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Expression of platelet derived growth factor-A and transforming growth factor β1 mRNA in heterotopic bone
Bone Vol. 17, No. 3 September 1995:31%331
Pl. Distribution of hyaluronan in normal and rachitic chick bone PG Genever, IR Dickson Department of Biology and Biochemistry, Brunei University, West London, Uxbridge, Middlesex UB8 3PH Hyaluronan (HA) is widely distributed throughout extracellular matrices. It is believed to be an important modulator of cell function, influencing cellular adhesion, migration, proliferation and differentiation. Little is known about the function of HA in bone. In the present work we investigated the distribution of HA in the proximal tibiotarsus of 4-week old normal (vitamin Dreplete) and rachitic (vitamin D-deficient) chicks using as a probe biotinylated HA-binding region of bovine aggrecan (generous gift from Dr. M. Bayliss, Kennedy Institute). In normal chicks, high levels of HA were present in early hypertrophic cartilage, predominantly in the immediate perilacunar matrix of chondrocytes. However the amount of HA was greatly reduced in late hypertrophic cartilage and the zone of provisional calcification. In the diaphysis, HA was present only on the nonbone-forming, basolateral surfaces of osteoblasts, and absent from the mineralised bone matrix. Rachitic chick bones displayed disorganised growth cartilage and poorly developed diaphyseal bone with marked marrow fibrosis. In these bones, perilacunar HA staining of hypertrophied chondrocytes was evident though the intensity of staining varied greatly throughout the hypertrophic zone. Rachitic diaphyseal bone exhibited intensely high levels of HA which appeared to be associated with abundant spindle-shaped cells occupying the marrow spaces. The finding that in normal bone HA is absent from areas undergoing mineralisation, and in rachitic bone the distribution and amount of HA are significantly altered, suggests that HA may play a significant, though as yet undefined role in the regulation of bone mineralisation.
P2. 1,24(S)-Dihydroxyvitamin Da a biologically active analogue of vitamin D, is a naturally occurring mctabolite in humans EB Mawer, M Davies, PE Still, G Jones*, JC Knutson’*, CW Bishop’* Department of Medicine, Manchester Royal Infirmary, Manchester, Ml3 9WL, *Department of Biochemistry, Queen’s University, Kingston, ON, Canada, and “Lunar Carp, Madison, WI, USA 1,24(S)-Dihydroxyvitamin 9 (1,24b) has been identified in rats given vitamin D2 and can also be synthesised in hepatoma cells in vitro by 24-hydroxylation of la-(0H)vitamin D2. This natural analogue is of interest because it binds avidly to the vitamin D receptor but has low calcaemic activity relative to l,2SD3. No similar pathway has been reported for 1,24&, perhaps because the 24-carbon in vitamin 9 is ally&z and so more reactive. We have investigated the production of 1,24D2 in four vitamin D-deficient human subjects given 1061U vitamin D2, by following the time-course of metabolites in serum for up to 91 days. Monohydroxy-metabolites (24D2, 25D2, 25D3) were measured by quantitative HPLC and dihydroxy-metabolites (1,24D2, 1,25&, 1,25D3) by RIA after HPLC separation. A novel RIA, 850 13pg/tube, was devised for 1,24D2 using MAb 5F2. Results are shown as median (range). 24& was maximal on day peaked on day 2, 80.5 (76-114)ng/ml. 1, 9.5 (7-21) ng/ml; 25b 1,24D2, 17 (15-Sl)pg/ml and 1,25D2, 235 (65-483)pg/ml both peaked on day 2 but could be detected up to day 91. There were no changes in endogenous D3 metabolites. A large volume of serum from one subject was extracted and used to confirm the identity of 24D2, 25D2 and 1,25D2 by HPLC comparison with standards and the generation of typical vitamin D chromophores by photodiode array. We have shown therefore that vitamin 9 can be activated in humans by an alternative pathway in which 24D2 is formed, and then la-hydroxylated to 1,249. P3. A comparison between biochemical and histomorphometric marken of bone turnover P L Selby, AM Davis’, S Bowles’, MW France*, A.J. Freemont” Manchester University Bone Disease Reasenrch Centre and Departments of Medicine, ‘Chemical Pathology and “Hisropathology, Manchester Royal Infrimary, Manchester Ml3 9WL
