Enhancement of BMP activity in vivo by heparin and analysis of its molecular mechanisms.

尽管骨形态发生蛋白（BMP）在临床上对骨再生有用，但在猴子和人类中需要大量的BMP来诱导新骨形成。我们最近发现，肝素刺激BMP活性在体外。在本研究中，我们研究了肝素是否可以增强骨形成蛋白诱导的骨形成在体内，并试图确定肝素刺激BMP活性的分子机制，使用C2 C12成肌细胞。肝素增强BMP-2诱导的基因表达和Smad 1/5/8磷酸化在24小时和之后，但不是在12小时内。肝素酶处理不影响细胞对BMP-2的反应。在肝素存在下，培养基中BMP-2的半衰期延长了10倍以上。尽管不管肝素的存在，头蛋白mRNA在1小时内被BMP-2诱导，但头蛋白在肝素存在下不能抑制BMP-2活性。此外，BMP-2和肝素在体内剂量依赖性地同时给药诱导比单独BMP-2更大量的矿化骨组织。这些发现清楚地表明，肝素不仅在体外而且在体内增强BMP诱导的成骨细胞分化。他们还表明，肝素保护BMP免受靶细胞周围的抑制性微环境的影响。

Although bone morphogenetic proteins (BMPs) are clinically useful for bone regeneration, large amounts of them are required to induce new bone formation in monkeys and humans. We recently found that heparin stimulated BMP activity in vitro. In the present study, we examined whether heparin can enhance the bone formation induced by BMPs in vivo and attempted to determine the molecular mechanisms by which heparin stimulates BMP activity using C2C12 myoblasts. Heparin enhanced BMP-2-induced gene expression and Smad1/5/8 phosphorylation at 24 hours and thereafter, though not within 12 hours. Heparitinase treatment did not affect the response of cells to BMP-2. The half-life of BMP-2 in culture media was prolonged more that 10-fold in the presence of heparin. Although noggin mRNA was induced by BMP-2 within 1 hour regardless of the presence of heparin, noggin failed to inhibit BMP-2 activity in the presence of heparin. Furthermore, simultaneous administration of BMP-2 and heparin in vivo dose-dependently induced larger amounts of mineralized bone tissue than BMP-2 alone. These findings clearly indicate that heparin enhances BMP-induced osteoblast differentiation not only in vitro but also in vivo. They also suggest that heparin protects BMPs from the inhibitory microenvironment around target cells.