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IntroductionGPR30 is an intracellular transmembrane G protein-coupled receptor that mediates non-genomic estrogen signaling. The GPR30 agonist G-1 modulates glucose homeostasis and vascular function. However, its impact on vascular inflammation and atherogenesis has not yet been investigated in the atherosclerotic apolipoprotein E-deficient(ApoE-/-) mouse model.Material and methodsApoE-/- mice were fed a high-cholesterol diet for 7 weeks while being treated with the selective GPR30 agonist G-1 (n=6-7). After the treatment period, vascular relaxation capacity, vascular oxidative stress, and atherosclerotic plaque burden were assessed. In vitro, reactive oxygen species, expression levels of the angiotensin II type1(AT1) receptor, and proliferation rate were quantified in human coronary artery smooth muscle cells(HCASMC).ResultsG-1 significantly improved glucose tolerance in vivo (142.2±8.1mg/dl vs. 204.6±13.3mg/dl), G-1 reduced vascular oxidative stress (221±88RLU/s/mg vs.1,983±885RLU/s/mg) and improved endothelium-dependent vasodilation (relaxation to 35.1±4.5% vs.63.0±4.6%). Furthermore, treatment with G-1 significantly reduced the atherosclerotic plaque burden of female ApoE-/- mice (56.5±3.7% vs.75.5±2.9%). In vitro, G-1 provoked a significant downregulation of the AT1 receptor in HCASMC (0.67±0.09-fold). Furthermore, G-1 blunted angiotensin II-induced ROS production by HCASMC (817±7RLU/s/mg vs.1,625±105 RLU/s/mg) and diminished HCASMC proliferation (-26.8±2.7% vs.+50.4±1.7%).ConclusionsSelective GPR30 activation improves glucose tolerance in vivo and decreases vascular ROS production in vitro and in vivo. In vitro, the antioxidant effect might be mediated by downregulation of the AT1 receptor. In vivo, the antioxidant effect of G-1 is associated with an improved endothelial function and a reduced atherosclerotic plaque burden in ApoE-deficient mice, indicating beneficial vascular effects of GPR30 activation. GPR30 agonism might thus be a compelling treatment strategy against atherosclerosis.

Background 17β-estradiol (E2) mediates vasculoprotection in various preclinical and clinical models of atherosclerosis and neointimal hyperplasia. However, the molecular mechanisms underlying these effects are still not fully elucidated. Previous studies have demonstrated the essential role of the peroxisome-proliferator-activated-receptor-γ (PPARγ) in mediating vasculoprotective effects of E2 in vivo. The aim of the current study was to investigate whether PPARγ is implicated in mediating vasculoprotective mechanisms of E2 in human coronary artery smooth muscle cells (HCASMC). Methods Primary HCASMC were purchased and stimulated with E2 [10 nM], the selective estrogen receptor α (ERα) agonist propylpyrazole triol (PPT) [50 nM] and the selective ERα antagonist methyl-piperidino-pyrazole (MPP) [1 μM], respectively. Changes in PPARγ mRNA and protein expression upon stimulation of ERα were assessed by qPCR and Western blot analyses. Nuclear PPARγ protein expression and DNA binding affinity was assessed after the isolation of the nuclear protein fraction. Hereafter, HCASMC were incubated with E2, PPARγ-antagonist GW9662 [1 μM – 30 μM], or both. HCASMC proliferation was assessed by nuclear BrdU staining and reactive oxygen species (ROS) formation was assessed by L-012- and DCF-DA assays. Results E2 significantly increased PPARγ expression in HCASMC (1.95±0.41 –fold; n=5; p=0.0335). This effect was mimicked by ERα agonist PPT (1.63±0.27 –fold; n=7; p=0.0489) and was abrogated by co-incubation with ERα antagonist MPP (1.17±0.18 –fold; n=3; pvs. control >0.05). Nuclear PPARγ expression was enhanced by E2 (1.53±0.16 –fold; n=4; pvs. control = 0.0074; Fig. 2A) whereas PPARγ's DNA binding activity to PPRE remained unchanged upon stimulation with E2 (0.94±0.11 –fold; n=4; pvs. control >0.05). Pharmacological inhibition of PI3K/Akt by LY294002 abrogated E2-induced expression of PPARγ (0.24±0.09 –fold; n=3; pvs. E2 = 0.0017), arguing for a PI3K/Akt-dependent activation by E2. The role of PPARγ in mediating vasculoprotective effects of E2 was assessed in functional assays using PPARγ-antagonist GW9662. E2 diminished HCASMC proliferation which was restored by GW9662. While E2 only slightly decreased ROS production by HCASMC, GW9662 significantly increased ROS levels (1,036±169 RLU x s–1 x cell–1 versus 561±99 RLU x s–1 x cell–1; n=5–6; p=0.0287). Conclusion In summary, the present study identifies PPARγ as a downstream mediator of E2-related atheroprotective effects in HCASMC. 17β-estradiol regulates vascular PPARγ-expression in HCASMC via the ERα receptor and the PI3K/Akt pathway. PPARγ agonism might be a promising therapeutic strategy to prevent cardiovascular events in postmenopausal women with depleted E2 plasma levels. Acknowledgement/Funding This work was supported by the Bonfor program of the University of Bonn [grant number O-109.0057 to JJ].