Abstracts
Biochemical markers of bone turnover are increasingly being used in research and clinical practice although their relationship to other measures of bone turnover is unclear. We have previously shown a relationship between some aspects of trabecular architecture on bone biopsy and the markers of collagen turnover PICP and ICTP in women with postmenopausal osteoporosis. In order to see if this is the case in other conditions we have looked at the relationship between biochemical markers of bone -turnover and histomorphometric indices in a variety of different metabolic bone diseases. 50 subjects with a variety of different generalised metabolic bone diseases (osteoporosis, osteomalacia and hyperparathyroidism) had transiliac bone biopsies taken following tetracycline labelling. At the same time the concentrations of ICTP, PICP, PIIINP, and total and bone specific alkaline phosphatase were measured in the blood together with calcium, hydroxyproline and collagen crosslinks in the urine. There was a positive relationship between ICTP concentration and trabecular volume (r=0.4O;p=0.02) and trabecular number (r=OSO;p<0.01). There was a positive relationship between bone specific alkaline phosphatase and osteoid surface (r=0.37;p=0.03) but not with any other histological marker of bone formation. Taking combinations of the various turnover markers did not improve tJw relationship with histological markers of turnover. We conclude that although the correspondence between biochemical and histological markers of bone turnover is poor there is evidence to suggest that some of these markers do relate to histological turnover and that others may reflect microarchitecture.
P4. Expression of platelet derived growth factor-h and transforming growth factor pl mRNA in heterotopic bone A Homer, S Bard, P Kemp’, J Compston Department of Medicine, University of Cambridge Clinical School, Cambtidge, CBZ 2QQ, and *Department of Biochemistry, University of Cambridge, Cambridge The role of growth factors in bone formation is poorly understood. We have investigated expression of platelet derived growth factor-A (PDGF-A) and transforming growth factor ~1 (TGFpl) mRNA in heterotopic bone. 10pm thick sections were placed onto RNase free Vectabond coated slides, and fixed in 4% paraformaldehyde. Specific mRNAs were detected using digoxigenin labelled anti-sense riboprobes. Bound probe was detected using an antidigoxigenin FabZ alkaline phosphatase complex. PDGF-A mRNA was expressed by most cells within the cartilaginous tissue. However, within the mineralising zones of cartilage decreased intensity or absence of staining was observed, suggesting down-regulation of expression. Within bone, PDGFA mRNA was not detected at sites of bone formation but was expressed by some lining cells and osteocytes. In contrast, TGFel mRNA was readily detected within cells at modelling and remodelling sites but not in osteocytes. TGFpl mRNA was also expressed by cells in the cartilaginous tissue. Our results indicate that locally produced PDGF-A and TGFpl may play important roles in the development of heterotopic bone. The different sites of expression of the two growth factors within the mineralised tissue suggest that they perform different regulatory functions in bone modelling and remodelling.
P5.
