Early life stress induces dysregulation of the heme pathway in adult mice.

Early life stress (ELS) is associated with cardiovascular disease (CVD) risk in adulthood, but the underlying vascular mechanisms are poorly understood. Increased hemoglobin and heme have recently been implicated to mediate endothelial dysfunction in several vascular diseases. Chronic physiological stress is associated with alterations in the heme pathway that have been well-described in the literature. However, very little is known about the heme pathway with exposure to ELS or chronic psychosocial stress. Utilizing a mouse model of ELS, maternal separation with early weaning (MSEW), we previously reported that MSEW induces endothelial dysfunction via increased superoxide production. We reasoned that heme dysregulation may be one of the culprits induced by MSEW and sustained throughout adulthood; thus, we hypothesized that MSEW induces heme dysfunction. We investigated whether circulating levels of heme, a circulating pro-oxidant mediator, are increased by MSEW and examined the role of the heme metabolic pathway and heme homeostasis in this process. We found that circulating levels of heme are increased in mice exposed to MSEW and that plasma from MSEW mice stimulated higher superoxide production in cultured mouse aortic endothelial cells (MAECs) compared to plasma from normally reared mice. The heme scavenger hemopexin blunted this enhanced superoxide production. Splenic haptoglobin abundance was significantly lower and hemoglobin levels per red blood cell were significantly higher in MSEW versus control mice. These findings lead us to propose that ELS induces increased circulating heme through dysregulation of the haptoglobin-hemoglobin system representing a mechanistic link between ELS and CVD risk in adulthood.

High salt induces autocrine actions of ET-1 on inner medullary collecting duct NO production via upregulated ETB receptor expression.

The collecting duct endothelin-1 (ET-1), endothelin B (ETB) receptor, and nitric oxide synthase-1 (NOS1) pathways are critical for regulation of fluid-electrolyte balance and blood pressure control during high-salt feeding. ET-1, ETB receptor, and NOS1 are highly expressed in the inner medullary collecting duct (IMCD) and vasa recta, suggesting that there may be cross talk or paracrine signaling between the vasa recta and IMCD. The purpose of this study was to test the hypothesis that endothelial cell-derived ET-1 (paracrine) and collecting duct-derived ET-1 (autocrine) promote IMCD nitric oxide (NO) production through activation of the ETB receptor during high-salt feeding. We determined that after 7 days of a high-salt diet (HS7), there was a shift to 100% ETB expression in IMCDs, as well as a twofold increase in nitrite production (a metabolite of NO), and this increase could be prevented by acute inhibition of the ETB receptor. ETB receptor blockade or NOS1 inhibition also prevented the ET-1-dependent decrease in ion transport from primary IMCDs, as determined by transepithelial resistance. IMCD were also isolated from vascular endothelial ET-1 knockout mice (VEETKO), collecting duct ET-1 KO (CDET-1KO), and flox controls. Nitrite production by IMCD from VEETKO and flox mice was similarly increased twofold with HS7. However, IMCD NO production from CDET-1KO mice was significantly blunted with HS7 compared with flox control. Taken together, these data indicate that during high-salt feeding, the autocrine actions of ET-1 via upregulation of the ETB receptor are critical for IMCD NO production, facilitating inhibition of ion reabsorption.

Early life stress in male mice induces superoxide production and endothelial dysfunction in adulthood.

Early life stress (ELS) is a risk for cardiovascular disease in adulthood although very little mechanistic insight is available. Because oxidative stress and endothelial dysfunction are major contributors to cardiovascular risk, we hypothesized that ELS induces endothelial dysfunction in adult male mice via increased superoxide production. Studies employed a mouse model of ELS, maternal separation with early weaning (MSEW), in which litters were separated from the dam for 4 h/day [postnatal days (PD) 2-5] and 8 h/day (PD6-16), and weaned at PD17. Control litters remained undisturbed until weaning at PD21. When compared with control mice, thoracic aortic rings from adult male MSEW mice displayed significant endothelial dysfunction that was reversed by the superoxide scavenger, polyethylene glycol-superoxide dismutase (PEG-SOD). PEG-SOD-inhibitable superoxide production by aortae from MSEW mice was significantly greater than observed in control aortae, although unaffected by nitric oxide synthase inhibition, suggesting that uncoupled nitric oxide synthase was not responsible for the accelerated superoxide production. Aortic SOD expression, plasma SOD activity, and total antioxidant activity were similar in MSEW and control mice, indicating unaltered antioxidant capacity in MSEW mice. Increased expression of the NADPH oxidase subunits, NOX2 and NOX4, was evident in the aortae of MSEW mice. Moreover, endothelial dysfunction and superoxide production in MSEW mice was reversed with the NADPH oxidase inhibitor, apocynin, indicating increased NADPH oxidase-dependent superoxide production and endothelial dysfunction. The finding that MSEW induces superoxide production and endothelial dysfunction in adult mice may provide a mechanistic link between ELS and adult cardiovascular disease risk.