Enhanced chondrogenic potential in GelMA-based 3D cartilage model via Wnt3a surface immobilization.

Cartilage tissue engineering aims to develop functional substitutes for treating cartilage defects and osteoarthritis. Traditional two-dimensional (2D) cell culture systems lack the complexity of native cartilage, leading to the development of 3D regenerative cartilage models. In this study, we developed a 3D model using Gelatin Methacryloyl (GelMA)-based hydrogels seeded with Y201 cells, a bone marrow mesenchymal stem cell line. The model investigated chondrogenic differentiation potential in response to Wnt3a stimulation within the GelMA scaffold and validated using known chondrogenic agonists. Y201 cells demonstrated suitability for the model, with increased proteoglycan content and upregulated chondrogenic marker expression under chondrogenic conditions. Wnt3a enhanced cell proliferation, indicating activation of the Wnt/ß-catenin pathway, which plays a role in cartilage development. GelMA hydrogels provided an optimal scaffold, supporting cell viability and proliferation. The 3D model exhibited consistent responses to chondrogenic agonists, with TGF-ß3 enhancing cartilage-specific extracellular matrix (ECM) production and chondrogenic differentiation. The combination of Wnt3a and TGF-ß3 showed synergistic effects, promoting chondrogenic differentiation and ECM production. This study presents a 3D regenerative cartilage model with potential for investigating cartilage biology, disease mechanisms, and drug screening. The model provides insights into complex cartilage regeneration mechanisms and offers a platform for developing therapeutic approaches for cartilage repair and osteoarthritis treatment.

WNT Signaling Cascade Proteins and LRP6 in the Formation of Various Types of Coronary Lesions in Patients With Coronary Artery Disease.

AIM: Assessment of WNT1, WNT3a, and LRP6 concentrations in patients with ischemic heart disease (IHD) and obstructive and non-obstructive coronary artery (CA) disease. MATERIAL AND METHODS: This cross-sectional observational study included 50 IHD patients (verified by coronary angiography, CAG), of which 25 (50%) were men, mean age 64.9±8.1 years; 20 patients had non-obstructive CA disease (stenosis <50%), and 30 patients had hemodynamically significant stenosis. Concentrations of WNT1, WNT3a and LRP6 were measured in all patients. RESULTS: The concentrations of WNT1 and WNT3a proteins were significantly higher in patients with IHD and obstructive CA disease (p < 0.001), while the concentration of LRP6 was higher in the group with non-obstructive CA disease (p = 0.016). Data analysis of the group with obstructive CA disease showed a moderate correlation between WNT1 and LRP6 (ρ=0.374; p=0.042). Correlation analysis of all groups of patients with CA disease revealed a moderate association between the concentrations of WNT1 and uric acid (ρ=0.416; p=0.007). Regression analysis showed that risk factors for the development of IHD, such as increased body mass index, age, smoking, dyslipidemia, and hypertension, did not significantly influence the type of CA disease in IHD patients. According to ROC analysis, the obstructive form of IHD was predicted by a WNT3a concentration higher than 0.155 ng/ml and a LRP6 concentration lower than 12.94 ng/ml. CONCLUSION: IHD patients with non-obstructive CA disease had the greatest increase in LRP6, while patients with obstructive CA disease had significantly higher concentrations of the canonical WNT cascade proteins, WNT1 and WNT3a. According to the ROC analysis, a WNT3a concentration >0.155 ng/ml can serve as a predictor for the presence of hemodynamically significant CA stenosis in IHD patients (sensitivity 96.7%; specificity 70%), whereas a LRP6 concentration >12.94 ng/ml can predict the development of non-obstructive CA disease (sensitivity 76.7%; specificity 65%).

rESWT promoted angiogenesis via Bach1/Wnt/ß-catenin signaling pathway.

