Effect of Circulating EGF‐like Ligands on NADPH Oxidase Expression in Vascular Cells

The mechanisms responsible for the increased risk of cardiovascular disease in patients with chronic inflammatory conditions are not clear. We and others have shown that the vascular NADPH oxidases have an important role in mediating vascular disease. Expression of the pro‐atherogenic Nox1 NADPH oxidase is regulated in cultured smooth muscle cells (SMC) by activation of the epidermal growth factor receptor (EGFR). We previously reported that epidermal growth factor‐like ligands (EGF‐LL) are increased in serum and vessels of a non‐human primate model of atherosclerosis. We hypothesized that increased circulating EGF‐LL in chronic inflammatory conditions enhance vascular disease via increased NADPH oxidase expression and activation. Cultured primary human aortic SMC were serum deprived and treated with the EGF‐LL amphiregulin (AREG), epiregulin (EREG), epidermal growth factor (EGF) and heparin‐binding EGF‐like growth factor (HB‐EGF) and NADPH oxidase expression measured by qPCR. Among the EGF‐LL, AREG was most effect at increasing Nox1 expression (4.5‐fold increase compared to control) in SMC, followed by HB‐EGF (1.7‐fold increase compared to control). We observed marked variability or no change of expression of the other NADPH oxidase subunits, including Nox4, Nox5, p22phox, NoxA1, NoxO1, p47phox, p67phox. EGF‐LL had no significant effect on the expression of NADPH oxidase in human EC, although there was variability in experiments. Because we previously showed that Nox1 activation contributes to cytokine activation of NFκB in SMC, we used immunohistochemistry to evaluate TNFα stimulated nuclear translocation of p65 in mouse and human SMCs exposed to EGF‐LLs. TNFα stimulated p65 activity to a much greater degree in human SMCs compared to mouse, but activity was unchanged by pretreatment with EGF‐LL. To better emulate in vivo conditions, we harvested aortic segments from C57BL/6 (WT) and Nox1 −/− mice. Segments were exposed to either AREG, a pro‐inflammatory cytokine mix of tumor necrosis factor (TNFα) and interleukin 1 beta (IL‐1β), or both. Exposure to AREG for 24 hours increased Nox1 expression in WT (2.3‐fold). Ongoing studies are examining the response in Nox1 −/− mice. In summary, these data suggest that circulating AREG may contribute to development of vascular disease by increasing SMC Nox1 expression and identify circulating EGF‐LL as potential therapeutic target in patients with chronic inflammation. Support or Funding Information FJM is supported by the Office of Research and Development, Department of Veterans Affairs [2I01BX001729] and the National Institutes of Health [HL130039]. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .