Differential effects of heparin and low molecular weight heparin on osteoblastogenesis and adipogenesis in vitro

Summary

We have previously demonstrated that heparin produces cancellous bone loss in rats due in part to a decrease in the number of osteoblasts lining the trabecular bone surface. In the present study, we use a stromal-derived cell culture system together with measurements of alkaline phosphatase (ALP) activity, to compare the effects of heparin and the low molecular weight heparin (LMWH), Fragmin, on osteoblast differentiation in vitro. In addition, we examined the possibility that both heparin and LMWH can induce adipogenesis in our stromal cell culture system. Both heparin and LMWH were found to produce a statistically significant (P <0.01) and concentration-dependent decrease in the number of osteoblasts while increasing the number of adipocytes. When the effects of gravimetrically equivalent amounts of heparin and LMWH were compared, heparin had a 4-fold greater effect than LMWH. In contrast to heparin, N-desulfated heparin was found to have minimal effects on both osteoblast and adipocyte differentiation indicating that the heparin effect is not only chain-length dependent but also charge-dependent. The observation that LMWH has less of an effect on bone formation than heparin is compatible with the results of clinical trials indicating that LMWH produces less bone loss after long-term administration.

Keywords

Heparin - low molecular weight heparin - bone - osteoblastogenesis - adipogenesis

