Geeta Hampson

Orthosilicic acid stimulates collagen type 1 synthesis and osteoblastic differentiation in human osteoblast-like cells in vitro

Silicon deficiency in animals leads to bone defects. This element may therefore play an important role in bone metabolism. Silicon is absorbed from the diet as orthosilicic acid and concentrations in plasma are 5-20 microM. The in vitro effects of orthosilicic acid (0-50 microM) on collagen type 1 synthesis was investigated using the human osteosarcoma cell line (MG-63), primary osteoblast-like cells derived from human bone marrow stromal cells, and an immortalized human early osteoblastic cell line (HCC1). Collagen type 1 mRNA expression and prolyl hydroxylase activity were also determined in the MG-63 cells. Alkaline phosphatase and osteocalcin (osteoblastic differentiation) were assessed both at the protein and the mRNA level in MG-63 cells treated with orthosilicic acid. Collagen type 1 synthesis increased in all treated cells at orthosilicic acid concentrations of 10 and 20 microM, although the effects were more marked in the clonal cell lines (MG-63, HCCl 1.75- and 1.8-fold, respectively, P < 0.001, compared to 1.45-fold in the primary cell lines). Treatment at 50 microM resulted in a smaller increase in collagen type 1 synthesis (MG-63 1.45-fold, P = 0.004). The effect of orthosilicic acid was abolished in the presence of prolyl hydroxylase inhibitors. No change in collagen type 1 mRNA level was seen in treated MG-63 cells. Alkaline phosphatase activity and osteocalcin were significantly increased (1.5, 1.2-fold at concentrations of 10 and 20 microM, respectively, P < 0.05). Gene expression of alkaline phosphatase and osteocalcin also increased significantly following treatment. In conclusion, orthosilicic acid at physiological concentrations stimulates collagen type 1 synthesis in human osteoblast-like cells and enhances osteoblastic differentiation.

Characterization of GATA3 Mutations in the Hypoparathyroidism, Deafness, and Renal Dysplasia (HDR) Syndrome

The hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder caused by mutations of the dual zinc finger transcription factor, GATA3. The C-terminal zinc finger (ZnF2) binds DNA, whereas the N-terminal finger (ZnF1) stabilizes this DNA binding and interacts with other zinc finger proteins, such as the Friends of GATA (FOG). We have investigated seven HDR probands and their families for GATA3 abnormalities and have identified two nonsense mutations (Glu-228 → Stop and Arg-367 → Stop); two intragenic deletions that result in frameshifts from codons 201 and 355 with premature terminations at codons 205 and 370, respectively; one acceptor splice site mutation that leads to a frameshift from codon 351 and a premature termination at codon 367; and two missense mutations (Cys-318 → Arg and Asn-320 → Lys). The functional effects of these mutations, together with a previously reported GATA3 ZnF1 mutation and seven other engineered ZnF1 mutations, were assessed by electrophoretic mobility shift, dissociation, yeast two-hybrid and glutathione S-transferase pull-down assays. Mutations involving GATA3 ZnF2 or adjacent basic amino acids resulted in a loss of DNA binding, but those of ZnF1 either lead to a loss of interaction with specific FOG2 ZnFs or altered DNA-binding affinity. These findings are consistent with the proposed three-dimensional model of ZnF1, which has separate DNA and protein binding surfaces. Thus, our results, which expand the spectrum of HDR-associated GATA3 mutations and report the first acceptor splice site mutation, help to elucidate the molecular mechanisms that alter the function of this zinc finger transcription factor and its role in causing this developmental anomaly.

The relationship between inhibitors of the Wnt signalling pathway (Dickkopf-1(DKK1) and sclerostin), bone mineral density, vascular calcification and arterial stiffness in post-menopausal women

