts express N-methyl-d-aspartate-receptor (NMDA-R), we hypothesized that in osteoclasts NMDA-R may be involved in nitric oxide production as in neuronal cells. We confirmed that blockade of NMDA-R with specific non-competitive antagonists, MK801 and DEP, strongly inhibited bone resorption. As for carboxy-PTIO, we showed that blockade of NMDA-R by both antagonists induced osteoclast apoptosis in a dose dependent manner by a mechanism dependent on caspase 3 activation. Intracellular calcium concentration in osteoclasts decreased within minutes in the presence of both antagonists. Finally, MK801-induced osteoclast apoptosis was partially reversed in the presence of small amount of SNAP (100 nM), a nitric oxide donor, suggesting that the effect of NMDA-R on osteoclast apoptotic cell death could be due to a decrease in nitric oxide production. Taken together, our results are consistent with the hypothesis that NMDA-R on osteoclasts could have a similar function as those in neuronal cells, i.e., to allow a calcium influx, which in turn activates a constitutive neuronal nitric oxide synthase. Nitric oxide generated by this pathway may be essential for osteoclast survival and hence for normal bone resorption.

Evaluation of in vitro bone resorption: high-performance liquid chromatography measurement of the pyridinolines released in osteoclast cultures.

None of the currently used methods to evaluate bone resorption by osteoclasts cultured on bone substrate measures directly the amounts of degraded bone collagen, which is a direct reflection of the osteoclast "work done." We therefore propose a reliable biochemical method to evaluate the in vitro collagenolysis process. Bone-resorbing activity was evaluated, after HPLC separation, by fluorimetric measurement of hydroxylysylpyridinoline (HP), a collagen cross-link molecule, released in culture supernatants. We first confirm previous data reporting that HP is released in the culture medium in a peptide-conjugated form. After acid hydrolysis, we show that HP is highly correlated with the lacunae area (r = 0.68, P<0.0001) and with the amounts of antigenic collagen fragments (Cross-laps for culture) released in culture medium (r = 0.77, P<0.0002). Using a cysteine protease inhibitor, we observed that lacunae areas are dramatically less inhibited (35% inhibition) than the release of bone-degraded products, including HP and antigenic collagen fragments (96 and 92% inhibition, respectively). Coupled to the resorbed area measurement, biochemical evaluations offer both quantitative and qualitative complementary measurements of the osteoclastic bone-resorbing process.