Thrombospondin-1: a proatherosclerotic protein augmented by hyperglycemia

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.