Iva Orlić

Systemically Administered Bone Morphogenetic Protein-6 Restores Bone in Aged Ovariectomized Rats by Increasing Bone Formation and Suppressing Bone Resorption

Although recombinant human bone morphogenetic proteins (BMPs) are used locally for treating bone defects in humans, their systemic effect on bone augmentation has not been explored. We have previously demonstrated that demineralized bone (DB) from ovariectomized (OVX) rats cannot induce bone formation when implanted ectopically at the subcutaneous site. Here we showed in vitro that 17β-estradiol (E2) specifically induced expression of Bmp6 mRNA in MC3T3-E1 preosteoblastic cells and that bone extracts from OVX rats lack BMPs. Next we demonstrated that 125I-BMP-6 administered systemically accumulated in the skeleton and also restored the osteoinductive capacity of ectopically implanted DB from OVX rats. BMP-6 applied systemically to aged OVX rats significantly increased bone volume and mechanical characteristics of both the trabecular and cortical bone, the osteoblast surface, serum osteocalcin and osteoprotegerin levels, and decreased the osteoclast surface, serum C-telopeptide, and interleukin-6. E2 was significantly less effective, and was not synergistic with BMP-6. Animals that discontinued BMP-6 therapy maintained bone mineral density gains for another 12 weeks. BMP-6 increased in vivo the bone expression of Acvr-1, Bmpr1b, Smad5, alkaline phosphatase, and collagen type I and decreased expression of Bmp3 and BMP antagonists, chordin and cerberus. These results show, for the first time, that systemically administered BMP-6 restores the bone inductive capacity, microarchitecture, and quality of the skeleton in osteoporotic rats.

Gene Expression Profiling in Bone Tissue of Osteoporotic Mice

Gene Expression Profiling in Bone Tissue of Osteoporotic Mice Ovaricetomized (OVX) animals represent an optimal model to investigate bone loss in osteoporosis. To further elucidate the underlying mechanisms of decreased bone formation and increased bone resorption following OVX, we conducted gene expression profiling experiments using bone samples of ovariectomized C57BL/6J mice. Following OVX, genes involved in immune response, cell cycle regulation, growth, apoptosis and bone resorption were upregulated, while genes that are important for regular cell processes, mitosis, metabolism of carbohydrates, extracellular matrix structure, angiogenesis, skeletal development and morphogenesis were downregulated. Among bone specific genes we observed upregulation of interleukin 7 (IL-7), IL-7 receptor and matrix metallopeptidase 8, while genes such as transforming growth factor-beta 3, procollagen type I and procollagen type VI exhibited marked decrease in expression. We also observed downregulation of two genes, parathyroid hormone receptor 1 and WD repeat domain 5, that are involved in skeletal development but were not previously reported to be altered in osteoporosis. We further performed gene set enrichment analysis (GSEA) in order to calculate enrichment of pathways specifically altered in murine bones following ovariectomy. In conclusion, OVX greatly influences expression of various genes involved in diverse biological processes confirming the notion that numerous pathways play an important role in pathophysiology of osteoporosis. Analiza Ekspresije Gena U Koštanom Tkivu Osteoporotičnih Miševa Osteoporoza je najčešća metabolička bolest kostiju, obilježena smanjenom koštanom masom i poremećenom koštanom mikroarhitekturom. Prikladan životinjski model za istraživanje osteoporoze su ovarijektomirani miševi. Kako bismo pobliže istražili mehanizme smanjene koštane formacije i pojačane koštane razgradnje nakon ovarijektomije, proveli smo pokuse ekspresijskog profiliranja koristeći se uzorcima kostiju ovarijektomiranih miševa soja C57BL/6J. Nakon ovarijektomije pojačava se izražaj gena uključenih u imunološki odgovor, regulaciju staničnog ciklusa, apoptozu i koštanu razgradnju, dok se ekspresija gena bitnih za mitozu, metabolizam ugljikohidrata, razvoj kosti, strukturu izvanstaničnog matriksa i angiogenezu smanjuje. Od koštano specifičnih gena, interleukin 7 (IL-7), receptor za IL-7 i matriks metalopeptidazu 8 imali su pojačan izražaj, dok je za transformirajući čimbenik rasta -beta 3, prokolagen tipa I i tipa VI uočen smanjen genski izražaj. Također smo otkrili smanjen izražaj dvaju gena, i to receptora 1 za paratireoidni hormon i WD ponavljajuću domenu 5, koji su bitni za koštani razvoj, a promjena njihove regulacije nije do sada primijećena u osteoporozi. Kako bismo dodatno istražili obogaćenost pojedinih funkcionalnih skupina gena u kostima ovarijetkomiranih životinja, podatke smo analizirali s pomoću algoritma gene set enrichment analysis (GSEA). Zaključno, ovarijektomija značajno utječe na izražaj brojnih gena uključenih u različite biološke procese, što potvrđuje pretpostavku da mnogi molekularni putovi imaju važnu ulogu u patofiziologiji osteoporoze.