Previous reports have established that rESWT fosters angiogenesis, yet the mechanism by which rESWT promotes cerebral angiogenesis remains elusive. rESWT stimulated HUVECs proliferation as evidenced by the CCK-8 test, with an optimal dosage of 2.0 Bar, 200 impulses, and 2 Hz. The tube formation assay of HUVECs revealed that tube formation peaked at 36 h post-rESWT treatment, concurrent with the lowest expression level of Bach1, as detected by both Western blot and immunofluorescence. The expression level of Wnt3a, ß-catenin, and VEGF also peaked at 36 h. A Bach1 overexpression plasmid was transfected into HUVECs, resulting in a decreased expression level of Wnt3a, ß-catenin, and VEGF. Upon treatment with rESWT, the down-regulation of Wnt3a, ß-catenin, and VEGF expression in the transfected cells was reversed. The Wnt/ß-catenin inhibitor DKK-1 was utilized to suppress Wnt3a and ß-catenin expression, which led to a concurrent decrease in VEGF expression. However, rESWT treatment could restore the expression of these three proteins, even in the presence of DKK-1. Moreover, in the established OGD model, it was observed that rESWT could inhibit the overexpression of Bach1 and enhance VEGF and VEGFR-2 expression under the OGD environment.

Expression and potential molecular mechanism of TOP2A in metastasis of non-small cell lung cancer.

DNA topoisomerase II alpha (TOP2A) expression, gene alterations, and enzyme activity have been studied in various malignant tumors. Abnormal elevation of TOP2A expression is considered to be related to the development of non-small cell lung cancer (NSCLC). However, its association with tumor metastasis and its mode of action remains unclear. Bioinformatics, real-time quantitative PCR, immunohistochemistry and immunoblotting were used to detect TOP2A expression in NSCLC tissues and cells. Cell migration and invasion assays as well as cytoskeletal staining were performed to analyze the effects of TOP2A on the motility, migration and invasion ability of NSCLC cells. Cell cycle and apoptosis assays were used to verify the effects of TOP2A on apoptosis as well as cycle distribution in NSCLC. TOP2A expression was considerably upregulated in NSCLC and significantly correlated with tumor metastasis and the occurrence of epithelial-mesenchymal transition (EMT) in NSCLC. Additionally, by interacting with the classical ligand Wnt3a, TOP2A may trigger the canonical Wnt signaling pathway in NSCLC. These observations suggest that TOP2A promotes EMT in NSCLC by activating the Wnt/ß-catenin signaling pathway and positively regulates malignant events in NSCLC, in addition to its significant association with tumor metastasis. TOP2A promotes the metastasis of NSCLC by stimulating the canonical Wnt signaling pathway and inducing EMT. This study further elucidates the mechanism of action of TOP2A, suggesting that it might be a potential therapeutic target for anti-metastatic therapy.

A small molecule compound 759 inhibits the wnt/beta-catenin signaling pathway via increasing the Axin protein stability.

Wnt/ß-catenin signaling plays important role in cancers. Compound 759 is one of the compounds previously screened to identify inhibitors of the Wnt/ß-catenin pathway in A549 cells [Lee et al. in Bioorg Med Chem Lett 20:5900-5904, 2010]. However, the mechanism by which Compound 759 induces the inhibition of the Wnt/ß-catenin pathway remains unknown. In our study, we employed various assays to comprehensively evaluate the effects of Compound 759 on lung cancer cells. Our results demonstrated that Compound 759 significantly suppressed cell proliferation and Wnt3a-induced Topflash activity and arrested the cell cycle at the G1 stage. Changes in Wnt/ß-catenin signaling-related protein expression, gene activity, and protein stability including Axin, and p21, were achieved through western blot and qRT-PCR analysis. Compound 759 treatment upregulated the mRNA level of p21 and increased Axin protein levels without altering the mRNA expression in A549 cells. Co-treatment of Wnt3a and varying doses of Compound 759 dose-dependently increased the amounts of Axin1 in the cytosol and inhibited ß-catenin translocation into the nucleus. Moreover, Compound 759 reduced tumor size and weight in the A549 cell-induced tumor growth in the in vivo tumor xenograft mouse model. Our findings indicate that Compound 759 exhibits potential anti-cancer activity by inhibiting the Wnt/ß-catenin signaling pathway through the increase of Axin1 protein stability.

Wnt signaling regulates chemokine production and cell migration of circulating human monocytes.

