Mueller H K, Fritsche U, Haslinger A, Landgraf R
Medical Clinic, Innenstad, University of Munich, Germany.
Exp Clin Endocrinol Diabetes. 1997;105(1):32-8. doi: 10.1055/s-0029-1211724.
One of the crucial pathophysiological changes in diabetic angiopathy might be the glucose-induced synthesis of components of the extracellular matrix-like fibronectin. This effect has been described for endothelial and mesangial cells. In order to gain further insight into the mechanisms how glucose-induced fibronectin expression is regulated, confluent monolayers of human umbilical vein endothelial cells (HUVEC) were incubated with varying concentrations of D-glucose (5-30 mM) in a time-dependent manner. Using Westernblotting techniques with a monoclonal antibody, a 3 +/- 0.2-fold increase of the 220 kDa-signal corresponding to human fibronectin was visible after an incubation time of 6 h indicating de-novo synthesis. Using incubation times of 6 or 10 days, glucose-mediated fibronectin overexpression was still visible, reaching a maximum at 15 mM D-glucose. The corresponding maximum values were 2.4 +/- 0.3 (6 d) and 2.3 +/- 0.2 (10 d). There was a concomitant glucose-dependent onset of expression of Protein Kinase C (PKC)-isoforms PKC-delta (2.5 +/- 0.3-fold), PKC-epsilon (2 +/- 0.2-fold) and PKC-zeta (1.8 +/- 0.2-fold), which reached a maximum after 12 h and was still visible during long-term culture. Induction of fibronectin expression was also obtained using the PKC-activating phorbolester Phorbol-12-myristat-13-acetat (PMA) which mimicks glucose-induced PKC activation. Glucose-induced fibronectin expression was decreased by the PKC-inhibitor H-7 (1- [5-isoquinolinylsulfonyl]-2-methylpiperazine). These experiments suggest that: 1. Short-term induction of fibronectin expression mediated by glucose is probably PKC-triggered since concomitant induction of PKC-isoforms PKC-delta, PKC-epsilon and PKC-zeta was observed. 2. Long-term incubation with D-glucose leads to an ongoing concentration-dependent coexpression of fibronection and various PKC-isoforms. If glucose-induced, PKC-mediated de-novo synthesis of components of the connective tissue or the extracellular matrix may be important in the pathomechanism of diabetic angiopathy, has to be proven.
糖尿病性血管病关键的病理生理变化之一可能是葡萄糖诱导合成细胞外基质成分如纤连蛋白。这种效应在内皮细胞和系膜细胞中已有描述。为了进一步深入了解葡萄糖诱导纤连蛋白表达的调控机制，将汇合的人脐静脉内皮细胞（HUVEC）单层培养物与不同浓度的D -葡萄糖（5 - 30 mM）进行时间依赖性孵育。使用针对单克隆抗体的蛋白质印迹技术，孵育6小时后，对应于人纤连蛋白的220 kDa信号增加了3±0.2倍，表明有从头合成。使用6天或10天的孵育时间，葡萄糖介导的纤连蛋白过表达仍然可见，在15 mM D -葡萄糖时达到最大值。相应的最大值分别为2.4±0.3（6天）和2.3±0.2（10天）。同时出现了葡萄糖依赖性的蛋白激酶C（PKC）同工型PKC -δ（2.5±0.3倍）、PKC -ε（2±0.2倍）和PKC -ζ（1.8±0.2倍）的表达开始，在12小时后达到最大值，并且在长期培养过程中仍然可见。使用模拟葡萄糖诱导PKC活化的PKC激活剂佛波酯佛波醇 - 12 -肉豆蔻酸酯 - 13 -乙酸酯（PMA）也可诱导纤连蛋白表达。葡萄糖诱导的纤连蛋白表达被PKC抑制剂H - 7（1 - [5 -异喹啉磺酰基]-2 -甲基哌嗪）降低。这些实验表明：1. 葡萄糖介导的纤连蛋白表达短期诱导可能由PKC触发，因为观察到PKC同工型PKC -δ、PKC -ε和PKC -ζ同时被诱导。2. 用D -葡萄糖长期孵育导致纤连蛋白和各种PKC同工型持续的浓度依赖性共表达。葡萄糖诱导的、PKC介导的结缔组织或细胞外基质成分的从头合成在糖尿病性血管病发病机制中是否重要，还有待证实。
