Miller Rachel E, Grodzinsky Alan J, Cummings Kiersten, Plaas Anna H K, Cole Ada A, Lee Richard T, Patwari Parth
Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Arthritis Rheum. 2010 Dec;62(12):3686-94. doi: 10.1002/art.27709.
Insulin-like growth factor 1 (IGF-1) stimulates cartilage repair but is not a practical therapy due to its short half-life. We have previously modified IGF-1 by adding a heparin-binding domain and have shown that this fusion protein (HB-IGF-1) stimulates sustained proteoglycan synthesis in cartilage. This study was undertaken to examine the mechanism by which HB-IGF-1 is retained in cartilage and to test whether HB-IGF-1 provides sustained growth factor delivery to cartilage in vivo and to human cartilage explants.
Retention of HB-IGF-1 and IGF-1 was analyzed by Western blotting. The necessity of heparan sulfate (HS) or chondroitin sulfate (CS) glycosaminoglycans (GAGs) for binding was tested using enzymatic removal and cells with genetic deficiency of HS. Binding affinities of HB-IGF-1 and IGF-1 proteins for isolated GAGs were examined by surface plasmon resonance and enzyme-linked immunosorbent assay.
In cartilage explants, chondroitinase treatment decreased binding of HB-IGF-1, whereas heparitinase had no effect. Furthermore, HS was not necessary for HB-IGF-1 retention on cell monolayers. Binding assays showed that HB-IGF-1 bound both CS and HS, whereas IGF-1 did not bind either. After intraarticular injection in rat knees, HB-IGF-1 was retained in articular and meniscal cartilage, but not in tendon, consistent with enhanced delivery to CS-rich cartilage. Finally, HB-IGF-1 was retained in human cartilage explants but IGF-1 was not.
Our findings indicate that after intraarticular injection in rats, HB-IGF-1 is specifically retained in cartilage through its high abundance of CS. Modification of growth factors with heparin-binding domains may be a new strategy for sustained and specific local delivery to cartilage.
胰岛素样生长因子1（IGF-1）可刺激软骨修复，但因其半衰期短而并非一种实用的治疗方法。我们之前通过添加一个肝素结合结构域对IGF-1进行了修饰，并已表明这种融合蛋白（HB-IGF-1）可刺激软骨中蛋白聚糖的持续合成。本研究旨在探讨HB-IGF-1保留在软骨中的机制，并测试HB-IGF-1在体内以及对人软骨外植体是否能为软骨提供持续的生长因子递送。
通过蛋白质印迹法分析HB-IGF-1和IGF-1的保留情况。使用酶促去除法和硫酸乙酰肝素（HS）基因缺陷细胞测试硫酸乙酰肝素（HS）或硫酸软骨素（CS）糖胺聚糖（GAGs）结合的必要性。通过表面等离子体共振和酶联免疫吸附测定法检测HB-IGF-1和IGF-1蛋白对分离出的GAGs的结合亲和力。
在软骨外植体中，软骨素酶处理降低了HB-IGF-1的结合，而肝素酶则无影响。此外，HS对于HB-IGF-1保留在细胞单层上并非必需。结合试验表明，HB-IGF-1与CS和HS均结合，而IGF-1两者均不结合。在大鼠膝关节内注射后，HB-IGF-1保留在关节软骨和半月板软骨中，但不在肌腱中，这与向富含CS的软骨中递送增强一致。最后，HB-IGF-1保留在人软骨外植体中，但IGF-1则不然。
我们的研究结果表明，在大鼠膝关节内注射后，HB-IGF-1通过其高丰度的CS特异性保留在软骨中。用肝素结合结构域修饰生长因子可能是一种向软骨进行持续且特异性局部递送的新策略。