PTHrP
secretion
in
HCM:
metaat;rses in 08 patients WD Fraser, J Robinson, R Lawton, Department of Clinical Chemistry, Hospital, Liverpool, L7 8XP
analysis
of mortality
and
J Dutton, B Durham Royal Liverpwl University
138 patients with HCM (Adj Ca > 2.65 mmol/L on 2 occasions) were studied. Plasma PTHrP (Nichols Institute, WMA), total urinary Pyr and Dpyr excretion, where possible (fasting second void morning urine) were assayed (HPLC). Mean survival from diagnosis of hypercalcaemta was 42 d (range 2-203 d excluding 6 surviving z 650 d). Where PTHrP P 2.6 pmol/L mean survival was 34 d (2-197 d, no survivors) and PTHrP < 2.6 pmol/L mean survival was 57 d (2-203 d) (p = 0.008). Metastatic disease was assessed in 7.5 patients by radiology, ultrasound, isotope bone
321
Pl. Distribution of hyaluronan in normal and rachitic chick bone PG Genever, IR Dickson Department of Biology and Biochemistry, Brunei University, West London, Uxbridge, Middlesex UB8 3PH Hyaluronan (HA) is widely distributed throughout extracellular matrices. It is believed to be an important modulator of cell function, influencing cellular adhesion, migration, proliferation and differentiation. Little is known about the function of HA in bone. In the present work we investigated the distribution of HA in the proximal tibiotarsus of 4-week old normal (vitamin Dreplete) and rachitic (vitamin D-deficient) chicks using as a probe biotinylated HA-binding region of bovine aggrecan (generous gift from Dr. M. Bayliss, Kennedy Institute). In normal chicks, high levels of HA were present in early hypertrophic cartilage, predominantly in the immediate perilacunar matrix of chondrocytes. However the amount of HA was greatly reduced in late hypertrophic cartilage and the zone of provisional calcification. In the diaphysis, HA was present only on the nonbone-forming, basolateral surfaces of osteoblasts, and absent from the mineralised bone matrix. Rachitic chick bones displayed disorganised growth cartilage and poorly developed diaphyseal bone with marked marrow fibrosis. In these bones, perilacunar HA staining of hypertrophied chondrocytes was evident though the intensity of staining varied greatly throughout the hypertrophic zone. Rachitic diaphyseal bone exhibited intensely high levels of HA which appeared to be associated with abundant spindle-shaped cells occupying the marrow spaces. The finding that in normal bone HA is absent from areas undergoing mineralisation, and in rachitic bone the distribution and amount of HA are significantly altered, suggests that HA may play a significant, though as yet undefined role in the regulation of bone mineralisation.
P2. 1,24(S)-Dihydroxyvitamin Da a biologically active analogue of vitamin D, is a naturally occurring mctabolite in humans EB Mawer, M Davies, PE Still, G Jones*, JC Knutson’*, CW Bishop’* Department of Medicine, Manchester Royal Infirmary, Manchester, Ml3 9WL, *Department of Biochemistry, Queen’s University, Kingston, ON, Canada, and “Lunar Carp, Madison, WI, USA 1,24(S)-Dihydroxyvitamin 9 (1,24b) has been identified in rats given vitamin D2 and can also be synthesised in hepatoma cells in vitro by 24-hydroxylation of la-(0H)vitamin D2. This natural analogue is of interest because it binds avidly to the vitamin D receptor but has low calcaemic activity relative to l,2SD3. No similar pathway has been reported for 1,24&, perhaps because the 24-carbon in vitamin 9 is ally&z and so more reactive. We have investigated the production of 1,24D2 in four vitamin D-deficient human subjects given 1061U vitamin D2, by following the time-course of metabolites in serum for up to 91 days. Monohydroxy-metabolites (24D2, 25D2, 25D3) were measured by quantitative HPLC and dihydroxy-metabolites (1,24D2, 1,25&, 1,25D3) by RIA after HPLC separation. A novel RIA, 850 13pg/tube, was devised for 1,24D2 using MAb 5F2. Results are shown as median (range). 24& was maximal on day peaked on day 2, 80.5 (76-114)ng/ml. 1, 9.5 (7-21) ng/ml; 25b 1,24D2, 17 (15-Sl)pg/ml and 1,25D2, 235 (65-483)pg/ml both peaked on day 2 but could be detected up to day 91. There were no changes in endogenous D3 metabolites. A large volume of serum from one subject was extracted and used to confirm the identity of 24D2, 25D2 and 1,25D2 by HPLC comparison with standards and the generation of typical vitamin D chromophores by photodiode array. We have shown therefore that vitamin 9 can be activated in humans by an alternative pathway in which 24D2 is formed, and then la-hydroxylated to 1,249. P3. A comparison between biochemical and histomorphometric marken of bone turnover P L Selby, AM Davis’, S Bowles’, MW France*, A.J. Freemont” Manchester University Bone Disease Reasenrch Centre and Departments of Medicine, ‘Chemical Pathology and “Hisropathology, Manchester Royal Infrimary, Manchester Ml3 9WL
