Heterogeneity in the response of vascular smooth muscle to heparin: altered signaling in heparin-resistant cells.
OBJECTIVE
Vascular smooth muscle cells show phenotypic heterogeneity in vivo that affects the extent to which they respond to the antimitogenic effects of heparin. In vitro, heparin-resistant cells are readily selected. This study was undertaken to determine whether differences in the antiproliferative response to heparin involve differences in activity of heparin-sensitive signal transduction pathways.


METHODS
Rat thoracic aorta smooth muscle cells (ASMC) at early passage together with two established vascular smooth muscle lines, PAC-1 and A10, were examined before and after selection for growth in the presence of heparin (10 micrograms/ml). Cells were rendered quiescent and then stimulated with serum.


RESULTS
The three cell types showed different sensitivities to the antimitogenic effects of heparin. With respect to [3H]thymidine incorporation, A10 cells were insensitive to 1 microgram/ml heparin whereas PAC-1 cells responded down to 0.05 microgram/ml and ASMC were of intermediate sensitivity. ASMC and PAC-1 cells but not A10 showed a decrease in c-fos mRNA in response to 1 microgram/ml heparin, and a decrease in the c-Fos content of AP-1 DNA binding activity. None of the cells had decreased c-jun mRNA in the presence of heparin. Although induction of c-fos by serum is thought to signal through the Erk mitogen activated protein kinase family, Erk activity was decreased more by 1 microgram/ml heparin in A10 cells than in PAC-1 or ASMC. When cells were selected by growth in the presence of 10 micrograms/ml heparin, A10 cells were unaffected but PAC-1 and ASMC showed a blunted effect of heparin on serum stimulation. In contrast to A10 and their controls not exposed to continuous heparin, heparin-selected PAC-1 and ASMC showed a diminished ability to induce c-fos in response to serum.


CONCLUSIONS
Smooth muscle cell lines show different responses to the antimitogenic effects of heparin that correlate with the heparin sensitivity of c-Fos/c-Jun expression. Although Erk is implicated in c-fos induction, cells comparatively resistant to heparin still show heparin-dependent inhibition of Erk activation, suggesting that other pathways may be more important for heparin resistance. Furthermore, cells selected for heparin resistance may develop c-fos-independent pathways for proliferation.
Templeton
D M
DM
Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada. doug.templeton@utoronto.ca
Zhao
Y
Y
Fan
M Y
MY
D000818:Animals; D001013:Aorta, Thoracic; D015152:Blotting, Northern; D002460:Cell Line; D002478:Cells, Cultured; D004789:Enzyme Activation; D015870:Gene Expression; D016762:Genes, fos; D016758:Genes, jun; D006493:Heparin / Q000494:pharmacology*; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D011651:Pulmonary Artery; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
D006493:Heparin; D020928:Mitogen-Activated Protein Kinases