Transgene enhancement of hyaluronan in human osteoarthritic cartilage

描述（由申请人提供）：骨性关节炎（OA）进展中的一个关键事件是关节软骨细胞外基质中蛋白多糖聚集物的丢失。除了聚集蛋白外，与正常人软骨相比，OA患者软骨中透明质酸（HA）的含量也显著降低，透明质酸是软骨蛋白聚糖聚集体的核心纤维。这些损失是由于常驻软骨细胞代谢状态的改变。在过去的研究中，我们已经验证了HA转换主要通过HA受体CD44介导的内吞事件发生的假设。上调聚集蛋白和HA损失的实验条件也会影响CD44表达的增强和HA内吞作用的相应增加。此外，我们还测试了软骨细胞是否也使用CD44通过启动信号级联来“感知”和响应蛋白多糖的损失。直到最近，我们只能通过软骨细胞培养研究来检测CD44的功能。这个新提议的中心假设是，受损软骨细胞外基质（ECM）内的HA是有限的。因此，基质修复和聚集蛋白保留是无效的。我们最近已经成功地通过使用人腺病毒和腺相关病毒构建体将人转基因引入完整的人、牛和小鼠软骨外植体中。该建议包括若干创新方法。我们将使用基因转移方法将HA合成酶（HAS2）转基因引入完整的人OA软骨外植体。这种方法将在ECM内产生选择性和大量的HA，以解决一个问题，即当驻留的软骨细胞被诱导进入静止状态时，局部合成的HA原位沉积的增加是否提供软骨修复和聚集蛋白溶解生物标志物的减少。我们的第二个目标是确定HA诱导的恢复稳态的机制，以及HA受体如CD44是否起作用。我们的长期目标是确定透明质酸对软骨稳态的重要性，以及如何改变体内特别是人体关节软骨内的透明质酸代谢。

DESCRIPTION (provided by applicant): A key event in the progression of osteoarthritis (OA) is the loss of proteoglycan aggregate from the extracellular matrix of articular cartilage. In addition to aggrecan, a significant decrease in hyaluronan (HA), the core filament of the cartilage proteoglycan aggregate, is also observed in cartilage derived from OA patients as compared to normal human cartilage. These losses are due to changes in the metabolic state of resident chondrocytes. In past studies we have tested the hypothesis that HA turnover occurs primarily through an endocytosis event, mediated by the HA receptor CD44. Experimental conditions that upregulate the loss of aggrecan and HA, also affect an enhancement in CD44 expression and a coordinate increase in HA endocytosis. In addition we have tested whether chondrocytes also use CD44 to "sense" and respond to a loss of proteoglycan by initiating signaling cascades. Until recently we have only been able to examine CD44 functions by using chondrocyte cell-culture studies. The central hypothesis of this new proposal is that HA within the extracellular matrix (ECM) of damaged cartilage is limiting. As such, matrix repair and aggrecan retention are ineffective. We have been recently successful at introducing human transgenes into intact human, bovine and mouse cartilage explants through the use of human adenoviral and adeno-associated virus constructs. This proposal includes several innovative approaches. We will use a gene transfer approach to introduce a HA synthase (HAS2) transgene into intact human OA cartilage explants. This approach will generate a selective and substantial increase in HA within the ECM to address the question of whether an increased deposition of locally-synthesized HA in situ provides for cartilage repair and a diminution of aggrecanolysis biomarkers as resident chondrocytes are coaxed into a state of quiescence. Our second goal will be to determine the mechanism for the HA-induced return to homeostasis and whether HA receptors such as CD44 are responsible. Our long-term goal is to determine the importance of HA to cartilage homeostasis and, how to alter HA metabolism in vivo especially within human articular cartilage.