Grant M B, Caballero S, Tarnuzzer R W, Bass K E, Ljubimov A V, Spoerri P E, Galardy R E
Department of Medicine, University of Florida, Gainesville 32610-0226, USA.
Diabetes. 1998 Aug;47(8):1311-7. doi: 10.2337/diab.47.8.1311.
The degree of hyperglycemia correlates with the development of diabetic retinopathy. We investigated the effect of glucose on the expression of matrix metalloproteinase (MMP)-2 and MMP-9 (72-kDa and 92-kDa type IV collagenases, respectively) by human retinal microvascular endothelial cells (HRECs). Cultured HRECs from nondiabetic and diabetic donors were exposed to 5 or 30 mmol/l glucose. Using gelatin zymography, conditioned medium (CM) from all cultures revealed a gelatinolytic band migrating at 65 kDa (representing the proform of MMP-2 that runs at 72 kDa under reducing conditions). This band was unchanged by glucose exposure or the disease state of the donors. CM from nondiabetic HREC cultures demonstrated an additional proteolytic activity migrating at 90 kDa when cells were exposed to 30 mmol/l glucose, but not when they were exposed to 5 mmol/l glucose. This same activity was seen in CM from HREC cultures of diabetic origin in the presence of both 5 and 30 mmol/l glucose. Western analysis confirmed the identity of the 65-kDa band as MMP-2. The anomalous activity at 90 kDa was identified as MMP-2 associated and co-migrating with a fibronectin fragment. Competition-based reverse transcription-polymerase chain reaction revealed that nondiabetic and diabetic HRECs expressed constitutively mRNA for MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, and fibronectin. After exposure to 5 or 30 mmol/l glucose, no changes were detected in mRNA levels in MMP-2 or MMP-9, their inhibitors TIMP-1 and TIMP-2, or fibronectin in either nondiabetic or diabetic HREC cultures. These results support the notion that modulation of MMP function by extracellular matrix components occurs in response to glucose and may be relevant to the development of diabetic retinopathy.
高血糖程度与糖尿病视网膜病变的发展相关。我们研究了葡萄糖对人视网膜微血管内皮细胞（HRECs）基质金属蛋白酶（MMP）-2和MMP-9（分别为72-kDa和92-kDa的IV型胶原酶）表达的影响。将来自非糖尿病和糖尿病供体的培养HRECs暴露于5或30 mmol/l葡萄糖中。使用明胶酶谱法，所有培养物的条件培养基（CM）显示出一条迁移在65 kDa的明胶水解带（代表在还原条件下迁移为72 kDa的MMP-2前体形式）。该条带不受葡萄糖暴露或供体疾病状态的影响。当非糖尿病HRECs培养物的细胞暴露于30 mmol/l葡萄糖而非5 mmol/l葡萄糖时，CM显示出另一种迁移在90 kDa的蛋白水解活性。在5和30 mmol/l葡萄糖存在下，糖尿病来源的HRECs培养物的CM中也观察到相同的活性。蛋白质印迹分析证实65-kDa条带为MMP-2。90 kDa处的异常活性被鉴定为与纤连蛋白片段相关并共迁移的MMP-2。基于竞争的逆转录-聚合酶链反应显示，非糖尿病和糖尿病HRECs组成性表达MMP-2、MMP-9、金属蛋白酶组织抑制剂（TIMP）-1、TIMP-2和纤连蛋白的mRNA。在暴露于5或30 mmol/l葡萄糖后，非糖尿病或糖尿病HRECs培养物中MMP-2或MMP-9、其抑制剂TIMP-1和TIMP-2或纤连蛋白的mRNA水平均未检测到变化。这些结果支持这样一种观点，即细胞外基质成分对MMP功能的调节是对葡萄糖的反应，可能与糖尿病视网膜病变的发展有关。