• References

• 1 Dahlman TC. Osteoporotic fractures and the recurrence of thromboembolism during pregnancy and the puerperium in 184 women undergoing thromboprophylaxis with heparin. Am J Obstet Gynecol 1993; 168: 1265-70.
• 2 Monreal M, Lafoz E, Olive A. et al. Comparison of subcutaneous unfractionated heparin with a low molecular weight heparin (Fragmin) in patients with venous thromboembolism and contraindications to coumarin. Thromb Heamost 1994; 71: 7-11.
• 3 Douketis JD, Ginsberg JS, Burrows RF. et al. The effects of long-term heparin therapy during pregnancy on bone density. Thromb Haemost 1996; 75: 254-7.
• 4 Pettila V, Leinonen P, Markkola A. et al. Postpartum bone mineral density in women treated for thromboprophylaxis with unfractionated heparin or LMW heparin. Thromb Haemost 2002; 87: 182-6.
• 5 Wawrzynska L, Tomkowski WZ, Przedlacki J. et al. Changes in bone density during longterm administration of low-molecular-weight heparins or acenocoumarol for secondary prophylaxis of venous thromboembolism. Pathophysiol Haemost Thromb 2003; 33: 64-7.
• 6 Muir JM, Weitz JI, Andrew M. et al. A histomorphometric comparison of the effects of heparin and low molecular weight heparin on cancellous bone in rats. Blood 1997; 89: 3236-42.
• 7 Kajkenova O, Lecka-Czernik B, Gubrij I. et al. Increased adipogenesis and myelopoeisis in the bone marrow of samp6, a murine model of defective osteoblastogenesis and low turnover osteopenia. J Bone Miner Res 1997; 12: 1772-9.
• 8 Hicok K, Thomas T, Gori F. et al. Development and characterization of conditionally immortalized osteoblast precursor cell lines from human bone marrow stroma. J Bone Miner Res 1998; 13: 205-17.
• 9 Thompson D, Lum K, Nygaard S. et al. The derivation and characterization of stromal cell lines from the bone marrow of p53-/-mice: new insights into osteoblast and adipocyte differentiation. J Bone Miner Res 1998; 13: 195-204.
• 10 Nuttal ME, Patton A, Olivera D. et al. Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype:implications for osteopenic disorders. J Bone Miner Res 1998; 13: 371-82.
• 11 Lecka-Czernik B, Moerman EJ, Grant DF. et al. Divergent effects of selective peroxisome proliferator-activated receptor-γ2 ligands on adipocyte versus osteoblast differentiation. Endocrinology 2002; 143: 2376-84.
• 12 Diascro DD, Vogel R, Johnson T. et al. High fatty acid content in rabbit serum is responsible for the differentiation of osteoblasts into adipocyte-like cells. J Bone Miner Res 1998; 13: 96-106.
• 13 Poliard A, Nifuji A, Lamblin D. et al. Controlled conversion of an immortalized mesodermal progenitor cell towards osteogenic, chondrogenic, or adipogenic pathways. J Cell Biol 1995; 130: 1461-72.
• 14 Beresford J, Bennett J, Devlin C. et al. Evidence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J Cell Sci 1992; 102: 341-51.
• 15 Tholpady SS, Katz AJ, Ogle RC. Mesenchymal stem cells from rat visceral fat exhibit multipotential differentiation in vitro. Anat Rec 2003; 272A: 398-402.
• 16 Noth U, Osyczka AM, Tuli R. et al. Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells. J Orthop Res 2002; 20: 1060-9.
• 17 Ducy P, Zhang R, Geoffroy V. et al. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 1997; 89: 747-54.
• 18 Nakashima K, Zhou X, Kunkel G. et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 2002; 108: 17-29.
• 19 Shui C, Spelsberg TC, Riggs BL. et al. Changes in Runx2/Cbfa1 expression and activity during osteoblastic differentiation of human bone marrow stromal cells. J Bone Miner Res 2003; 18: 213-21.
• 20 Zheng H, Guo Z, Ma Q. et al. Cbfa1/osf2 transduced bone marrow stromal cells facilitate bone formation in vitro and in vivo . Calcif Tissue Int 2004; 74: 194-203.
• 21 Bellows CG, Wang YH, Heersche JNM. et al. 1,25-dihydroxyvitamin D stimulates adipocyte differentiation in cultures of fetal rat calvaria cells: comparison with the effects of dexamethasone. Endocrinology 1994; 134: 2221-9.
• 22 Shaughnessy SG, Young E. Deschamps et al. The effects of low molecular weight and standard heparin on calcium loss from fetal rat calvaria. Blood 1995; 86: 1368-73.
• 23 Bhandari M, Hirsh J, Weitz JI. et al. The effects of standard and low molecular weight heparin on bone nodule formation in vitro . Thromb Haemost 1998; 80: 413-7.
• 24 Mishina Y, Starbuck MW, Gentile MA. et al. BMP type IA receptor signaling regulates postnatal osteoblast function and bone remodeling. J Biol Chem 2004; 10: 1074.
• 25 Skillington J, Choy L, Derynck R. Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes. J Cell Biol 2002; 159: 135-46.
• 26 Gimble JM, Morgan C, Kelly K. et al. Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells. J Cell Biochem 1995; 58: 393-402.
• 27 Doerrier W, Feingold KR, Grunfeld C. Cytokines induce catabolic effects in cultured adipocytes by multiple mechanisms. Cytokine 1994; 06: 478-84.
• 28 Hurley MM, Kessler M, Gronowicz G. et al. The interaction of heparin and basic fibroblast growth factor on collagen synthesis in 21 day fetal rat calvariae. Endrocrinology 1992; 130: 2675-82.
• 29 Aubin JE. Regulation of osteoblast formation and function. Rev Endocr Metab Disord 2001; 02: 81-94.
• 30 Gregoire FM, Smas CM, Sul HS. Understanding adipocyte differentiation. Physiol Rev 1998; 78: 783-809.
• 31 Knock HJ, Handschin AE. Inhibition of human osteoblast growth by unfractionated heparin and by low molecular weight heparins in vitro . Thromb Haemost 2002; 88: 361-2.
• 32 Cosmi B, Fredenburgh JC, Rischke J. et al. Effect of nonspecific binding to plasma proteins on the antithrombin activities of unfractionated heparin, low-molecular-weight heparin, and dermatan sulfate. Circulation 1997; 95: 118-24.
• 33 Young E, Wells P, Holloway S. et al. Ex-vivo and in-vitro evidence that low molecular weight heparins exhibit less binding to plasma proteins than unfractionated heparin. Thromb Haemost 1994; 71: 300-4.
• 34 Barzu T, Molho P, Tobelem G. et al. Heparin degradation in the endothelial cells. Thromb Res 1987; 47: 601-9.
• 35 Hurley MM, Kream BE, Raisz LG. Structural determinants of the capacity of heparin to inhibit collagen synthesis in 21-day fetal rat calvariae. J Bone Min Res 1990; 05: 1127-32.
• 36 Levine MN, Hirsh J, Gent M. et al. A randomized trial comparing activated thromboplastin time with heparin assay in patients with acute venous thromboembolism requiring large daily doses of heparin. Arch Intern Med 1994; 154: 49-56.