Objectives Inflammatory responses play a pivotal role in the pathogenesis of hypertensive cardiac remodelling. Macrophage infiltration and polarisation contribute to the development of cardiac fibrosis. Although serum-glucocorticoid regulated kinase 1 (SGK1) is a key mediator of fibrosis, its role in regulating macrophage function leading to cardiac fibrosis has been investigated. We aimed to determine the mechanism by which SGK1 regulates the cardiac inflammatory process, thus contributing to hypertensive cardiac fibrosis.
Methods The SGK1-/- mouse strain was backcrossed onto the genetic background of WT (B6/129S) mice for >10 generations, and littermates were used as controls. We infused 10- to 12-week-old male mice (n = 6-10 per group) for 7 days with vehicle (saline) or a pressor dose of angiotensin II (Ang II) (1500 ng.kg−1.min−1) by osmotic mini-pumps implanted subcutaneously. We examined cardiac fibrosis of WT and SGK1-/- hearts after Ang II infusion by Masson trichrome staining, and cardiac structure and function were evaluated by small animal ultrasound to evaluate the effect of infused Ang II. In addition, the inflammatory responses of hearts were assayed by immunohistochemistry and flow cytometry. The TUNEL assay and immunofluorescence analyses of macrophage apoptosis in cardiac tissue of WT and SGK1-/- mice after 7 days of Ang II infusion. We further investigated macrophage phenotype by flow cytometry and double immunofluorescence analysis of M2 macrophages (anti-F4/80 and anti-CD206) in hearts. In vitro, co-culture of WT or SGK-/- macrophage and cardiac fibroblast in 3-dimensional peptide gels were used to evaluate the effects of macrophage-mediated fibroblast-to-myofibroblast differentiation.
Results After Ang II infusion in mice, cardiac hypertrophy and fibrosis developed in wild-type but SGK1 knockout mice, with equal levels of hypertension in both groups. Compared with wild-type hearts, SGK1 knockout hearts showed less infiltration of leukocytes and macrophages. Importantly, SGK1 deficiency led to increased macrophages apoptosis and decreased proportion of alternatively activated (M2) macrophages. Ang II infusion induced phosphorylation and nuclear localisation of signal transducer and activator of transcription 3 (STAT3) whereas SGK1 knockout hearts showed this effect attenuated. Furthermore, SGK1 deficiency enhances macrophage apoptosis in Ang II-infused heart through inhibiting FOXO3a phosphorylation and enhancement of caspase-3 activation. In a 3-dimensional peptide gel culture, inhibition of STAT3 suppressed differentiation into M2 macrophages. Coculture of macrophages with cardiac fibroblasts in 3-dimensional peptide gel stimulated the expression of α-smooth muscle actin and collagen in cardiac fibroblasts. However, SGK1 knockout mice with macrophage deficiency showed reduced fibroblast-to-myofibroblast transition.
Conclusions SGK1 may play an important role in macrophage survival and M2 macrophage activation by activating STAT3 pathway, which leads to Ang II-induced cardiac fibrosis.