Patients with fibro-calcific aortic valve disease (FCAVD) have lipid depositions in their aortic valve that engender a proinflammatory impetus toward fibrosis and calcification and ultimately valve leaflet stenosis. Although the lipoprotein(a)-autotaxin (ATX)-lysophosphatidic acid axis has been suggested as a potential therapeutic target to prevent the development of FCAVD, supportive evidence using ATX inhibitors is lacking. We here evaluated the therapeutic potency of an ATX inhibitor to attenuate valvular calcification in the FCAVD animal models. ATX level and activity in healthy participants and patients with FCAVD were analyzed using a bioinformatics approach using the Gene Expression Omnibus datasets, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and western blotting. To evaluate the efficacy of ATX inhibitor, interleukin-1 receptor antagonist-deficient (Il1rn-/-) mice and cholesterol-enriched diet-induced rabbits were used as the FCAVD models, and primary human valvular interstitial cells (VICs) from patients with calcification were employed. The global gene expression profiles of the aortic valve tissue of patients with severe FCAVD demonstrated that ATX gene expression was significantly upregulated and correlated with lipid retention (r = 0.96) or fibro-calcific remodeling-related genes (r = 0.77) in comparison to age-matched non-FCAVD controls. Orally available ATX inhibitor, BBT-877, markedly ameliorated the osteogenic differentiation and further mineralization of primary human VICs in vitro. Additionally, ATX inhibition significantly attenuated fibrosis-related factors’ production, with a detectable reduction of osteogenesis-related factors, in human VICs. Mechanistically, ATX inhibitor prohibited fibrotic changes in human VICs via both canonical and non-canonical TGF-β signaling, and subsequent induction of CTGF, a key factor in tissue fibrosis. In the in vivo FCAVD model system, ATX inhibitor exposure markedly reduced calcific lesion formation in interleukin-1 receptor antagonist-deficient mice (Il1rn-/-, P = 0.0210). This inhibition ameliorated the rate of change in the aortic valve area (P = 0.0287) and mean pressure gradient (P = 0.0249) in the FCAVD rabbit model. Moreover, transaortic maximal velocity (Vmax) was diminished with ATX inhibitor administration (mean Vmax = 1.082) compared to vehicle control (mean Vmax = 1.508, P = 0.0221). Importantly, ATX inhibitor administration suppressed the effects of a high-cholesterol diet and vitamin D2-driven fibrosis, in association with a reduction in macrophage infiltration and calcific deposition, in the aortic valves of this rabbit model. ATX inhibition attenuates the development of FCAVD while protecting against fibrosis and calcification in VICs, suggesting the potential of using ATX inhibitors to treat FCAVD.

中文翻译：

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自分泌运动因子抑制通过抑制炎症驱动的瓣膜间质细胞纤维钙化重塑来减轻主动脉瓣钙化

纤维钙化性主动脉瓣疾病（FCAVD）患者的主动脉瓣内有脂质沉积，导致纤维化和钙化，最终导致瓣叶狭窄。尽管脂蛋白(a)-自分泌运动因子 (ATX)-溶血磷脂酸轴已被认为是预防 FAVD 发展的潜在治疗靶点，但缺乏使用 ATX 抑制剂的支持证据。我们在这里评估了 ATX 抑制剂在 FCAVD 动物模型中减轻瓣膜钙化的治疗效力。使用生物信息学方法，使用基因表达综合数据集、酶联免疫吸附测定 (ELISA)、免疫组织化学和蛋白质印迹分析健康参与者和 FAVD 患者的 ATX 水平和活性。为了评估ATX抑制剂的疗效，白细胞介素1受体拮抗剂缺陷（Il1rn-/-）小鼠和高胆固醇饮食诱导的兔子被用作FCAVD模型，并使用来自钙化患者的原代人瓣膜间质细胞（VIC）被雇用。严重 FCAVD 患者主动脉瓣组织的整体基因表达谱表明，与年龄相比，ATX 基因表达显着上调，并与脂质潴留 (r = 0.96) 或纤维钙化重塑相关基因 (r = 0.77) 相关-匹配的非 FAVD 控件。口服 ATX 抑制剂 BBT-877 在体外显着改善原代人 VIC 的成骨分化和进一步矿化。此外，在人类 VIC 中，ATX 抑制显着减弱了纤维化相关因子的产生，并可检测到成骨相关因子的减少。从机制上讲，ATX 抑制剂通过经典和非经典 TGF-β 信号传导以及随后诱导 CTGF（组织纤维化的关键因素）来抑制人类 VIC 的纤维化变化。在体内 FAVD 模型系统中，ATX 抑制剂暴露显着减少了白细胞介素 1 受体拮抗剂缺陷小鼠的钙化病变形成（Il1rn-/-，P = 0.0210）。这种抑制改善了 FAVD 兔模型中主动脉瓣面积的变化率 (P = 0.0287) 和平均压力梯度 (P = 0.0249)。此外，与媒介物对照相比（平均Vmax = 1.508，P = 0.0221），ATX抑制剂给药后经主动脉最大速度（Vmax）降低（平均Vmax = 1.082）。重要的是，在该兔模型的主动脉瓣中，ATX抑制剂的施用抑制了高胆固醇饮食和维生素D2驱动的纤维化的影响，与巨噬细胞浸润和钙化沉积的减少相关。ATX 抑制可减弱 FCAVD 的发展，同时防止 VIC 中的纤维化和钙化，这表明使用 ATX 抑制剂治疗 FCAVD 的潜力。