Epidemiological studies have shown an association between bone loss/osteoporosis and vascular calcification (VC). Recent studies have implicated the Wnt signalling pathway in the pathogenesis of VC. We investigated the association between circulating concentrations of Wnt inhibitors; DKK1 and sclerostin with bone mineral density (BMD), abdominal aortic calcification (AAC) and arterial stiffness in post-menopausal women. One hundred and forty six post-menopausal women aged (mean [SD]) 61.5[6.5] years were studied. Sclerostin and DKK1 were measured in serum. BMD was measured at the lumbar spine (LS), femoral neck (FN), total hip (TH). AAC was detected by Vertebral Fracture Assessment (VFA) imaging and quantified using an 8- and 24- point scoring methods. Arterial stiffness was determined by aortic pulse wave velocity (PWV). A significant positive correlation was observed between sclerostin and BMD at the FN (r = 0.166, p = 0.043) and TH (r = 0.165, p = 0.044). The association remained significant at the FN (p = 0.045) and TH (p = 0.026) following adjustment for confounders. No significant correlation was observed between DKK1 and BMD. In contrast, there was a significant negative correlation between log DKK1 and AAC (24-point score: r = -0.25, p = 0.008 and 8-point score: r = -0.21, p = 0.024). Subjects with AAC score of 1 or less had significantly higher DKK1 (p = 0.01). The association between DKK1 and AAC remained significant following correction for age, blood pressure, cholesterol (24-point score: p = 0.017, 8-point score: p = 0.044). In adjusted linear regression analysis, sclerostin was positively associated with AAC (24-point score: p = 0.048, 8-point score: p = 0.031). Subjects with a PWV>9 m/s had significantly higher sclerostin than those with PWV <9 m/s: 23.8[12.3], vs 29.7 [14] pmol/l, p = 0.03). No association was observed between DKK1 and PWV. The opposite association between AAC and the 2 Wnt signaling inhibitors is of interest and merits further investigations. Future longitudinal studies are needed to establish the precise role of sclerostin and DKK1 in the pathogenesis of VC.

Fibroblast growth factor-23 is associated with C-reactive protein, serum phosphate and bone mineral density in chronic kidney disease

SummaryWe investigated the association between fibroblast growth factor-23 (FGF-23) and (1) the biochemical parameters implicated in chronic kidney disorder (CKD)-bone and mineral disorder (CKD-MBD) and (2) bone mineral density (BMD) in patients with CKD 1-4. C-reactive protein (CRP) and serum phosphate correlated with FGF-23. A significant association was seen between FGF-23 and BMD at the hip.IntroductionCirculating FGF-23 is elevated in CKD, although the primary stimulus remains unclear. Moreover, it is still unknown whether increase in FGF-23 has a biological effect on bone metabolism. The aim of the study was to investigate the association of FGF-23 with (1) the biochemical parameters linked with CKD-bone and mineral disorder (CKD-MBD) and (2) bone mineral density in CKD.MethodsWe studied 145 patients (74 M, 71 F) aged (mean [SD]) 53 [14] years with CKD 1–4. Serum calcium, phosphate, parathyroid hormone, FGF-23, 25 (OH) vitamin D, 1, 25 (OH)2 vitamin D, bone turnover markers, CRP were determined. BMD was measured at the lumbar spine, femoral neck (FN), forearm, and total hip (TH). Multivariate analysis was undertaken to explore the association between (1) the biochemical variables and FGF-23 and (2) FGF-23 and BMD.ResultsElevations in FGF-23 occurred in CKD stage 3 compared to CKD stage 1/2, although no significant differences in serum phosphate were observed. Serum phosphate (p < 0.001), CRP (p < 0.001) and diabetes mellitus (p < 0.05) were associated with FGF-23. BMD Z-score was significantly lower at the TH and FN in CKD 4 (p < 0.05). A significant association was seen between BMI, FGF-23, bone specific alkaline phosphatase and BMD at the TH (p < 0.05).ConclusionsThe data suggest that FGF-23 may be associated with parameters implicated in the complications of CKD. Longitudinal studies are required for further clinical evaluation.

Abnormal sulfate metabolism in vitamin D-deficient rats.

To explore the possibility that vitamin D status regulates sulfate homeostasis, plasma sulfate levels, renal sulfate excretion, and the expression of the renal Na-SO4 cotransporter were evaluated in vitamin D-deficient (D-D-) rats and in D-D- rats rendered normocalcemic by either vitamin D or calcium/lactose supplementation. D-D- rats had significantly lower plasma sulfate levels than control animals (0.93+/-0.01 and 1.15+/-0.05 mM, respectively, P < 0.05), and fractional sulfate renal excretion was approximately threefold higher comparing D-D- and control rats. A decrease in renal cortical brush border membrane Na-SO4 cotransport activity, associated with a parallel decrease in both renal Na-SO4 cotransport protein and mRNA content (78+/-3 and 73+/-3% decreases, respectively, compared with control values), was also observed in D-D- rats. Vitamin D supplementation resulted in a return to normal of plasma sulfate, fractional sulfate excretion, and both renal Na-SO4 cotransport mRNA and protein. In contrast, renal sulfate excretion and renal Na-SO4 cotransport activity, protein abundance, and mRNA remained decreased in vitamin D-depleted rats fed a diet supplemented with lactose and calcium, despite that these rats were normocalcemic, and had significantly lower levels of parathyroid hormone and 25(OH)- and 1,25(OH)2-vitamin D levels than the vitamin D-supplemented groups. These results demonstrate that vitamin D modulates renal Na-SO4 sulfate cotransport and sulfate homeostasis. The ability of vitamin D status to regulate Na-SO4 cotransport appears to be a direct effect, and is not mediated by the effects of vitamin D on plasma calcium or parathyroid hormone levels. Because sulfate is required for synthesis of essential matrix components, abnormal sulfate metabolism in vitamin D-deficient animals may contribute to producing some of the abnormalities observed in rickets and osteomalacia.

