Division of Vascular Surgery and Endovascular Services, SUNY Upstate Medical University, Syracuse, NY, USA.
J Vasc Surg. 2010 May;51(5):1238-47. doi: 10.1016/j.jvs.2009.11.073. Epub 2010 Mar 29.
Diabetes is associated with a more aggressive form of atherosclerosis. Thrombospondin-1 (TSP-1), an extracellular matrix protein, is an acute-phase reactant that induces vascular smooth muscle (VSMC) migration and proliferation in areas of vascular injury and is also up-regulated in VSMCs exposed to hyperglycemia. This study tested the hypothesis that hyperglycemia amplifies the expression of genes induced by TSP-1 in VSMCs.
Human aortic VSMCs were cultured in Dulbecco Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. Cells were used between passages three and five. VSMCs were preincubated in DMEM containing 0.2% FBS with 5 mM glucose (normoglycemia), 25 mM glucose (hyperglycemia), 25 mM mannose (osmotic control), TSP-1 (20 microg/mL), 25 mM glucose + TSP-1 (20 microg/mL), or 25 mM mannose + TSP-1 (20 microg/mL). Total RNA was extracted. Microarray analysis was performed and analyzed by analysis of variance. P < .05 was considered significant. Quantitative real-time polymerase chain reaction (rtPCR) was used to confirm selected up-regulated genes.
Microarray analysis revealed: (1) hyperglycemia altered 30 genes; (2) TSP-1 altered 212 genes, of which 8 were altered similarly to VSMCs exposed to 25 mM glucose; (3) TSP-1 up-regulated 10 genes associated with atherosclerosis and 4 others with diabetic vascular disease; (4) hyperglycemia combined with TSP-1 altered expression of 2822 genes. The three genes most up-regulated by TSP-1 in a normoglycemic environment were uridine 5'-diphosphoglucose (UDP-glucose) dehydrogenase (UGDH, 127%), transforming growth factor beta-2 (TGFbeta2, 116%), and hyaluronan synthase 2 (HAS2, 113%). Further, TSP-1 altered the expression of genes in 13 canonical pathways; however, when combined with hyperglycemia, 53 canonical pathways were affected.
Quantitative rtPCR confirmed that genes in several of these pathways for TSP-1 and hyperglycemia combined with TSP-1 were up-regulated. These findings suggest that TSP-1 may be germane to the progression of atherosclerosis and may have a large effect with concurrent hyperglycemia.
糖尿病与动脉粥样硬化的侵袭形式有关。血小板反应蛋白-1（TSP-1）是一种细胞外基质蛋白，是一种急性期反应物，可诱导血管损伤部位的血管平滑肌（VSMC）迁移和增殖，并且在暴露于高血糖的 VSMC 中也上调。本研究检验了以下假设：高血糖可放大 TSP-1 诱导的 VSMC 中基因的表达。
将人主动脉 VSMC 在补充有 10％胎牛血清（FBS）和 1％抗生素的 Dulbecco 改良 Eagle 培养基（DMEM）中培养。细胞使用三到五代之间。将 VSMC 用含 0.2％FBS 的 DMEM 在 5 mM 葡萄糖（正常血糖），25 mM 葡萄糖（高血糖），25 mM 甘露糖（渗透对照），TSP-1（20μg/mL），25 mM 葡萄糖+TSP-1（20μg/mL）或 25 mM 甘露糖+TSP-1（20μg/mL）中预孵育。提取总 RNA。进行微阵列分析，并通过方差分析进行分析。P <.05 被认为具有统计学意义。使用实时定量聚合酶链反应（rtPCR）确认所选上调基因。
微阵列分析显示：（1）高血糖改变了 30 个基因；（2）TSP-1改变了 212 个基因，其中 8 个基因与暴露于 25 mM 葡萄糖的 VSMC 相似；（3）TSP-1 上调了 10 个与动脉粥样硬化有关的基因和 4 个与糖尿病血管疾病有关的基因；（4）高血糖与 TSP-1 共同改变了 2822 个基因的表达。在正常血糖环境下 TSP-1 上调最明显的三个基因是尿苷二磷酸葡萄糖（UDP-葡萄糖）脱氢酶（UGDH，127％），转化生长因子β2（TGFbeta2，116％）和透明质酸合酶 2（HAS2，113％）。此外，TSP-1 改变了 13 个经典途径中的基因表达；但是，当与高血糖结合时，有 53 个经典途径受到影响。
实时定量 rtPCR 证实，这些途径中的某些基因与 TSP-1 以及与 TSP-1 联合的高血糖均上调。这些发现表明 TSP-1 可能与动脉粥样硬化的进展有关，并且与同时发生的高血糖症可能有很大的影响。
