Chen J, Brodsky S, Li H, Hampel D J, Miyata T, Weinstein T, Gafter U, Norman J T, Fine L G, Goligorsky M S
Department of Medicine and Physiology and Biophysics, State University of New York, Stony Brook, New York 11794-8152, USA.
Am J Physiol Renal Physiol. 2001 Jul;281(1):F71-80. doi: 10.1152/ajprenal.2001.281.1.F71.
Development of micro- and macrovascular disease in diabetes mellitus (DM) warrants a thorough investigation into the repertoire of endothelial cell (EC) responses to diabetic environmental cues. Using human umbilical vein EC (HUVEC) cultured in three-dimensional (3-D) native collagen I (NC) or glycated collagen I (GC), we observed capillary cord formation that showed a significant reduction in branching when cells were cultured in GC. To gain insight into the molecular determinants of this phenomenon, HUVEC subjected to GC vs. NC were studied using a PCR-selected subtraction approach. Nine different genes were identified as up- or downregulated in response to GC; among those, plasminogen activator inhibitor-1 (PAI-1) mRNA was found to be upregulated by GC. Western blot analysis of HUVEC cultured on GC showed an increase in PAI-1 expression. The addition of a neutralizing anti-PAI-1 antibody to HUVEC cultured in GC restored the branching pattern of formed capillary cords. In contrast, supplementation of culture medium with the constitutively active PAI-1 reproduced defective branching patterns in HUVEC cultured in NC. Ex vivo capillary sprouting in GC was unaffected in PAI-1 knockout mice but was inhibited in wild-type mice. This difference persisted in diabetic mice. In conclusion, the PCR-selected subtraction technique identified PAI-1 as one of the genes characterizing an early response of HUVEC to the diabetic-like interstitial environment modeled by GC and responsible for the defective branching of endothelial cells. We propose that an upregulation of PAI-1 is causatively linked to the defective formation of capillary networks during wound healing and eventual vascular dropout characteristic of diabetic nephropathy.
糖尿病（DM）中微血管和大血管疾病的发展促使人们深入研究内皮细胞（EC）对糖尿病环境信号的反应机制。我们将人脐静脉内皮细胞（HUVEC）培养在三维（3-D）天然I型胶原蛋白（NC）或糖化I型胶原蛋白（GC）中，观察到在GC中培养时，细胞形成的毛细血管索分支显著减少。为深入了解这一现象的分子决定因素，我们采用PCR筛选消减方法研究了GC与NC培养条件下的HUVEC。结果鉴定出9个因GC而表达上调或下调的不同基因；其中，纤溶酶原激活物抑制剂-1（PAI-1）mRNA被发现受GC上调。对GC上培养的HUVEC进行蛋白质免疫印迹分析显示PAI-1表达增加。向GC中培养的HUVEC添加中和性抗PAI-1抗体可恢复形成的毛细血管索的分支模式。相反，在培养基中添加组成型活性PAI-1会使NC中培养的HUVEC出现有缺陷的分支模式。在PAI-1基因敲除小鼠中，GC中的体外毛细血管发芽不受影响，但在野生型小鼠中受到抑制。这种差异在糖尿病小鼠中持续存在。总之，PCR筛选消减技术确定PAI-1是表征HUVEC对由GC模拟的糖尿病样间质环境早期反应的基因之一，并导致内皮细胞分支缺陷。我们认为，PAI-1的上调与伤口愈合过程中毛细血管网络形成缺陷以及糖尿病肾病最终的血管丢失存在因果关系。
