Markway Brandon D, Cho Holly, Johnstone Brian
Arthritis Res Ther. 2013 Aug 21;15(4):R92. doi: 10.1186/ar4272.
Hypoxia is considered to be a positive influence on the healthy chondrocyte phenotype and cartilage matrix formation. However, hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of osteoarthritis (OA). Thus, we assessed whether healthy and OA chondrocytes have distinct responses to oxygen, particularly with regard to hypertrophy and degradation during redifferentiation.
Monolayer-expanded healthy and OA chondrocytes were redifferentiated for 14 days in pellet cultures under standard (20% oxygen) or hypoxic (2% oxygen) conditions. Cartilage matrix gene expression, matrix quality and quantity, degradative enzyme expression and HIF expression were measured.
In hypoxia, both healthy and OA chondrocytes had higher human collagen type II, α1 gene (COL2A1), and aggrecan (ACAN) expression and sulfated glycosaminoglycan (sGAG) accumulation, concomitant with lower human collagen type X, α1 gene (COL10A1), and human collagen type I, α1 gene (COL1A1), expression and collagen I extracellular accumulation. OA chondrocytes had significantly lower sGAGs/DNA than healthy chondrocytes, but only in high oxygen conditions. Hypoxia also caused significantly greater sGAG retention and hyaluronic acid synthase 2 (HAS2) expression by OA chondrocytes. Both healthy and OA chondrocytes had significantly lower expression of matrix metalloproteinases (MMPs) MMP1, MMP2, MMP3 and MMP13 in hypoxia and less active MMP2 enzyme, consistent with lower MMP14 expression. However, aggrecanase (ADAMTS4 and ADAMTS5) expression was significantly lowered by hypoxia only in healthy cells, and COL10A1 and MMP13 remained significantly higher in OA chondrocytes than in healthy chondrocytes in hypoxic conditions. HIF-1α and HIF-2α had similar expression profiles in healthy and OA cells, increasing to maximal levels early in hypoxia and decreasing over time.
Hypoxic culture of human chondrocytes has long been acknowledged to result in increased matrix accumulation, but still little is known of its effects on catabolism. We show herein that the increased expression of matrix proteins, combined with decreased expression of numerous degradative enzymes by hypoxia, minimizes but does not abolish differences between redifferentiated healthy and OA chondrocytes. Hypoxia-induced HIF expression is associated with hypertrophic marker and degradative enzyme downregulation and increased measures of redifferentiation in both healthy and OA chondrocytes. Therefore, though HIFs may be involved in the pathogenesis of OA, conditions that promote HIF expression in vitro promote matrix accumulation and decrease degradation and hypertrophy, even in cells from OA joints.
缺氧被认为对健康软骨细胞表型和软骨基质形成具有积极影响。然而，缺氧诱导因子（HIFs）与骨关节炎（OA）的发病机制有关。因此，我们评估了健康软骨细胞和OA软骨细胞对氧气是否有不同反应，特别是在再分化过程中的肥大和降解方面。
将单层扩增的健康软骨细胞和OA软骨细胞在标准（20%氧气）或缺氧（2%氧气）条件下的微团培养中再分化14天。检测软骨基质基因表达、基质质量和数量、降解酶表达以及HIF表达。
在缺氧条件下，健康软骨细胞和OA软骨细胞的人II型胶原蛋白α1基因（COL2A1）和聚集蛋白聚糖（ACAN）表达均较高，硫酸化糖胺聚糖（sGAG）积累增加，同时人X型胶原蛋白α1基因（COL10A1）和人I型胶原蛋白α1基因（COL1A1）表达降低，胶原蛋白I细胞外积累减少。OA软骨细胞的sGAGs/DNA显著低于健康软骨细胞，但仅在高氧条件下如此。缺氧还导致OA软骨细胞的sGAG保留和透明质酸合酶2（HAS2）表达显著增加。在缺氧条件下，健康软骨细胞和OA软骨细胞的基质金属蛋白酶（MMPs）MMP1、MMP2、MMP3和MMP13表达均显著降低，MMP2酶活性降低，这与MMP14表达降低一致。然而，缺氧仅使健康细胞中的聚集蛋白聚糖酶（ADAMTS4和ADAMTS5）表达显著降低，并且在缺氧条件下，OA软骨细胞中的COL10A1和MMP13仍显著高于健康软骨细胞。HIF-1α和HIF-2α在健康细胞和OA细胞中的表达谱相似，在缺氧早期增加至最高水平，随后随时间下降。
长期以来，人们一直认为人软骨细胞的缺氧培养会导致基质积累增加，但对其在分解代谢方面的影响仍知之甚少。我们在此表明，缺氧导致的基质蛋白表达增加，同时多种降解酶表达降低，使再分化的健康软骨细胞和OA软骨细胞之间差异最小化，但并未消除差异。缺氧诱导的HIF表达与肥大标志物和降解酶下调以及健康软骨细胞和OA软骨细胞再分化指标增加有关。因此，尽管HIFs可能参与OA的发病机制，但在体外促进HIF表达的条件会促进基质积累并减少降解和肥大，即使是来自OA关节的细胞也是如此。