Abstracts
Biochemical markers of bone turnover are increasingly being used in research and clinical practice although their relationship to other measures of bone turnover is unclear. We have previously shown a relationship between some aspects of trabecular architecture on bone biopsy and the markers of collagen turnover PICP and ICTP in women with postmenopausal osteoporosis. In order to see if this is the case in other conditions we have looked at the relationship between biochemical markers of bone -turnover and histomorphometric indices in a variety of different metabolic bone diseases. 50 subjects with a variety of different generalised metabolic bone diseases (osteoporosis, osteomalacia and hyperparathyroidism) had transiliac bone biopsies taken following tetracycline labelling. At the same time the concentrations of ICTP, PICP, PIIINP, and total and bone specific alkaline phosphatase were measured in the blood together with calcium, hydroxyproline and collagen crosslinks in the urine. There was a positive relationship between ICTP concentration and trabecular volume (r=0.4O;p=0.02) and trabecular number (r=OSO;p<0.01). There was a positive relationship between bone specific alkaline phosphatase and osteoid surface (r=0.37;p=0.03) but not with any other histological marker of bone formation. Taking combinations of the various turnover markers did not improve tJw relationship with histological markers of turnover. We conclude that although the correspondence between biochemical and histological markers of bone turnover is poor there is evidence to suggest that some of these markers do relate to histological turnover and that others may reflect microarchitecture.
P4. Expression of platelet derived growth factor-h and transforming growth factor pl mRNA in heterotopic bone A Homer, S Bard, P Kemp’, J Compston Department of Medicine, University of Cambridge Clinical School, Cambtidge, CBZ 2QQ, and *Department of Biochemistry, University of Cambridge, Cambridge The role of growth factors in bone formation is poorly understood. We have investigated expression of platelet derived growth factor-A (PDGF-A) and transforming growth factor ~1 (TGFpl) mRNA in heterotopic bone. 10pm thick sections were placed onto RNase free Vectabond coated slides, and fixed in 4% paraformaldehyde. Specific mRNAs were detected using digoxigenin labelled anti-sense riboprobes. Bound probe was detected using an antidigoxigenin FabZ alkaline phosphatase complex. PDGF-A mRNA was expressed by most cells within the cartilaginous tissue. However, within the mineralising zones of cartilage decreased intensity or absence of staining was observed, suggesting down-regulation of expression. Within bone, PDGFA mRNA was not detected at sites of bone formation but was expressed by some lining cells and osteocytes. In contrast, TGFel mRNA was readily detected within cells at modelling and remodelling sites but not in osteocytes. TGFpl mRNA was also expressed by cells in the cartilaginous tissue. Our results indicate that locally produced PDGF-A and TGFpl may play important roles in the development of heterotopic bone. The different sites of expression of the two growth factors within the mineralised tissue suggest that they perform different regulatory functions in bone modelling and remodelling.
P5.
PTHrP
secretion
in
HCM:
metaat;rses in 08 patients WD Fraser, J Robinson, R Lawton, Department of Clinical Chemistry, Hospital, Liverpool, L7 8XP
analysis
of mortality
and
J Dutton, B Durham Royal Liverpwl University
138 patients with HCM (Adj Ca > 2.65 mmol/L on 2 occasions) were studied. Plasma PTHrP (Nichols Institute, WMA), total urinary Pyr and Dpyr excretion, where possible (fasting second void morning urine) were assayed (HPLC). Mean survival from diagnosis of hypercalcaemta was 42 d (range 2-203 d excluding 6 surviving z 650 d). Where PTHrP P 2.6 pmol/L mean survival was 34 d (2-197 d, no survivors) and PTHrP < 2.6 pmol/L mean survival was 57 d (2-203 d) (p = 0.008). Metastatic disease was assessed in 7.5 patients by radiology, ultrasound, isotope bone
321