Transgene enhancement of hyaluronan in human osteoarthritic cartilage

描述(申请人提供)：骨关节炎(OA)进展过程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体的丢失。除聚集素外，与正常人软骨相比，骨性关节炎患者软骨中的透明质酸(HA)显著减少，透明质酸(HA)是软骨蛋白多糖聚合体的核心纤维。这些损失是由于常驻软骨细胞代谢状态的变化造成的。在过去的研究中，我们已经验证了这样的假设，即HA周转主要通过HA受体CD44介导的内吞作用发生。上调聚集素和透明质酸丢失的实验条件，也影响CD44表达的增强和透明质酸内吞作用的协调增加。此外，我们还测试了软骨细胞是否也使用CD44来“感知”蛋白多糖的丢失，并通过启动信号级联反应来响应。直到最近，我们还只能通过软骨细胞培养研究来检测CD44的功能。这一新提议的中心假设是受损软骨的细胞外基质(ECM)中的HA是有限的。因此，基质修复和聚集素保留是无效的。最近，我们通过使用人腺病毒和腺相关病毒的构建，成功地将人类转基因引入完整的人、牛和小鼠软骨移植中。这项提议包括几种创新的方法。我们将使用基因转移的方法将HA合成酶(HAS2)转基因导入完整的人骨关节炎软骨移植中。这种方法将在ECM内产生选择性和实质性的HA增加，以解决本地合成的HA在原位沉积的增加是否有助于软骨修复，以及当常驻软骨细胞被诱使进入静止状态时聚集溶解生物标志物的减少。我们的第二个目标将是确定HA诱导的内稳态恢复的机制，以及是否HA受体如CD44起作用。我们的长期目标是确定HA对软骨动态平衡的重要性，以及如何改变体内尤其是人关节软骨中的HA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.