Inhibition of phosphatidylinositol 3-kinase and protein kinase C attenuates extracellular matrix protein-induced vascular smooth muscle cell chemotaxis.
OBJECTIVE
Intimal hyperplasia (IH), a significant cause of vascular reconstructive failure, is characterized by abnormal vascular smooth muscle cell (VSMC) migration, proliferation, and extracellular matrix (ECM) deposition. The ECM proteins, thrombospondin-1 (TSP-1), fibronectin (Fn), and vitronectin (Vn) can induce VSMC migration; however, the cellular signaling pathways involved are not identical for each ECM protein. Phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) are two enzymes that have been associated with VSMC migration. We sought to elucidate the roles of these enzymes in TSP-1-, Fn-, and Vn-stimulated VSMC migration.


METHODS
Chemotaxis assays were performed by using a modified Boyden Chamber. TSP-1, Fn, or Vn (20 microg/mL) or serum-free media (SFM) was placed in the bottom wells of the chamber. Quiescent bovine aortic VSMC were preincubated with LY 294002 (100 micromol/L), a PI3K inhibitor, bisindolylmaleimide I (GF 109203X, 1 micromol/L), a PKC inhibitor, or in SFM alone for 30 minutes. VSMCs (50,000 cells per well) were then placed in the top wells of the chamber, and the assay was conducted for 4 hours at 37 degrees C. Results were recorded as the number of cells migrated per five fields (400x) and analyzed by means of the paired t test, with P value less than.05 considered to be significant (n = 3).


RESULTS
The VSMC migration was significantly increased by TSP-1, Fn, and Vn. LY 294002 inhibited TSP-1-, Fn-, and Vn-stimulated VSMC migration (85% to 89%, P <.05). GF 109203X inhibited only TSP-1-stimulated migration (65%, P <.05).


CONCLUSIONS
These results suggest that TSP-1-, Fn-, and Vn-stimulated migration is at least partially dependent on PI3K. However, only TSP-1 stimulated migration is at least partially dependent on PKC.
Willis
A I
AI
Yale University School of Medicine, New Haven, CT 06520, USA.
Fuse
S
S
Wang
X J
XJ
Chen
E
E
Tuszynski
G P
GP
Sumpio
B E
BE
Gahtan
V
V
D000818:Animals; D002417:Cattle; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects*; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016326:Extracellular Matrix Proteins / Q000378:metabolism / Q000494:pharmacology*; D005353:Fibronectins / Q000494:pharmacology; D006965:Hyperplasia; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D015398:Signal Transduction / Q000187:drug effects; D013223:Statistics as Topic; D019700:Thrombospondin 1 / Q000494:pharmacology; D017539:Tunica Intima / Q000187:drug effects / Q000473:pathology; D019096:Vitronectin / Q000494:pharmacology
D002867:Chromones; D004791:Enzyme Inhibitors; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D007211:Indoles; D008301:Maleimides; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019700:Thrombospondin 1; D019096:Vitronectin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D011493:Protein Kinase C; C070515:bisindolylmaleimide I