Heparin has long been known to slow the growth of vascular smooth muscle cells. However, the mechanism(s) by which heparin acts has yet to be resolved. The identification of a putative heparin receptor in endothelial cells with antibodies that blocked heparin binding to the cells provided the means to further examine the possible involvement of a heparin receptor in smooth muscle cell responses to heparin. Immunoprecipitation of a smooth muscle cell protein with the anti-heparin receptor antibodies provided evidence that the protein was present in smooth muscle cells. Experiments with the anti-heparin receptor antibodies indicate that the antibodies can mimic heparin in decreasing PDGF induced thymidine and BrdU incorporation. The anti-heparin receptor antibodies were also found to decrease MAPK activity levels after activation similarly to heparin. These results support the identification of a heparin receptor and its role in heparin effects on vascular smooth muscle cell growth.

Published data provide strong evidence that heparin treatment of proliferating vascular smooth muscle cells results in decreased signaling through the ERK pathway and decreases in cell proliferation. In addition, these changes have been shown to be mimicked by antibodies that block heparin binding to the cell surface. Here we provide evidence that the activity of protein kinase G is required for these heparin effects. Specifically, a chemical inhibitor of protein kinase G, Rp-8-pCPT-cGMS, eliminates heparin and anti-heparin receptor antibody effects on bromodeoxyuridine incorporation into growth factor stimulated cells. In addition, protein kinase G inhibitors decrease heparin effects on ERK activity, phosphorylation of the transcription factor ELK-1, and heparin induced MKP-1 synthesis. Although transient, the levels of cGMP increase in heparin treated cells. Finally, knock down of protein kinase G also significantly decreases heparin effects in growth factor activated vascular smooth muscle cells. Together, these data indicate that heparin effects on vascular smooth muscle cell proliferation depend, at least in part, on signaling through protein kinase G.

The ability of heparin to block proliferation of vascular smooth muscle cells has been well documented. It is clear that heparin treatment can decrease the level of ERK activity in vascular smooth muscle cells that are sensitive to heparin. In this study, the mechanism by which heparin induces decreases in ERK activity was investigated by evaluating the dual specificity phosphatase, MKP-1, in heparin treated cells. Heparin induced MKP-1 synthesis in a time and concentration dependent manner. The time-course of MKP-1 expression correlated with the decrease in ERK activity. Over the same time frame, heparin treatment did not result in decreases in MEK-1 activity which could have, along with constitutive phosphatase activity, accounted for the decrease in ERK activity. Antibodies against a heparin receptor also induced the synthesis of MKP-1 along with decreasing ERK activity. Blocking either phosphatase activity or synthesis also blocked heparin-induced decreases in ERK activity. Consistent with a role for MKP-1, a nuclear phosphatase, heparin treated cells exhibited decreases in nuclear ERK activity more rapidly than cells not treated with heparin. The data support MKP-1 as a heparin-induced phosphatase that dephosphorylates ERK, decreasing ERK activity, in vascular smooth muscle cells.