A Biased Relaxin Receptor Agonist, ML290, Attenuates Atherosclerotic Calcification in Apolipoprotein E‐deficient Mice

Abstract

Vascular calcification is the most significant predictor of cardiovascular morbidity and mortality, but therapeutic options are unavailable. Relaxin has emerged as a vasoprotective molecule, but several drawbacks prevent therapeutic translation. Targeting the relaxin receptor, RXFP1, is safe and well-tolerated in animal models of vascular disease and humans. We identified a non-peptide biased allosteric agonist of human RXFP1, ML290, and aimed to test the hypothesis that ML290 arrests the progression of vascular calcification in a mouse model of atherosclerosis. Atherosclerotic calcification was induced in male humanized (hRXFP1/hRXFP1) mice crossbred with the ApoE-/- mice (humanized ApoE-/-) using a custom diet that contains 42% calorie fat for 25 weeks starting at 10 weeks of age. Age-matched humanized ApoE-/- mice on normal chow diet were used as negative control and received vehicle treatment starting at 10 weeks of age. Atherogenic mice were randomly assigned to either receive vehicle or ML290 (30 mg/kg) at the beginning (preventative treatment) or following 15 weeks of the atherogenic diet (reversal treatment). Vehicle and ML290 treatments were administered on alternate days via oral gavage. ML290 treatment significantly prevented (P = 0.0422, n = 8) and reversed (P = 0.0099, n = 10) atherosclerotic calcification in the aorta of humanized ApoE-/- mice fed an atherogenic diet. Longitudinal tracing of mineral formation in the aortic arch revealed accelerated mineral growth in vehicle-treated mice, and this was reduced by ML290 treatment. The anti-calcification effects of ML290 were associated with a significant (P = 0.0411, n = 5) reduction in plaque burden, but not serum cholesterol level. In vitro, ML290 reduced (P = 0.0005, n = 3) superoxide production under osteogenic conditions in vascular smooth muscle cells (VSMCs). Osteogenic changes in VSMC phenotype associate with a release of alkaline phosphatase (ALP) in extracellular vesicles (EVs), which promote mineralization. ML290 treatment significantly (P = 0.0001, n = 3) suppressed the formation of ALP-loaded EVs. BMP4, an inducer of osteogenic transition, and caveolin-1, a scaffolding protein required for calcifying EV formation, were significantly (P = 0.0059, n = 4) downregulated after 24 h treatment with ML290 compared to vehicle-treated VSMCs under osteogenic conditions. In summary, we demonstrate the therapeutic potential for ML290 to mitigate and reverse atherosclerosis-induced vascular calcification in vivo. The actions of ML290 to attenuate calcification are in part attributed to its ability to limit the release of calcifying EVs as a result of osteogenic differentiation, and to reduce vascular superoxide production. The outcomes of this study can lead to the discovery of clinically-viable non-peptide-based therapies for the treatment of vascular calcification that can benefit the rapidly increasing patient population.