Proliferative synergy of ANG II and EGF in porcine aortic vascular smooth muscle cells

To test erowth effects of anriotensin II (ANG II) in porcine vascular smooth muscle cells (VSMC) and potential ANG II synergy with epidermal growth factor (EGF), we exposed subconfluent, near-quiescent porcine aortic VSMC to ANG II, EGF, or ANG II + EGF (each 10^-9 M) in Dulbecco's modified Eagle's-Ham's F-12 medium with insulin + 0.4% fetal calf serum (PCS) selected for minimal ANG II-degrading capacity. Cell number and DNA and protein synthesis (by [³H]-thymidine and [³⁵S]methionine incorporation, respectively) were determined serially over 1-6 days. ANG II alone induced an early 20% increase and then a plateau in cell number over the 0.4% PCS control (P < 0.01; n = 8), thus without sustained increase in proliferation rate. Yet ANG II alone did not increase fractional DNA or protein synthesis (each as cpm/10³ cells) and, by flow cytometry, reduced S phase fraction without increase in cell size. EGF alone induced brisk DNA synthesis yet minimal cell division over days 0-4, thus late-cycle arrest. ANG II + EGF, despite no increase in fractional DNA or protein synthesis rates over EGF alone, induced significant indomethacin-resistant dose-dependent (P < 0.001) increase in cell proliferation rate over EGF alone with a median effective dose of 5 × 10^-10 M ANG II, thus proliferative synergy. We propose that 1) ANG II induces a subpopulation of cells arrested in or beyond S phase to proceed through mitosis but does not influence G₁ traversal or S phase entry and 2) ANG II + EGF achieve proliferative synergy by complementary actions at sequential cell cycle loci, with EGF supporting progression from G₀/G₁ to S phase and ANG II inducing completion of mitosis by cells already in or beyond S phase ("late-cycle completion").