Freise Christian, Kretzschmar Nadja, Querfeld Uwe
Center for Cardiovascular Research, Charité - University Medicine, Campus Mitte, Hessische Str. 3-4, 10115, Berlin, Germany.
Department of Pediatric Nephrology, Charité - University Medicine, Campus Virchow Clinic, 13353, Berlin, Germany.
BMC Cardiovasc Disord. 2016 Sep 30;16(1):185. doi: 10.1186/s12872-016-0362-8.
Vascular calcifications such as arteriosclerosis, which is characterized by a calcificiation of the tunica media, represent major comorbidities e.g. in patients with chronic kidney disease (CKD). An essential step during the development of arteriosclerosis is the transdifferentiation/calcification of vascular smooth muscle cells (VSMC) resembling osteogenesis. The matrix metalloproteinases (MMP)-2 and -9 were shown to promote these VSMC calcifications and their inhibition is of therapeutic value to prevent arteriosclerosis in preclinical studies. Aiming for an understanding of the underlying regulatory mechanisms of MMPs we here investigated, if the MMP-mediated VSMC calcification involves altered signaling of the Wnt pathway, which is known to impact osteogenesis.
We used an experimental in vitro model of vascular calcification. Transdifferentiation/calcification of murine VSMC was induced by elevated calcium and phosphorus levels. Calcification was assessed by calcium and alkaline phosphatase measurements. Activation/activity of the gelatinases MMP-2 and MMP-9 was assessed by conversion of fluorescence-labelled substrates. Activation of the Wnt pathway was analysed by a reporter gene assay.
Besides pro-calcifying culture conditions, also activation of Wnt signaling by a specific agonist (under normal culture conditions) stimulated VSMC-calcification accompanied by enhanced expression and secretion of the gelatinases MMP-2 and -9. Vice versa, recombinant MMP-2 and -9 induced a time-delayed activation of Wnt signaling after 72 h in VSMC but showed no direct effects after 24-48 h. These effects were blocked by pharmacological inhibition of MMPs or of Wnt signaling.
Our study suggests that the pro-calcifying environment in CKD induces Wnt signaling in VSMC which in turn contributes to the induction of MMPs which then foster the development of arteriosclerosis. Thus, besides MMP inhibition, the inhibition of Wnt signaling in VSMC might represent a therapeutic target for the prevention of vascular calcifications.
血管钙化，如以中膜钙化为特征的动脉硬化，是慢性肾病（CKD）等患者的主要合并症。动脉硬化发展过程中的一个关键步骤是血管平滑肌细胞（VSMC）发生类似骨生成的转分化/钙化。基质金属蛋白酶（MMP）-2和-9已被证明可促进这些VSMC钙化，在临床前研究中，抑制它们对预防动脉硬化具有治疗价值。为了理解MMP的潜在调控机制，我们在此研究了MMP介导的VSMC钙化是否涉及Wnt信号通路的改变，已知该信号通路会影响骨生成。
我们使用了血管钙化的体外实验模型。通过提高钙和磷水平诱导小鼠VSMC的转分化/钙化。通过测量钙和碱性磷酸酶评估钙化情况。通过荧光标记底物的转化评估明胶酶MMP-2和-9的激活/活性。通过报告基因检测分析Wnt信号通路的激活情况。
除了促钙化培养条件外，特定激动剂激活Wnt信号（在正常培养条件下）也刺激了VSMC钙化，同时伴有明胶酶MMP-2和-9表达及分泌增加。反之，重组MMP-2和-9在VSMC中72小时后诱导Wnt信号的延迟激活，但在24-48小时后无直接作用。这些作用可被MMP或Wnt信号的药理学抑制所阻断。
我们的研究表明，CKD中的促钙化环境诱导VSMC中的Wnt信号，这反过来又有助于诱导MMP，进而促进动脉硬化的发展。因此，除了抑制MMP外，抑制VSMC中的Wnt信号可能是预防血管钙化的治疗靶点。
