Introduction

Though vascular smooth muscle cells adopt an osteogenic phenotype during pathological vascular calcification, clinical studies note an inverse correlation between bone mineral density and arterial mineral—also known as the calcification paradox. Both processes are mediated by extracellular vesicles (EVs) that sequester calcium and phosphate. Calcifying EV formation in the vasculature requires caveolin-1 (CAV1), a membrane scaffolding protein that resides in membrane invaginations (caveolae). Of note, caveolin-1-deficient mice, however, have increased bone mineral density. We hypothesized that caveolin-1 may play divergent roles in calcifying EV formation from vascular smooth muscle cells (VSMCs) and osteoblasts (HOBs).

Methods

Primary human coronary artery VSMCs and osteoblasts were cultured for up to 28 days in an osteogenic media. CAV1 expression was knocked down using siRNA. Methyl β-cyclodextrin (MβCD) and a calpain inhibitor were used, respectively, to disrupt and stabilize the caveolar domains in VSMCs and HOBs.

Results

CAV1 genetic variation demonstrates significant inverse relationships between bone-mineral density (BMD) and coronary artery calcification (CAC) across two independent epidemiological cohorts. Culture in osteogenic (OS) media increased calcification in HOBs and VSMCs. siRNA knockdown of CAV1 abrogated VSMC calcification with no effect on osteoblast mineralization. MβCD-mediated caveolae disruption led to a 3-fold increase of calcification in VSMCs treated with osteogenic media (p < 0.05) but hindered osteoblast mineralization (p < 0.01). Conversely, stabilizing caveolae by calpain inhibition prevented VSMC calcification (p < 0.05) without affecting osteoblast mineralization. There was no significant difference in CAV1 content between lipid domains from HOBs cultured in OS and control media.

Conclusion

Our data indicate fundamental cellular-level differences in physiological and pathophysiological mineralization mediated by CAV1 dynamics. This is the first study to suggest that divergent mechanisms in calcifying EV formation may play a role in the calcification paradox.

中文翻译：

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Caveolin-1 动力学的改变导致骨和血管钙化中不同的矿化反应

介绍

尽管血管平滑肌细胞在病理性血管钙化过程中采用成骨表型，但临床研究注意到骨矿物质密度和动脉矿物质之间存在负相关性，也称为钙化悖论。这两个过程都是由隔离钙和磷酸盐的细胞外囊泡 (EV) 介导的。脉管系统中钙化 EV 的形成需要 Caveolin-1 (CAV1)，这是一种存在于膜内陷（小窝）中的膜支架蛋白。然而值得注意的是，caveolin-1 缺陷小鼠的骨矿物质密度有所增加。我们假设caveolin-1可能在血管平滑肌细胞（VSMC）和成骨细胞（HOB）钙化EV形成中发挥不同的作用。

方法

原代人冠状动脉 VSMC 和成骨细胞在成骨培养基中培养长达 28 天。使用 siRNA 敲低 CAV1 表达。分别使用甲基 β-环糊精 (MβCD) 和钙蛋白酶抑制剂来破坏和稳定 VSMC 和 HOB 中的小凹结构域。

结果

CAV1 遗传变异表明两个独立的流行病学队列中骨矿物质密度 (BMD) 和冠状动脉钙化 (CAC) 之间存在显着的负相关关系。成骨 (OS) 培养基中的培养增加了 HOB 和 VSMC 的钙化。CAV1 的 siRNA 敲除消除了 VSMC 钙化，但对成骨细胞矿化没有影响。MβCD 介导的小窝破坏导致经成骨介质处理的 VSMC 钙化增加 3 倍 ( p  < 0.05)，但阻碍成骨细胞矿化 ( p  < 0.01)。相反，通过抑制钙蛋白酶稳定小窝可防止 VSMC 钙化 ( p  < 0.05)，而不影响成骨细胞矿化。OS 和对照培养基中培养的 HOB 的脂质结构域之间的 CAV1 含量没有显着差异。

结论

我们的数据表明 CAV1 动力学介导的生理和病理生理矿化的基本细胞水平差异。这是第一项表明钙化 EV 形成的不同机制可能在钙化悖论中发挥作用的研究。