FIGURE 3.

An EGFR blocking antibody prevented the HCM‐induced increase in hPASMC Arg2 protein and viable cell numbers. Either an IgG control (IgG‐HCM) antibody or an antibody against EGFR (EGFRbAb‐HCM) was added to the media during the generation of hypoxic conditioned media. NCM was generated as described in Figure 1. An additional set of hPASMC were treated with HCM that had EGFR blocking antibody added to it just prior to placing on the hPASMC (HCM + EGFRbAb) The NCM, IgG‐HCM, EGFRbAb‐HCM, or HCM + EGFRbAb was then placed on hPASMC with smooth muscle media in a 50:50 ratio and the hPASMC were incubated in room air with 5% CO₂ for 24 h. (a) Protein was harvested for western blotting for Arg2 and β‐actin. Representative western blot images. (b) Arg2 protein levels were greater in hPASMC incubated with IgG‐HCM than in those incubated with NCM, EGFRbAb‐HCM, or HCM + EGFRbAb. Densitometry for the Arg2 western blots normalized to β‐actin. *Different from NCM p < 0.005. #Different from IgG‐HCM, p < 0.001. n = 6 for each group. (c) To measure viable hPASMC numbers, 1 × 10⁴ hPASMC were plated in each well of 6‐well plates, NCM, IgG‐HCM, EGFRbAb‐HCM, or HCM + EGFRbAb were placed on the cells, and after 48 h in normoxia viable cell numbers were determined using trypan blue exclusion. Viable hPASMC numbers were significantly greater after incubation with IgG‐HCM than after incubation with NCM, EGFRbAb‐HCM, or HCM + EGFRbAb. *Different from NCM p < 0.001, #Different from IgG‐HCM, p < 0.001. n = 6 for each group.