Fibrosis plays a major role in the progression of heart failure. Angiotensin II (Ang II) is a potent inducer of cardiac remodeling involving hypertrophy and fibrosis, which may be mediated in part through increases in reactive oxygen species (ROS). There is currently no drug treatment that can reverse fibrosis, however angiotensin converting enzyme inhibitors (ACEI) are able to slow the progression of fibrosis. Moreover, we have shown that protection against fibrotic remodeling persists even after ACEI treatment has been stopped. The mechanism underlying this long‐term protection has not yet been elucidated. In this study, we aimed to determine whether the beneficial effects of prior ACEI treatment are related to a more favorable oxidative stress profile in response to Ang II treatment. We hypothesize that hearts from hypertensive rats previously treated with ACEI will show decreased pro‐oxidant and increased antioxidant enzymes in response to Ang II. Adult (11‐week‐old) male and female spontaneously hypertensive rats were treated with vehicle or ACEI (enalapril, 30mg/kg/day) for two weeks, followed by a two‐week washout period. A subset of vehicle treated rats and all of the ACEI treated rats were then administered Ang II (200ng/kg/min) for 2 or 4 weeks (n=3/group per sex). Protein levels of pro‐oxidant (NOX2 and NOX4) and antioxidant (SOD1, SOD2, catalase) enzymes in the left ventricle (LV) were measured by western blotting. Ang II treatment caused a significant increase in LV hypertrophy after 4 weeks of Ang II. Overall, pro‐oxidant NOX4 levels were higher, while NOX2 levels were lower, in females compared to males regardless of treatment (p<0.05). This suggests alternative sources of ROS are implicated in the male vs. female hypertensive hearts. Additionally, Ang II treatment significantly increased NOX4 expression in males but not females (p<0.05) highlighting a sex‐specific responsiveness to Ang II. Antioxidant protein catalase expression is lower in females compared to males (p<0.05), while both sexes exhibited significant increases in response to Ang II (p<0.05). Ang II significantly decreased SOD1 expression in males with or without ACEI (p<0.05), while SOD2 tended to increase in females. ACEI treatment in males prevented Ang II‐induced effects in catalase and NOX2, but not other proteins, suggesting ACEI‐sensitive pathways. In summary, our findings reveal sex differences in both overall levels of NOX2, NOX4, and catalase, as well as responsiveness to Ang II and ACEI. Altered modulation of antioxidants may contribute to or compensate for sex‐ and Ang II‐dependent inflammation with hypertension. Determining whether oxidative stress is altered by prior ACEI treatment will allow for a better understanding of the mechanisms, and therefore therapeutic targets for slowing or preventing cardiac remodeling and heart failure. The unexpected finding of sex differences in oxidative protein profiles builds on a growing body of evidence that sex‐specific targeted therapies may be necessary for cardiovascular disease....

Hypertension promotes fibrotic cardiac remodeling that involves oxidative stress and inflammation that contribute to heart failure. Transient angiotensin converting enzyme inhibitor (ACEi) treatment in male hypertensive rats (SHR) produces persistent changes in the left ventricle (LV) that render it resistant to future fibrosis and inflammation. Oxidative stress produced by NADPH oxidase (NOX) enzymes is linked to angiotensin II (Ang II)‐mediated fibrotic signaling. Ang II also promotes secretion of macrophage‐recruiting cytokines including monocyte chemoattractant protein 1 (MCP1) which can perpetuate oxidative stress. Thus the present study investigated the impact of transient ACEi‐induced cardio‐protection on subsequent Ang II‐induced inflammatory and oxidative stress responses. After a 2‐week ACEi treatment (enalapril, 30mg/kg/day) followed by a 2‐week washout period male and female SHRs (11 wk old) were infused with Ang II (400ng/kg/min, s.c.) or vehicle (saline, n=5‐11/group/sex) for 2 weeks. At time of euthanasia, a mid‐myocardial section was fixed and paraffin embedded and remaining LV tissue was collected for protein, RNA and DNA extraction. LV macrophage infiltration was determined histologically by staining for ED‐1. MCP1 gene expression was evaluated by RTqPCR, and pro‐oxidants (NOX2 and NOX4) and antioxidants (catalase and superoxide dismutase 2; SOD2) were measured by immunoblotting. Oxidative DNA damage was quantified using an 8‐hydroxy‐2’‐deoxyguanosine (8‐OHdG) ELISA. Cardiac MCP1 gene expression was increased by Ang II in both sexes with no impact of transient ACEi. However, Ang II induced LV macrophage infiltration (ED‐1) in males only which was attenuated by prior transient ACEi. LV NOX2 was increased by Ang II and attenuated by prior transient ACEi in both sexes. NOX4 was reduced in females previously treated with an ACEi, but not males. The antioxidant, catalase, was increased by Ang II in both sexes, but attenuated by transient ACEi in females only. Further, both catalase and SOD2 were positively correlated to NOX2 in females, but not males. This suggests that Ang II‐induced pro‐oxidant responses may be more tightly coupled with antioxidant responses in females. DNA damage as measured by 8‐OHdG was not significantly impacted by Ang II in males or females. However 8‐OHdG was reduced in males previously treated with an ACEi suggesting that an alternative antioxidant mechanism may be active in males previously treated with an ACEi. At this dose and duration of Ang II there is not significant oxidative DNA damage, but MCP1 and pro‐oxidants are increased in both sexes. Males exhibit greater macrophage infiltration, while females exhibit greater antioxidant responses; both of which are prevented by prior transient ACEi. In conclusion, there are sex‐specific inflammatory and oxidative stress responses to Ang II and transient ACEi that may impact future cardiac remodeling. Current and future studies will aim to identify mechanisms involved in transient ACEi‐mediated cardio‐protection and how these ...