EGFR stimulation promotes proliferation in human pulmonary microvascular endothelial cells

In endothelial cells, L‐arginine can be metabolized to NO and citrulline via NOS or to urea and ornithine via arginase. Previously we have shown that Arginase and NOS compete for their common substrate and that EGFR activation selectively promotes arginase expression and activity. Enhanced arginase activity might be expected to increase proliferation via ornithine production and its subsequent metabolism to proline and/or polyamines. We hypothesized that EGFR activation would lead to pulmonary microvascular endothelial cell proliferation. Primary human pulmonary microvascular endothelial cells (hPMVECs) were treated with EGF and an EGFR inhibitor (AG1478) for 0, 2 and 4 hrs. After which, protein was isolated followed by western blotting using total EGFR and anti‐phosphotyrosine antibodies. Treated hPMVECs were also used in a proliferation assay and the number of viable cells was counted using trypan blue exclusion. Levels of total EGFR and total phosphotyrosine decreased in hPMVECs treated with AG1478 compared to those groups without treatment at 2 and 4 hrs. The proliferation assay revealed that EGF treated hPMVECs had an elevated level of proliferation compared to the AG1478 treated hPMVECs at 24 hrs. AG1478 inhibited EGFR in hPMVECs as demonstrated by decreased expression of EGFR and phosphotyrosine. Our data demonstrate that EGFR expression is critical in promoting proliferation of hPMVECs. We speculate that EGFR induced proliferation is central to the vascular remodeling in pulmonary hypertensive diseases.