Specific knockdown of WNT8b expression protects against phosphate-induced calcification in vascular smooth muscle cells by inhibiting the Wnt-β-catenin signaling pathway.

In the last 10 years, the prevalence, significance, and regulatory mechanisms of vascular calcification (VC) have gained increasing recognition. The aim of this study is to explore the action of WNT8b in the development of phosphate-induced VC through its effect on vascular smooth muscle cells (VSMCs) in vitro by inactivating the Wnt-β-catenin signaling pathway. To explore the effect of WNT8b on the Wnt-β-catenin signaling pathway and VC in vitro, β-glycerophosphate (GP)-induced T/G HA-VSMCs were treated with small interfering RNA against WNT8b (Si-WNT8b), Wnt-β-catenin signaling pathway activator (LiCl) and both, respectively. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to determine the messenger RNA and protein levels of WNT8b, α-smooth muscle actin (α-SMA), calcification-associated molecules, and molecules related to the Wnt signaling pathway. The TOP/FOP-Flash reporter assay was performed to detect the transcription activity mediated by β-catenin. Si-WNT8b reduced calcium deposition and the activity of alkaline phosphatase (ALP), increased the α-SMA level, and decreased bone morphogenetic protein 2, Pit1, MSX2, and Runt-related transcription factor 2 levels, whereas stimulation of LiCl worsened β-GP-induced calcium deposition, increased the activity of ALP, and reduced the α-SMA expression level. Si-WNT8b reduced the levels of WNT8b, frizzled-4, β-catenin, phospho-GSK-3β (p-GSK-3β), and cyclin-D, whereas it increased the levels of p-β-catenin and GSK-3β, indicating that si-WNT8b could alter the Wnt-β-catenin signaling pathway and thus hamper the VC in T/G HA-VSMC, which was further demonstrated by the TOP/FOP-Flash assay and detection of the β-catenin expression level in the nucleus. Altogether, we conclude that WNT8b knockdown terminates phosphate-induced VC in VSMCs by inhibiting the Wnt-β-catenin signaling pathway.

Tian BY ，Yao L ，Sheng ZT ，Wan PZ ，Qiu XB ，Wang J ，Xu TH ... -  《-》

RTEF-1 Inhibits Vascular Smooth Muscle Cell Calcification through Regulating Wnt/β-Catenin Signaling Pathway.

Vascular calcification (VC) is highly prevailing in cardiovascular disease, diabetes mellitus, and chronic kidney disease and, when present, is associated with cardiovascular events and mortality. The osteogenic differentiation of vascular smooth muscle cells (VSMCs) is regarded as the foundation for mediating VC. Related transcriptional enhancer factor (RTEF-1), also named as transcriptional enhanced associate domain (TEAD) 4 or transcriptional enhancer factor-3 (TEF-3), is a nuclear transcriptional factor with a potent effect on cardiovascular diseases, apart from its oncogenic role in the canonical Hippo pathway. However, the role and mechanism of RTEF-1 in VC, particularly in calcification of VSMCs, are poorly understood. Our results showed that RTEF-1 was reduced in calcified VSMCs. RTEF-1 significantly ameliorated β-glycerophosphate (β-GP)-induced VSMCs calcification, as detected by alizarin red staining and calcium content assay. Also, RTEF-1 reduced alkaline phosphatase (ALP) activity and decreased expressions of osteoblast markers such as Osteocalcin and Runt-related transcription factor-2 (Runx2), but increased expression of contractile protein, including SM α-actin (α-SMA). Additionally, RTEF-1 inhibited β-GP-activated Wnt/β-catenin pathway which plays a critical role in calcification and osteogenic differentiation of VSMCs. Specifically, RTEF-1 reduced the levels of Wnt3a, p-β-catenin (Ser675), glycogen synthase kinase-3β (GSK-3β), and p-GSK-3β (Ser9), but increased the levels of p-β-catenin (Ser33/37). Also, RTEF-1 increased the ratio of p-β-catenin (Ser33/37) to β-catenin proteins and decreased the ratio of p-GSK-3β (Ser9) to GSK-3β protein. LiCl, a Wnt/β-catenin signaling activator, was observed to reverse the protective effect of RTEF-1 overexpression on VSMCs calcification induced by β-GP. Accordingly, Dickkopf-1 (Dkk1), a Wnt antagonist, attenuated the role of RTEF-1 deficiency in β-GP-induced VSMCs calcification. Taken together, we concluded that RTEF-1 ameliorated β-GP-induced calcification and osteoblastic differentiation of VSMCs by inhibiting Wnt/β-catenin signaling pathway.

Cong J ，Cheng B ，Liu J ，He P ... -  《-》

Sclerostin/Receptor Related Protein 4 and Ginkgo Biloba Extract Alleviates β-Glycerophosphate-Induced Vascular Smooth Muscle Cell Calcification By Inhibiting Wnt/β-Catenin Pathway.

