Cellular communication network factor 3 (CCN3) has been implicated in the regulation of osteoblast differentiation. However, it is not known if CCN3 can regulate valvular calcification. While macrophages have been shown to regulate valvular calcification, the molecular and cellular mechanisms of this process remain poorly understood. In the present study, we investigated the role of macrophage-derived CCN3 in the progression of calcific aortic valve disease. Myeloid-specific knockout of CCN3 (Mye-CCN3-KO) and control mice were subjected to a single tail intravenous injection of AAV encoding mutant mPCSK9 (rAAV8/D377Y-mPCSK9) to induce hyperlipidemia. AAV-injected mice were then fed a high fat diet for 40 weeks. At the conclusion of high fat diet feeding, tissues were harvested and subjected to histologic and pathologic analyses. In vitro, bone marrow-derived macrophages (BMDM) were obtained from Mye-CCN3-KO and control mice and the expression of bone morphogenic protein signaling related gene were verified via quantitative real-time PCR and Western blotting. The BMDM conditioned medium was cocultured with human valvular intersititial cells which was artificially induced calcification to test the effect of the conditioned medium via Western blotting and Alizarin red staining. Echocardiography revealed that both male and female Mye-CCN3-KO mice displayed compromised aortic valvular function accompanied by exacerbated valve thickness and cardiac dysfunction. Histologically, Alizarin-Red staining revealed a marked increase in aortic valve calcification in Mye-CCN3-KO mice when compared to the controls. In vitro, CCN3 deficiency augmented BMP2 production and secretion from bone marrow-derived macrophages. In addition, human valvular interstitial cells cultured with conditioned media from CCN3-deficient BMDMs resulted in exaggerated pro-calcifying gene expression and the consequent calcification. Our data uncovered a novel role of myeloid CCN3 in the regulation of aortic valve calcification. Modulation of BMP2 production and secretion in macrophages might serve as a key mechanism for macrophage-derived CCN3’s anti-calcification function in the development of CAVD.

中文翻译：

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骨髓 CCN3 可防止主动脉瓣钙化

细胞通讯网络因子 3 (CCN3) 与成骨细胞分化的调节有关。然而，尚不清楚 CCN3 是否可以调节瓣膜钙化。虽然巨噬细胞已被证明可以调节瓣膜钙化，但对该过程的分子和细胞机制仍知之甚少。在本研究中，我们调查了巨噬细胞衍生的 CCN3 在钙化性主动脉瓣疾病进展中的作用。CCN3 (Mye-CCN3-KO) 的骨髓特异性敲除和对照小鼠接受单尾静脉注射 AAV 编码突变体 mPCSK9 (rAAV8/D377Y-mPCSK9) 以诱导高脂血症。注射 AAV 的小鼠随后被喂食高脂肪饮食 40 周。在高脂肪饮食喂养结束时，收获组织并进行组织学和病理学分析。体外，从Mye-CCN3-KO和对照小鼠获得骨髓源性巨噬细胞（BMDM），通过实时定量PCR和Western印迹验证骨形态发生蛋白信号相关基因的表达。将BMDM条件培养基与人工诱导钙化的人瓣膜间质细胞共培养，通过Western blotting和茜素红染色检测条件培养基的作用。超声心动图显示雄性和雌性 Mye-CCN3-KO 小鼠均表现出受损的主动脉瓣膜功能，并伴有瓣膜厚度加重和心脏功能障碍。组织学上，茜素红染色显示与对照组相比，Mye-CCN3-KO 小鼠的主动脉瓣钙化显着增加。体外，CCN3 缺陷增加了 BMP2 的产生和骨髓来源巨噬细胞的分泌。此外，用来自 CCN3 缺陷型 BMDM 的条件培养基培养的人瓣膜间质细胞会导致过度的促钙化基因表达和随之而来的钙化。我们的数据揭示了骨髓 CCN3 在调节主动脉瓣钙化中的新作用。调节巨噬细胞中 BMP2 的产生和分泌可能是巨噬细胞衍生的 CCN3 在 CAVD 发展中的抗钙化功能的关键机制。我们的数据揭示了骨髓 CCN3 在调节主动脉瓣钙化中的新作用。调节巨噬细胞中 BMP2 的产生和分泌可能是巨噬细胞衍生的 CCN3 在 CAVD 发展中的抗钙化功能的关键机制。我们的数据揭示了骨髓 CCN3 在调节主动脉瓣钙化中的新作用。调节巨噬细胞中 BMP2 的产生和分泌可能是巨噬细胞衍生的 CCN3 在 CAVD 发展中的抗钙化功能的关键机制。