Impact of vitamin D supplementation on arterial vasomotion, stiffness and endothelial biomarkers in chronic kidney disease patients.

Background Cardiovascular events are frequent and vascular endothelial function is abnormal in patients with chronic kidney disease (CKD). We demonstrated endothelial dysfunction with vitamin D deficiency in CKD patients; however the impact of cholecalciferol supplementation on vascular stiffness and vasomotor function, endothelial and bone biomarkers in CKD patients with low 25-hydroxy vitamin D [25(OH)D] is unknown, which this study investigated. Methods We assessed non-diabetic patients with CKD stage 3/4, age 17–80 years and serum 25(OH)D <75 nmol/L. Brachial artery Flow Mediated Dilation (FMD), Pulse Wave Velocity (PWV), Augmentation Index (AI) and circulating blood biomarkers were evaluated at baseline and at 16 weeks. Oral 300,000 units cholecalciferol was administered at baseline and 8-weeks. Results Clinical characteristics of 26 patients were: age 50±14 (mean±1SD) years, eGFR 41±11 ml/min/1.73 m2, males 73%, dyslipidaemia 36%, smokers 23% and hypertensives 87%. At 16-week serum 25(OH)D and calcium increased (43±16 to 84±29 nmol/L, p<0.001 and 2.37±0.09 to 2.42±0.09 mmol/L; p = 0.004, respectively) and parathyroid hormone decreased (10.8±8.6 to 7.4±4.4; p = 0.001). FMD improved from 3.1±3.3% to 6.1±3.7%, p = 0.001. Endothelial biomarker concentrations decreased: E-Selectin from 5666±2123 to 5256±2058 pg/mL; p = 0.032, ICAM-1, 3.45±0.01 to 3.10±1.04 ng/mL; p = 0.038 and VCAM-1, 54±33 to 42±33 ng/mL; p = 0.006. eGFR, BP, PWV, AI, hsCRP, von Willebrand factor and Fibroblast Growth Factor-23, remained unchanged. Conclusion This study demonstrates for the first time improvement of endothelial vasomotor and secretory functions with vitamin D in CKD patients without significant adverse effects on arterial stiffness, serum calcium or FGF-23. Trial Registration ClinicalTrials.gov NCT02005718

The impact of vitamin D status on changes in bone mineral density during treatment with bisphosphonates and after discontinuation following long-term use in post-menopausal osteoporosis

BackgroundIt is still unclear whether addition of calcium/vitamin D supplements leads to an incremental benefit in patients taking bisphosphonates and whether achievement of serum level of 25 (OH) vitamin D of at least 70 nmol/L has an impact on the skeletal response to bisphosphonates. Moreover the maintenance of BMD after bisphosphonates withdrawal with the continuation of calcium/vitamin D supplements only, remains uncertain. The aims were to assess the impact of vitamin D status on changes in bone mineral density (BMD) in firstly patients with post-menopausal osteoporosis on bisphosphonates and secondly following discontinuation of bisphosphonates after long-term use.MethodsTwo patient groups were recruited. The first study population comprised of 112 women treated with a bisphosphonate. The second study population consisted of 35 women who had been on bisphosphonates for > 5 years in whom the treatment agent was discontinued. Baseline BMD, changes in BMD following treatment, duration of treatment, serum 25 (OH) vitamin D, parathyroid hormone (PTH), urine C-terminal telopeptides of type 1 collagen (CTX) were obtained on the study participants.ResultsIn the first study group, subjects with serum vitamin D concentrations (> 70 nmol/L) had a significantly lower serum PTH level (mean [SEM] 41 [2] ng/L). PTH concentrations of 41 ng/L or less was associated with a significantly higher increase in BMD at the hip following treatment with bisphosphonates compared to patients with PTH > 41 ng/L (2.5% [0.9] v/s -0.2% [0.9], P = 0.04). In the second study group, discontinuation of bisphosphonate for 15 months after long-term treatment did not result in significant bone loss at the lumbar spine and total hip, although a trend towards gradual decline in BMD at the femoral neck was observed.Conclusionthe data suggest that optimal serum 25 (OH) vitamin D concentration may lead to further reduction in bone loss at the hip in patients on bisphosphonates. A prospective controlled trial is needed to evaluate whether the response to bisphosphonates is influenced by vitamin D status. BMD is preserved at the lumbar spine and total hip following discontinuation of bisphosphonate for a short period following long-term treatment, although a gradual loss occurs at the femoral neck.

