Specific knockdown of WNT8b expression protects against phosphate-induced calcification in vascular smooth muscle cells by inhibiting the Wnt-ß-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-ß-catenin signaling pathway. To explore the effect of WNT8b on the Wnt-ß-catenin signaling pathway and VC in vitro, ß-glycerophosphate (GP)-induced T/G HA-VSMCs were treated with small interfering RNA against WNT8b (Si-WNT8b), Wnt-ß-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 ß-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 ß-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, ß-catenin, phospho-GSK-3ß (p-GSK-3ß), and cyclin-D, whereas it increased the levels of p-ß-catenin and GSK-3ß, indicating that si-WNT8b could alter the Wnt-ß-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 ß-catenin expression level in the nucleus. Altogether, we conclude that WNT8b knockdown terminates phosphate-induced VC in VSMCs by inhibiting the Wnt-ß-catenin signaling pathway.

Restoration of microRNA-30b expression alleviates vascular calcification through the mTOR signaling pathway and autophagy.

Pathological calcification represents an event that consequently leads to a distinct elevation in the morbidity and mortality of patients with chronic kidney disease (CKD) in addition to strengthening its correlation with hyperphosphatemia. Epigenomic regulation by specific microRNAs (miRNAs) is reported to be involved in ectopic calcification. However, the finer molecular mechanisms governing this event remain unclear. Hence, this study aimed to identify the potential miRNAs involved in vascular calcification (VC) development and progression. Initially, mitochondrial membrane potential (MMP), autophagy-specific markers (LC3II/LC3I and Beclin1) and phenotype-specific markers of osteoblasts (runt-related transcription factor 2 and Msx2) were measured to evaluate autophagy and VC in ß-glycerophosphate-induced vascular smooth muscle cells (VSMCs) with either miR-30b restoration or miR-30b knockdown performed in vitro. The VC in vivo was represented by calcified nodule formation in the aorta of the rats undergoing 5/6 nephrectomy followed by a 1.2% phosphorus diet using Alizarin Red staining. SOX9 was verified as the target of miR-30b according to luciferase activity determination. Restoration of miR-30b was revealed to markedly diminish the expression of SOX9 while acting to inhibit activation of the mTOR signaling pathway. Knockdown of miR-30b reduced MMP and autophagy, elevated VC, and suppressed the presence of rapamycin (an inhibitor of the mTOR signaling pathway). In addition, upregulated expression of miR-30b attenuated VC in vivo. Taken together, the key findings of this study identified the inhibitory role of miR-30b in VC, presenting an enhanced understanding of miRNA as a therapeutic target to curtail progressive VC in hyperphosphatemia of CKD.

Activation of the Nrf2-ARE Signaling Pathway Prevents Hyperphosphatemia-Induced Vascular Calcification by Inducing Autophagy in Renal Vascular Smooth Muscle Cells.

This study investigates the effect of nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) signaling pathway in vascular calcification (VC) via inducing Autophagy in renal vascular smooth muscle cells (VSMCs). VSMCs were assigned into six experimental groups: the normal control, high phosphorus, si-negative control (si-NC), Nrf2-siRNA, over-expressed Nrf2, and negative control (NC) groups. RT-PCR was applied to detect the mRNA expressions of the desired Nrf2-ARE signaling pathway-related genes (Nrf2, NQO-1, HO-1, γ-GCS). The protein products of these genes: apoptosis-related genes (LC3I and LC3II), osteogenic marker proetins (Runt-related transcription factor 2) Runx2 and BMP2 were all detected by Western blotting. Autophagosomes in VSMCs were observed under a transmission electron microscope. We discovered an increased calcium ion concentration and upregulated Runx2, BMP2, Nrf2, HO-1, γ-GCS, NQO-1, and LC3II/LC3I expressions in the high phosphorous, si-NC and Nrf2-siRNA, and NC groups, compared with the normal control group. Compared to the high phosphorus and si-NC groups, higher levels of Runx2 and BMP2 but decreased Nrf2, HO-1, γ-GCS, NQO-1, and LC3II/LC3I expressions were detected in the Nrf2-siRNA group. The high phosphorus, si-NC and over-expressed Nrf2 experimental groups all had increased Nrf2, NQO-1, HO-1, γ-GCS, and LC3II/LC3I expressions as well as high numbers of autophagosomes compared with the normal control group. Finally, we detected a lower amount of autophagosomes presence and Nrf2, NQO-1, HO-1 γ-GCS, and LC3II/LC3 protein expression of Nrf2-siRNA group than that of the high phosphorus and si-NC groups. Activation of Nrf2-ARE signaling pathway may prevent hyperphosphatemia-induced VC by inducing autophagy in VSMCs. J. Cell. Biochem. 118: 4708-4715, 2017. © 2017 Wiley Periodicals, Inc.

