Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.
Curr Eye Res. 2011 Aug;36(8):747-53. doi: 10.3109/02713683.2011.585735.
The following study was designed to investigate early biosynthetic and ultrastructural changes that alter functional properties of the basement membrane (BM) and affect vascular permeability in diabetic retinopathy.
To determine whether altered matrix synthesis affects cell monolayer permeability, rat retinal endothelial cells (RRECs) were grown for 4 days to confluency in normal (N, 5 mM) or high glucose (HG, 30 mM) medium on transwell inserts and subjected to an in vitro cell monolayer permeability assay. Inserts were cut out and viewed under a transmission electron microscope to assess extracellular matrix (ECM) accumulation and cell morphology. In parallel cell cultures, fibronectin and collagen IV protein expression were determined using Western Blot analysis.
Electron microscopic analysis of cells exposed to short-term HG showed no difference in inter-endothelial cell tight junctions (TJs) or in the number of vesicles or coated pits compared to those of normal cells. However, ECM accumulation underlying HG cells was significantly increased compared to that of cells grown in N medium (139 ± 7% of control, p = 0.04), with areas of focal thickening. Western blot analysis showed increased fibronectin and collagen IV expression (152 ± 24% of control, p = 0.01; 146 ± 16% of control, p = 0.02, respectively) in cells grown in HG compared to those grown in N medium. Cell monolayers grown in HG exhibited increased permeability to FITC-dextran compared to cells grown in N medium (134 ± 15% of control, p = 0.02).
High glucose-induced excess ECM accumulation and altered composition underlies structural and functional changes that allow increased permeability. This finding provides evidence for the first time that the thickened vascular basement membrane contributes to the development of excess permeability seen in diabetic retinopathy.
本研究旨在探讨早期生物合成和超微结构变化，这些变化改变了基底膜的功能特性并影响糖尿病性视网膜病变中的血管通透性。
为了确定基质合成的改变是否会影响细胞单层通透性，将大鼠视网膜内皮细胞（RRECs）在正常（N，5mM）或高糖（HG，30mM）培养基中于 Transwell 插入物上培养 4 天至汇合，并进行体外细胞单层通透性测定。取出插入物并在透射电子显微镜下观察，以评估细胞外基质（ECM）的积累和细胞形态。在平行的细胞培养物中，使用 Western Blot 分析确定纤连蛋白和胶原 IV 蛋白的表达。
暴露于短期 HG 的细胞的电子显微镜分析显示，内皮细胞紧密连接（TJ）之间或囊泡或被覆陷窝的数量与正常细胞相比没有差异。然而，与在 N 培养基中生长的细胞相比，HG 细胞下的 ECM 积累显著增加（比对照增加 139±7％，p=0.04），并有局灶性增厚区域。Western blot 分析显示，与在 N 培养基中生长的细胞相比，在 HG 中生长的细胞中纤连蛋白和胶原 IV 的表达增加（分别比对照增加 152±24％，p=0.01；比对照增加 146±16％，p=0.02）。与在 N 培养基中生长的细胞相比，在 HG 中生长的细胞单层对 FITC-右旋糖酐的通透性增加（比对照增加 134±15％，p=0.02）。
高葡萄糖诱导的 ECM 过度积累和组成改变是结构和功能变化的基础，这些变化允许通透性增加。这一发现首次提供证据表明，增厚的血管基底膜有助于糖尿病性视网膜病变中所见的通透性增加的发展。