Functional characterization of calcium sensing receptor polymorphisms and absence of association with indices of calcium homeostasis and bone mineral density.

Objectives  Associations between calcium‐sensing receptor (CaSR) polymorphisms and serum calcium, PTH and bone mineral density (BMD) have been reported by six studies. However, three other studies have failed to detect such associations. We therefore further investigated three CaSR coding region polymorphisms (Ala986Ser, Arg990Gly and Gln1011Glu) for associations with indices of calcium homeostasis and BMD and for alterations in receptor function.

Changes in RANKL/OPG/RANK gene expression in peripheral mononuclear cells following treatment with estrogen or raloxifene.

The RANKL/OPG/RANK pathway is the key mediator of osteoclastogenesis. Mononuclear cells may be implicated in post-menopausal osteoporosis. The effect of estrogen or raloxifene on bone resorption and the expression of RANKL/OPG/RANK in peripheral blood mononuclear cells (PBMCs) was examined. Twenty-nine women with post-menopausal osteoporosis were treated with estrogen (HRT) or raloxifene for 12 months. Bone mineral density (BMD) was measured at baseline and at 12 months at the spine and hip. Serum C-terminal telopeptide (CTX) and OPG were measured at baseline and at 1, 3, 6 and 12 months. PBMCs were isolated from 17 women and changes in RANKL, OPG and RANK mRNA were determined. The effects of estrogen or raloxifene in PBMCs in vitro were also assessed. BMD increased following treatment (lumbar spine % change mean [S.E.M.]: 4.3% [0.9], p<0.001). Serum CTX decreased (6 months: -43.7% [6.0], p<0.0001). Serum OPG declined gradually (12 months: -26.4% [4.4], p<0.001). RANKL, OPG and RANK gene expression decreased (6 months: RANKL 50.0% [24.8] p<0.001, OPG: 21.7% [28] p<0.001, RANK: 76.6% [10.2] p=0.015). Changes in OPG mRNA correlated with changes in BMD (r=-0.53, p=0.027) and CTX (r=0.7, p=0.0044). Down-regulation in RANKL, OPG, RANK mRNA and reduction in bone resorption was also seen in vitro. These results suggest that the expression of RANKL/OPG/RANK in PBMCs are responsive to the slowing in bone turnover/remodeling associated with treatment with estrogen or raloxifene. Further confirmatory studies are needed.

Effect of a 300 000-IU Loading Dose of Ergocalciferol (Vitamin D2) on Circulating 1,25(OH)2-Vitamin D and Fibroblast Growth Factor-23 (FGF-23) in Vitamin D Insufficiency

CONTEXT Several trials have reported an increased risk of fractures and falls after intermittent high-dose vitamin D. Treatment with loading doses of vitamin D may increase 1,25(OH)(2) vitamin D catabolism through changes in calcium/phosphate homeostasis and fibroblast growth factor-23 (FGF-23). OBJECTIVE The aim was to determine the effects of high-dose vitamin D on circulating concentrations of 1,25(OH)(2) vitamin D and FGF-23 in patients with osteoporosis and vitamin D insufficiency. DESIGN, SETTING, PATIENTS, AND INTERVENTION We carried out a prospective study of 45 subjects with vitamin D deficiency/insufficiency treated with a bolus dose of 300 000 IU of vitamin D(2) im. Blood samples were obtained at baseline and 1, 2, and 3 months after treatment. OUTCOME MEASURES Changes in 1,25(OH)(2)-vitamin D and FGF-23 were measured. RESULTS Loading dose of vitamin D(2) increased 1,25(OH)(2)-vitamin D(2) at 3 months, with a mean [SD] of 41 [56] pmol/L at baseline and 162.3 [137.8] pmol/L at 3 months (P < .001). FGF-23 increased significantly at all time points with a peak at 3 months, with percent change from baseline (mean [SEM]) of 50% [48%] at 3 months (P < .01). There was a positive correlation between FGF-23 and serum phosphate (r = 0.36, P = .024) and calcium (r = 0.532, P < .001) and a negative correlation between total 1,25(OH)(2)-vitamin D and FGF-23 (r = -0.32, P = .036) at 3 months. CONCLUSIONS High-dose vitamin D increases 1,25(OH)(2)-vitamin D and FGF-23 concentration. Further studies are required to determine whether adjusting vitamin D dose and frequency to minimize increases in FGF-23 may prevent the adverse outcomes associated with high-dose intermittent vitamin D supplementation.