Hypoxic-induction of arginase II requires EGF-mediated EGFR activation in human pulmonary microvascular endothelial cells

Abstract

We have previously shown that hypoxia‐induced proliferation of human pulmonary microvascular endothelial cells (hPMVEC) depends on arginase II, and that epidermal growth factor receptor (EGFR) is necessary for hypoxic‐induction of arginase II. However, it remains unclear how hypoxia activates EGFR‐mediated signaling in hPMVEC. We hypothesized that hypoxia results in epidermal growth factor (EGF) production and that EGF binds to EGFR to activate the signaling cascade leading to arginase II induction and proliferation in hPMVEC. We found that hypoxia significantly increased the mRNA levels of EGF, EGFR, and arginase in hPMVEC. Hypoxia significantly increased pEGFR(Tyr845) protein levels and an EGF neutralizing antibody prevented the hypoxic induction of pEGFR. Inhibiting EGFR activation prevented hypoxia‐induced arginase II mRNA and protein induction. Treatment of hPMVEC with exogenous EGF resulted in greater levels of arginase II protein both in normoxia and hypoxia. An EGF neutralizing antibody diminished hypoxic induction of arginase II and resulted in fewer viable cells in hPMVEC. Similarly, siRNA against EGF prevented hypoxic induction of arginase II and resulted in fewer viable cells. Finally, conditioned media from hypoxic hPMVEC induced proliferation in human pulmonary artery smooth muscle cells (hPASMC), however, conditioned media from a group of hypoxic hPMVEC in which EGF were knocked down did not promote hPASMC proliferation. These findings demonstrate that hypoxia‐induced arginase II expression and cellular proliferation depend on autocrine EGF production leading to EGFR activation in hPMVEC. We speculate that EGF‐EGFR signaling may be a novel therapeutic target for pulmonary hypertensive disorders associated with hypoxia.

Hypoxic pulmonary endothelial cells release epidermal growth factor leading to vascular smooth muscle cell arginase‐2 expression and proliferation

“…In the present study, we provide further evidence that hypoxia results in release of EGF by showing that treatment of endothelial cells with an EGF neutralizing antibody during the generation of HCM prevented the subsequent HCM-induced increases in Arg2 protein and viable cell numbers in hPASMC (Figure 2). This is also consistent with our previous report in hPMVEC that culture in hypoxia-induced a robust induction of EGF mRNA, and that an EGF neutralizing antibody or siRNA transfection against EGF prevented the hypoxia-induced endothelial cell Arg2 protein expression and increase in viable endothelial cell numbers (Pool et al, 2018). It has been shown that hPASMC respond to EGF treatment with a robust proliferative response (Schultz et al, 2006;Tu et al, 2012).…”

“…Human PMVEC were cultured as previously described (Pool et al, 2018 ; Toby et al, 2010 ; White et al, 2017 ). Briefly, fetal hPMVEC were purchased from ScienCell (Carlsbad, CA) and were all from females including lot#5016, lot#15900, lot#17807, and lot#15902.…”

“…We have previously shown (figure 8 in Pool et al (2018)) that HCM-induced increases in hPASMC Arg2 and proliferation were prevented using EGF-deficient conditioned media collected after transfection with siRNA against EGF in hPMVEC that were placed in hypoxia for 24 h. To further delineate the role of released EGF into the conditioned media on the HCM-induced increases in Arg2 protein expression and viable cell numbers we used an EGF neutralizing antibody added to the EC media during the generation of conditioned media. The hPMVEC were treated with either EGF neutralizing antibody (EGFnAb) or an isotype IgG control antibody (IgG) during incubation in hypoxia for 24 h to generate either EGFnAb-HCM or IgG-HCM.…”

“…Furthermore, we have described that EGFR activation on hPMVEC leads to the downstream activation of extracellular signal‐regulated kinase (ERK) and that this EGFR‐ERK pathway is necessary for hypoxia‐induced Arg2 expression in hPMVEC (White et al, 2017 ). Recently, we demonstrated in hPMVEC incubated in hypoxic conditions that EGF‐mediated activation of EGFR was necessary for hypoxia‐induced Arg2 expression and proliferation (Pool et al, 2018 ). Furthermore, we demonstrated that conditioned media from hypoxic hPMVEC induced proliferation in hPASMC that was prevented by treating the hPMVEC with an siRNA against EGF prior to generating the hypoxic conditioned media (see figure 8 in Pool et al ( 2018 )).…”

“…Recently, we demonstrated in hPMVEC incubated in hypoxic conditions that EGF‐mediated activation of EGFR was necessary for hypoxia‐induced Arg2 expression and proliferation (Pool et al, 2018 ). Furthermore, we demonstrated that conditioned media from hypoxic hPMVEC induced proliferation in hPASMC that was prevented by treating the hPMVEC with an siRNA against EGF prior to generating the hypoxic conditioned media (see figure 8 in Pool et al ( 2018 )). These data support our postulate that hypoxia induces EGF production in pulmonary endothelial cells, which is released and can cause proliferation in vascular SMCs.…”