MicroRNA-30b Regulates High Phosphorus Level-Induced Autophagy in Vascular Smooth Muscle Cells by Targeting BECN1.

BACKGROUND/AIMS: Autophagy is an evolutionarily conserved mechanism that affects the survival and functions of vascular smooth muscle cells (VSMCs). We explored the role of microRNAs (miRNAs) in regulating autophagy in VSMCs exposed to high phosphorus (Pi) levels. METHODS: VSMCs were isolated from the thoracic aorta of rats and were cultured primarily. Real-time PCR was used to measure the mRNA expression of indicated genes. Western blotting was performed to detect the protein expression of autophagy-related markers. RESULTS: We found that treatment with high Pi levels (1 and 3 mM) activated LC3II expression and promoted autophagic flux in VSMCs. Conversely, treatment with an autophagy inhibitor decreased LC3II expression. Pi stimulation dysregulated the expression of several miRNAs such as miR-18a, miR-21, miR-23a, miR-30b, and miR-31a. However, miR-30b overexpression decreased Pi-induced expression of autophagy-related marker genes such as BECN1, ATG5, and LC3b, whereas miR-30b downregulation increased Pi-induced expression of these genes. In addition, we found that miR-30b directly targeted BECN1. CONCLUSIONS: These data suggest that miR-30b plays an important role in the regulation of high Pi level-induced autophagy in VSMCs by targeting BECN1.

Light intensity dependent photosynthetic electron transport in eelgrass (Zostera marina L.).

Responses of electron transport to three levels of irradiation (20, 200, and 1200 µmol photons m-2 s-1 PAR; exposures called LL, ML and HL, respectively) were investigated in eelgrass (Zostera marina L.) utilizing the chlorophyll a fluorescence technique. Exposure to ML and HL reduced the maximum quantum yield of photosystem II (PSII) (Fv/Fm) and the maximum slope decrease of MR/MRO (VPSI), indicating the occurrence of photoinhibition of both PSII and photosystem I (PSI). A comparatively slow recovery rate of Fv/Fm due to longer half-life recovery time of PSII and 40% lower descending amplitude compared to other higher plants implied the poor resilience of the PSII. Comparatively, PSI demonstrated high resilience and cyclic electron transport (CEF) around PSI maintained high activity. With sustained exposure, the amplitudes of the kinetic components (L1 and L2), the probability of electron transfer from PSII to plastoquinone pool (ψET2o), and the connectivity among PSII units decreased, accompanied by an enhancement of energy dissipation. Principle component analysis revealed that both VPSI and Fv/Fm contributed to the same component, which was consistent with high connectivity between PSII and PSI, suggesting close coordination between both photosystems. Such coordination was likely beneficial for the adaption of high light. Exposure to LL significantly increased the activity of both PSI and CEF, which could lead to increased light harvesting. Moreover, smooth electron transport as indicated by the enhancement of L1, L2, ψET2o and the probability of electron transport to the final PSI acceptor sides, could contribute to an increase in light utilization efficiency.

The alternation between PSII and PSI in ivy (Hedera nepalensis) demonstrated by in vivo chlorophyll a fluorescence and modulated 820 nm reflection.

To examine the coordination between photosystem II (PSII) and photosystem I (PSI) in response to varying environmental conditions, both diurnal fluctuations and seasonal variability of photosynthetic electron transport activity in ivy (Hedera nepalensis, Araliaceae) were investigated: by measuring prompt fluorescence, delayed fluorescence (DF) and modulated reflection of 820 nm light (MR). During diurnal fluctuations, the PSII electron donor side was damaged, as evidenced by decreases of the fast amplitude of DF decay kinetics at I1, although there was no significant change in relative variable fluorescence at K-step to amplitude of FJ - Fo. Decreases in the maximum photochemical efficiency (i.e., PSII photoinactivation) were accompanied by an increased maximum decrease in the slope of MR/MRo (i.e., PSI photoactivation). Subsequently, PSII recovery and PSI relaxation occurred in the afternoon. Throughout the season, alternations between PSII and PSI were also suggested by the down-regulation of PSII and the up-regulation of PSI from summer to winter. Significant negative linear correlations between the activity of PSII and PSI across both diurnal fluctuations and seasonal variability were verified by correlation analyses. In addition, PSI was active throughout the year, suggesting PSI is independent from high temperatures. High PSI activity may maintain the functional integrity of the photosynthetic apparatus in overwintering ivy. The alternation between PSII and PSI activity may regulate the distribution of excitation energy between the two photosystems and balance the redox state of the electron transport change, thereby enabling ivy to respond to varying environmental conditions.