High glucose increases lysyl oxidase expression and activity in retinal endothelial cells: mechanism for compromised extracellular matrix barrier function.
High glucose increases lysyl oxidase expression and activity in retinal endothelial cells: mechanism for compromised extracellular matrix barrier function.
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作者：
Chronopoulos A;Tang A;Beglova E;Trackman PC;Roy S
在糖尿病中，视网膜血管基底膜（BM）经历显著增厚并损害血管功能，包括血管通透性增加，这是早期糖尿病视网膜病变的显著病变。在这项研究中，我们确定是否改变赖氨酰氧化酶（LOX），交联酶的表达和活性，可能会损害血管基底膜功能的完整性在高糖（HG）条件下。通过Western印迹分析和LOX酶活性评估在正常（5 mmol/l）或HG（30 mmol/l葡萄糖）培养基中生长7天的大鼠视网膜内皮细胞（RREC）的LOX和proLOX表达。为了确定HG是否改变LOX和proLOX的细胞分布模式，用各自的抗体进行免疫染色。类似地，使在正常或HG培养基中生长的细胞经受用β-氨基丙腈（BAPN）和通过小干扰RNA敲低的LOX抑制，并分别检查细胞单层渗透性。此外，分析链脲佐菌素（STZ）诱导的糖尿病大鼠的视网膜，以确定糖尿病是否改变LOX表达。Western blot分析显示，与正常培养基中生长的细胞相比，HG培养基中生长的细胞中LOX和proLOX表达显著增加。糖尿病视网膜中LOX水平的增加与LOX上调非常相似。在HG培养基中生长的细胞中，LOX活性和细胞单层通透性显著增加，LOX和proLOX免疫染色也显著增加。小干扰RNA或BAPN诱导的LOX表达或活性的特异性阻断，分别降低细胞单层通透性。HG诱导的LOX表达和活性增加损害了屏障功能的完整性，这是糖尿病视网膜病变的一个突出病变。
In diabetes, retinal vascular basement membrane (BM) undergoes significant thickening and compromises vessel function including increased vascular permeability, a prominent lesion of early diabetic retinopathy. In this study we determined whether altered expression and activity of lysyl oxidase (LOX), a cross-linking enzyme, may compromise vascular basement membrane functional integrity under high-glucose (HG) conditions. Rat retinal endothelial cells (RRECs) grown in normal (5 mmol/l) or HG (30 mmol/l glucose) medium for 7 days were assessed for expression of LOX and proLOX by Western blot analysis and LOX enzyme activity. To determine whether HG alters cellular distribution patterns of LOX and proLOX, immunostaining with respective antibodies was performed. Similarly, cells grown in normal or HG medium were subjected to both LOX inhibition with β-aminopropionitrile (BAPN) and by small interfering RNA knockdown, and respectively examined for cell monolayer permeability. Additionally, retinas of streptozotocin (STZ)-induced diabetic rats were analyzed to determine if diabetes altered LOX expression. Western blot analysis revealed significantly increased LOX and proLOX expression in cells grown in HG medium compared with those grown in normal medium. The increased LOX level was strikingly similar to LOX upegulation in the diabetic retinas. In cells grown in HG medium, LOX activity and cell monolayer permeability was significantly increased, as were LOX and proLOX immunostaining. Small interfering RNA- or BAPN–induced-specific blockage of LOX expression or activity, respectively, reduced cell monolayer permeability. HG-induced increased LOX expression and activity compromises barrier functional integrity, a prominent lesion of diabetic retinopathy.