Following washout of angiotensin converting enzyme inhibitor (ACEi) treatment in hypertensive rats (SHR), there is an increase in the proportion of homeostatic cardiac fibroblasts (CFs) characterized by a less fibrogenic gene profile. The present study investigated the impact of this shift in CFs on subsequent Ang II-induced oxidative stress and fibrogenic responses in the left ventricle (LV). Male and female SHRs (11 wk old) underwent a 6-week treatment scheme: 2-week ACEi (enalapril, 30mg/kg/day) or vehicle treatment, followed by a 2-week washout period, and then a 2-week Ang II (400ng/kg/min, s.c.) or vehicle infusion (n=5-11/group/sex). RT-qPCR for collagens and immunoblotting for LOX, Postn, OPN, NOX2, SOD2, and catalase was performed. In males, Ang II-induced increases in ColI, III, and IV expression were significantly attenuated by prior ACEi. While in females, Ang II significantly increased the expression of ColI and III similarly, regardless of prior ACEi treatment; highlighting a sex-specific impact of ACEi. After Ang II infusion, both Postn and LOX are reduced in LV of male and female SHR previously treated with an ACEi - suggesting reduced collagen cross-linking. Oxidative stress induced by NOX2 has been linked to OPN in fibrotic tissue. Ang II-induced increases in NOX2 are attenuated in LV of both sexes, while OPN is only reduced in females previously treated with an ACEi. NOX2 is significantly positively correlated with OPN in males only suggesting a sex-specific link between OPN and oxidative stress. Antioxidant (SOD2, catalase) levels were positively correlated to NOX2 in females, but not males. This suggests that females more efficiently neutralize oxidative stress than males - regardless of prior ACEi. Taken together, the persistent genomic and phenotypic changes in CFs following transient ACEi render the heart resistant to future fibrotic and oxidative effects of Ang II, but the mechanisms of protection may differ by sex. Collectively, these data reveal that there are sex-specific processes that govern collagen production vs. cross-linking and oxidative stress. Future studies will elucidate the mechanistic underpinnings of sex-specific cellular memory following transient ACEi with a goal of identifying novel therapeutic targets.
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 ...
Hypertensive heart disease is characterized by cardiac fibrosis in the left ventricle (LV). Angiotensin II (Ang II) promotes cardiac fibrosis through direct actions on cardiac fibroblasts. Inhibition of endogenous Ang II production with angiotensin converting enzyme (ACE) inhibitors has been shown to reduce interstitial collagen deposition in the LV. We have previously shown that transient treatment of male spontaneously hypertensive rats (SHRs) with an ACE inhibitor suppresses the fibrogenic capacity of cardiac fibroblasts and this persists even after stopping treatment. In this study, the goal was to investigate the impact of transient ACE inhibition on subsequent Ang II‐stimulated fibrogenic responses in the LV of male and female SHR. Male and female SHR (11‐week‐old) were treated with an ACE inhibitor (enalapril, 30mg/kg/day) or vehicle for 2 weeks followed by a 2‐week washout period. At the end of the washout, rats were given Ang II (400ng/kg/min, s.c.) or vehicle (saline) for 2 weeks (n=8‐11 per group per sex). Collagen I, III, and IV gene expression was assessed via RT‐qPCR, total collagen was quantified using a hydroxyproline assay, and immunoblotting for lysyl oxidase (LOX) and periostin (Postn) was performed. In males, the Ang II‐stimulated increase in collagen gene expression (i.e. Col1a1, Col3a1, and Col4a1) was significantly attenuated in SHR previously treated with enalapril. In female rats, Ang II significantly increased the expression of Col1a1 and Col3a1 similarly, regardless of prior enalapril treatment. We found a positive correlation between the degree of increase in Col1a1 and Col3a1 as well as with Col1a1 and Col4a1 in male controls with Ang II stimulation. However, in Ang II‐stimulated male rats transiently treated with enalapril, there was a negative correlation between Col1a1 and Col3a1 and no correlation between changes in Col1a1 and Col4a1. In female rats, there were significant positive correlations in all cases regardless of prior transient ACE inhibition. Total collagen content in both male and female rats was significantly increased by Ang II in control animals, but not in those previously treated with enalapril. Additionally, in both male and female rats stimulated with Ang II, cross‐linking proteins Postn and LOX showed lower expression in SHR that were transiently treated with enalapril. These data reveal that even after stopping treatment, ACE inhibition produces persistent changes in the myocardium that render it resistant to the effects of Ang II. The effects seen in females suggests a possible dysregulation between collagen production and extracellular matrix cross‐linking events following transient ACE inhibition. Future studies will elucidate the mechanisms underlying the sex‐specific response to ACE inhibition and the long‐term protective effect in male LV, with a goal of identifying novel therapeutic targets.
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