title: Thrombospondin-1, -2 and -5 have differential effects on vascular smooth muscle cell physiology.
abstract: BACKGROUND
  The thrombospondins (TSPs) are matricellular proteins that exert multifunctional effects by binding cytokines, cell-surface receptors and other proteins. TSPs play important roles in vascular pathobiology and are all expressed in arterial lesions. The differential effects of TSP-1, -2, and -5 represent a gap in knowledge in vascular smooth muscle cell (VSMC) physiology. Our objective is to determine if structural differences of the TSPs imparted different effects on VSMC functions critical to the formation of neointimal hyperplasia. We hypothesize that TSP-1 and -2 induce similar patterns of migration, proliferation and gene expression, while the effects of TSP-5 are different.
  
  
  METHODS
  Human aortic VSMC chemotaxis was tested for TSP-2 and TSP-5 (1-40 μg/mL), and compared to TSP-1 and serum-free media (SFM) using a modified Boyden chamber. Next, VSMCs were exposed to TSP-1, TSP-2 or TSP-5 (0.2-40 μg/mL). Proliferation was assessed by MTS assay. Finally, VSMCs were exposed to TSP-1, TSP-2, TSP-5 or SFM for 3, 6 or 24 h. Quantitative real-time PCR was performed on 96 genes using a microfluidic card. Statistical analysis was performed by ANOVA or t-test, with p < 0.05 being significant.
  
  
  RESULTS
  TSP-1, TSP-2 and TSP-5 at 20 μg/mL all induce chemotaxis 3.1 fold compared to serum-free media. TSP-1 and TSP-2 induced proliferation 53% and 54% respectively, whereas TSP-5 did not. In the gene analysis, overall, cardiovascular system development and function is the canonical pathway most influenced by TSP treatment, and includes multiple growth factors, cytokines and proteases implicated in cellular migration, proliferation, vasculogenesis, apoptosis and inflammation pathways.
  
  
  CONCLUSIONS
  The results of this study indicate TSP-1, -2, and -5 play active roles in VSMC physiology and gene expression. Similarly to TSP-1, VSMC chemotaxis to TSP-2 and -5 is dose-dependent. TSP-1 and -2 induces VSMC proliferation, but TSP-5 does not, likely due conservation of N-terminal domains in TSP-1 and -2. In addition, TSP-1, -2 and -5 significantly affect VSMC gene expression; however, little overlap exists in the specific genes altered. This study further delineates TSP-1, -2 and -5's contributions to processes related to VSMC physiology.
authors.affiliation: SUNY Upstate Medical University, Division of Vascular Surgery and Endovascular Services, Syracuse, NY, USA; Department of Veterans Affairs VA Healthcare Network Upstate New York at Syracuse, Syracuse, NY, USA.
authors.forename: Alex
authors.initials: A
authors.lastname: Helkin
authors.affiliation: SUNY Upstate Medical University, Division of Vascular Surgery and Endovascular Services, Syracuse, NY, USA; Department of Veterans Affairs VA Healthcare Network Upstate New York at Syracuse, Syracuse, NY, USA.
authors.forename: Kristopher G
authors.initials: KG
authors.lastname: Maier
authors.affiliation: SUNY Upstate Medical University, Division of Vascular Surgery and Endovascular Services, Syracuse, NY, USA; Department of Veterans Affairs VA Healthcare Network Upstate New York at Syracuse, Syracuse, NY, USA. Electronic address: gahtanv@upstate.edu.
authors.forename: Vivian
authors.initials: V
authors.lastname: Gahtan
mesh_terms: D064236:Cartilage Oligomeric Matrix Protein / Q000235:genetics / Q000494:pharmacology / Q000502:physiology; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D058426:Neointima / Q000209:etiology; D019700:Thrombospondin 1 / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D019699:Thrombospondins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D000099262:Thrombospondin 2
chemical_list: D064236:Cartilage Oligomeric Matrix Protein; D019700:Thrombospondin 1; D019699:Thrombospondins; D000099262:Thrombospondin 2; C577553:TSP5 protein, human
keywords: Cartilage oligomeric matrix protein; Cell migration; Cellular proliferation; Gene expression profiling; Thrombospondins; Vascular smooth muscles