Abnormal mineral metabolism in patients with chronic kidney disease (CKD) may lead to vascular calcification, which is markedly associated with adverse events, including ischemic cardiac diseases and all-cause cardiovascular mortality. Thus, preventing and treating vascular calcification play an important role in improving the prognosis of CKD patients. To investigate the potential functions of sclerostin and low-density lipoprotein receptor-related protein 4 (Lrp4) in alleviating the β-glycerophosphate (β-GP)-induced vascular smooth muscle cell (VSMC) calcification, and the protective effect of Ginkgo biloba extract (GBE). VSMC were extracted from Sprague-Dawley rat aorta and cultured in medium. The VSMCs were divided into 3 groups: (1) Negative control group, (2) β-GP group, in which the VSMCs were treated with β-GP, and (3) GBE and β-GP group, where the VSMCs were treated with both β-GP and GBE. The calcium nodules within the cells were examined by using Alizarin red S staining. The mRNA expression levels of β-catenin and bone gamma-carboxyglutamic-acid-containing proteins (BGP) were detected by real-time PCR. The protein levels of sclerostin and Lrp4 were determined by Western blot. Alizarin red S staining showed that the VSMCs in β-GP group had a distinct orange-red precipitate when compared with VSMCs in the negative control group, while the orange-red precipitate of the GBE and β-GP group was significantly reduced compared to the β-GP group. Real-time PCR showed that the mRNA levels of β-catenin and BGP in VSMCs of β-GP group were significantly higher than those of the negative control group (p < 0.05); while they were significantly reduced in VSMCs of the GBE and β-GP group (p < 0.05). Western blot results showed that the expression of sclerostin in the β-GP group was significantly higher than that in the control group (p < 0.05), whereas Lrp4 was significantly lower than in control group (p < 0.05). Sclerostin in GBE and β-GP group was significantly reduced (p < 0.05), but Lrp4 was significantly elevated when compared with that of the β-GP group (p < 0.05). β-GP induced VSMC calcification by activating the Wnt/β-catenin signaling pathway. Sclerostin and Lrp4 were involved in β-GP-induced VSMC calcification and play an important role. GBE could alleviate VSMC calcification induced by β-GP through inhibiting the Wnt/β-catenin signaling pathway.

Wang J ，Qiu X ，Xu T ，Sheng Z ，Yao L ... -  《-》

Klotho/FGF23 axis mediates high phosphate-induced vascular calcification in vascular smooth muscle cells via Wnt7b/β-catenin pathway.

Vascular calcification (VC) plays as a critical role on cardiovascular disease (CVD) and acts as a notable risk factor in cardiovascular system. Vascular smooth muscle cells (VSMCs) calcification can be triggered by high phosphate treatment; however, the explicit mechanism remains unclear. In the present study, we isolated VSMCs from primary rat artery, applied β-GP (β-glycerophosphate) for inducing VSMCs calcification in vitro to explore the mechanism of phosphate-induced calcification in VSMCs. Alizarin red staining was performed to assess the mineralization in VSMCs. Calcium deposition experiment was taken to evaluate the calcium content. ALP staining was determined to assess the ALP activity. The recombinant adenoviruses were constructed for the overexpression of Klotho and FGF23, respectively. qRT-PCR and western blot analysis were subjected to measure the expression of Klotho/FGF23 and correlated genes among Wnt7b/β-catenin pathway. We found that the calcium content was obviously increased and Alizarin red staining was positive in calcification group exposure with high phosphate in a time-dependent manner. The expression of Klotho and FGF23 was significantly decreased in the calcification group. However, overexpression of Klotho and FGF23 markedly reversed VSMCs calcification stimulating with high phosphate treatment. Moreover, Wnt7b/β-catenin inhibitor DKK1 could partly attenuate the effect of high phosphate on calcified VSMCs. These findings demonstrated that Klotho/FGF23 axis could modulate high phosphate-induced VSMCs calcification via Wnt7b/β-catenin signaling pathway. Our findings unravel that Klotho/FGF23- Wnt7b/β-catenin axis functions as a crucial role in the VSMCs calcification.

Chen YX ，Huang C ，Duan ZB ，Xu CY ，Chen Y ... -  《-》

GALNT3 protects against phosphate-induced calcification in vascular smooth muscle cells by enhancing active FGF23 and inhibiting the wnt/β-catenin signaling pathway.

Vascular calcification (VC) acts as a notable risk factor in the cardiovascular system. Disorder of phosphorus (Pi) metabolism promotes VC. Recent findings show that polypeptide N-acetylgalactosaminyltransferase 3(GALNT3) is Pi responsive and with potent effects on Pi homeostasis. However, whether GALNT3 is involved in high Pi-induced VC remains unclear. The present study investigated the potential role of GALNT3 as a novel regulator of VC. In vitro, human aortic smooth muscle cells (HASMCs) calcification was induced by inorganic Pi, while in vivo, C57BL/6 J mice were used to determine the effects of GALNT3 on Vitamin D3-induced medial arterial calcification. Alizarin red staining, Von Kossa staining, calcium and alkaline phosphatase (ALP) activity were performed to test VC. We showed that expression of GALNT3 was increased in the calcified HASMCs and aortas of the calcified mice.In vitro, overexpression of GALNT3 increased the levels of active full-length FGF23, accompanied by suppression of the osteoblast-related factors (Runx2 and BMP2), and further inhibited the formation of calcified nodules. Moreover, the protein levels of Wnt3a and active β-catenin were determined and it was found that GALNT3 significantly inhibited their expression. LiCl, a Wnt/β-catenin signaling activator, was observed to reverse the protective effect of GALNT3 overexpression. The opposite results were observed in the GALNT3 knockdown cells. In vivo, overexpression of GALNT3 by adeno-associated virus decreased the serum Pi and slowed the formation of aortic calcification in the calcified mice. In conclusion, our results indicate that GALNT3 counteracts high Pi-induced osteoblastic differentiation of VSMCs and protects against the initiation and progression of VC by inhibiting the Wnt/β-catenin signaling pathway.

Guo L ，Wang Y ，Li S ，Zhou L ，Li D ... -  《-》