Vascular calcification (VC) is a hallmark of atherosclerotic plaques. Calcification of advanced plaques shares common features with endochondral ossification of long bones and appears to be protective. On the other hand, microcalcification of early plaques, which is poorly understood, is thought to be harmful. Tissue-nonspecific alkaline phosphatase (TNAP) and collagen are the two proteins necessary for physiological mineralization. Here, we demonstrate the presence of membrane-bound TNAP, detected by immunofluorescence, that seems to form clusters on the plasma membrane of vascular smooth muscle cells (VSMCs) cultured in mineralizing conditions. We observed that TNAP activity and mineralization were increased when VSMCs were cultured in the presence of ascorbic acid (AA) and β-glycerophosphate (β-GP). Increased TNAP activity was observed in whole cell lysates, total membrane fractions and, more particularly, in matrix vesicles (MVs). We have shown that TNAP-enriched MVs released from VSMCs subjected to collagenase contained more apatite-like mineral than the less TNAP-rich/TNAP-enriched vesicles isolated without collagenase treatment. These results suggest a role for collagen in promoting calcification induced by TNAP in atherosclerotic plaques.

血管钙化（VC）是动脉粥样硬化斑块的标志。晚期斑块的钙化与长骨的软骨内骨化具有共同特征，并且似乎具有保护作用。另一方面，人们知之甚少的早期斑块的微钙化被认为是有害的。组织非特异性碱性磷酸酶 (TNAP) 和胶原蛋白是生理矿化所必需的两种蛋白质。在这里，我们证明了通过免疫荧光检测到的膜结合 TNAP 的存在，它似乎在矿化条件下培养的血管平滑肌细胞 (VSMC) 的质膜上形成簇。我们观察到当 VSMC 在抗坏血酸 (AA) 和 β-甘油磷酸 (β-GP) 存在下培养时，TNAP 活性和矿化增加。在全细胞裂解物、总膜部分，更特别地，在基质囊泡 (MV) 中观察到增加的 TNAP 活性。我们已经表明，与未经胶原酶处理分离的较少富含 TNAP/富含 TNAP 的囊泡相比，经胶原酶处理的 VSMC 释放的富含 TNAP 的 MV 含有更多的磷灰石样矿物质。这些结果表明胶原蛋白在促进 TNAP 在动脉粥样硬化斑块中诱导的钙化中的作用。