Early‐life Stress Induces Increased Plasma Heme and Toll‐like Receptor 4 Activation Leading to Increased Superoxide Production in Aortic Endothelial Cells
Authors: Ho Dao H , Ring Ashley L , Pollock Jennifer S 
Published: November 2023

Epidemiological studies indicate that early‐life stress(ELS) is a significant, independent risk factor for cardiovascular disease. Our objective was to elucidate the potential mediators of ELS‐induced cardiovascular disease using a mouse model of ELS involving maternal separation and early weaning (MSEW). MSEW consists of separation of mice from dams during postnatal day (PD) 2 to 5 for 4h/day and PD 6 to 16 for 8h/day, and weaning at PD 17. Control mice remain undisturbed from birth until weaning at PD 21. Previously, we discovered that adult male mice subjected to MSEW exhibited aorticendothelial dysfunction due to increased aortic superoxide production compared to undisturbed control mice. Additionally, we found that MSEW induced increased free heme in plasma. Thus, we hypothesized that increased levels of heme in plasma resulting from MSEW stimulates superoxide production inaortic endothelial cells. Mouse aorticendothelial cells (MAECs) were incubated for 24 hours with plasma from controlmice or MSEW mice (25μl plasma/ml media), in the presence or absence of hemopexin (0.25μg/1μl plasma), a heme scavenger. We assessed superoxide production bydihydroethidium (DHE) assay. MAECsincubated in plasma from MSEW mice showed significant increase in superoxideproduction compared to MAECs incubated in plasma from control mice (344.1±58.0 versus157.8±27.1 pmol oxy‐ethidium/mg protein, p<0.05, n=10). Treatment of cells with hemopexin reduced superoxide generation in MAECs treated with MSEW plasma (344.1±58.0 versus193.5±47.8 pmol oxy‐ethidium/mg protein, p<0.05, n=10), whereas treatment with hemopexin did not reduce superoxide production in MAECs treated with control plasma (157.8±27.1 versus 171.5±38.2 mol oxy‐ethidium/mg protein, n=10). We further hypothesized that the heme‐induced superoxide production in MAECs is through a toll‐like receptor 4(TLR4)‐mediated pathway. MAECs were incubated for 24 hours in plasma from control mice or MSEW mice (25μl plasma/ml media), in the presence or absence of TAK‐242 (1μM), a TLR4 inhibitor. Treatment with TAK‐242 significantly reduced superoxide production in MAECs treated with MSEW plasma (141.8±16.0 versus77.20±20.1 pmol oxy‐ethidium/mg protein, p<0.05, n=10), whereas TAK‐242 had no effect on superoxide generation in MAECs treated with control plasma(73.9±9.1 versus 43.5±11.1 pmol oxy‐ethidium/mg protein, n=10). Furthermore, aortic tissue from MSEW mice displayed significantly greater mRNA expression of TLR4 compared to controlmice (1.65 fold increase from control, p=0.02, n=7–8). These findings indicate that elevated plasma heme in MSEW mice increased superoxide production in aorticendothelial cells through a TLR4‐mediated pathway. These observations suggest that MSEW induces activation of TLR4‐dependent oxidative stress and endothelial dysfunction in the aorta. In conclusion, ELS may lead to increased risk for cardiovascular disease through increased heme, oxidative stress, and TLR4 activation.Support or Funding InformationNIH P01 HL69999; NIH 1F32 HL116145