The ß-catenin dependent canonical Wnt signaling pathway plays a crucial role in maintaining normal homeostasis. However, when dysregulated, Wnt signaling is closely associated with various pathological conditions, including inflammation and different types of cancer.Here, we show a new connection between the leukocyte inflammatory response and the Wnt signaling pathway. Specifically, we demonstrate that circulating human primary monocytes express distinct Wnt signaling components and are susceptible to stimulation by the classical Wnt ligand-Wnt-3a. Although this stimulation increased the levels of ß-catenin protein, the expression of the classical Wnt-target genes was not affected. Intriguingly, treating circulating human monocytes with Wnt-3a induces the secretion of cytokines and chemokines, enhancing monocyte migration. Mechanistically, the enhanced monocyte migration in response to Wnt stimuli is mediated through CCL2, a strong monocyte-chemoattractant.To further explore the physiological relevance of these findings, we conducted ex-vivo experiments using blood samples of patients with rheumatic joint diseases (RJD) - conditions where monocytes are known to be dysfunctional. Wnt-3a generated a unique cytokine expression profile, which was significantly distinct from that observed in monocytes obtained from healthy donors.Thus, our results provide the first evidence that Wnt-3a may serve as a potent stimulator of monocyte-driven immune processes. These findings contribute to our understanding of inflammatory diseases and, more importantly, shed light on the role of a core signaling pathway in the circulation.

DKK1 Activates the PI3K/AKT Pathway via CKAP4 to Balance the Inhibitory Effect on Wnt/ß-Catenin Signaling and Regulates Wnt3a-Induced MSC Migration.

Wnt/ß-catenin signaling plays a crucial role in the migration of mesenchymal stem cells (MSCs). However, our study has revealed an intriguing phenomenon where Dickkopf-1 (DKK1), an inhibitor of Wnt/ß-catenin signaling, promotes MSC migration at certain concentrations ranging from 25 to 100 ng/mL while inhibiting Wnt3a-induced MSC migration at a higher concentration (400 ng/mL). Interestingly, DKK1 consistently inhibited Wnt3a-induced phosphorylation of LRP6 at all concentrations. We further identified cytoskeleton-associated protein 4 (CKAP4), another DKK1 receptor, to be localized on the cell membrane of MSCs. Overexpressing the CRD2 deletion mutant of DKK1 (ΔCRD2), which selectively binds to CKAP4, promoted the accumulation of active ß-catenin (ABC), the phosphorylation of AKT (Ser473) and the migration of MSCs, suggesting that DKK1 may activate Wnt/ß-catenin signaling via the CKAP4/PI3K/AKT cascade. We also investigated the effect of the CKAP4 intracellular domain mutant (CKAP4-P/A) that failed to activate the PI3K/AKT pathway and found that CKAP4-P/A suppressed DKK1 (100 ng/mL)-induced AKT activation, ABC accumulation, and MSC migration. Moreover, CKAP4-P/A significantly weakened the inhibitory effects of DKK1 (400 ng/mL) on Wnt3a-induced MSC migration and Wnt/ß-catenin signaling. Based on these findings, we propose that DKK1 may activate the PI3K/AKT pathway via CKAP4 to balance the inhibitory effect on Wnt/ß-catenin signaling and thus regulate Wnt3a-induced migration of MSCs. Our study reveals a previously unrecognized role of DKK1 in regulating MSC migration, highlighting the importance of CKAP4 and PI3K/AKT pathways in this process.

R-spondin-1 induces Axin degradation via the LRP6-CK1ε axis.

R-spondins (RSPOs) are secreted signaling molecules that potentiate the Wnt/ß-catenin pathway by cooperating with Wnt ligands. RSPO1 is crucial in tissue development and tissue homeostasis. However, the molecular mechanism by which RSPOs activate Wnt/ß-catenin signaling remains elusive. In this study, we found that RSPOs could mediate the degradation of Axin through the ubiquitin-proteasome pathway. The results of Co-IP showed that the recombinant RSPO1 protein promoted the interaction between Axin1 and CK1ε. Either knockout of the CK1ε gene or treatment with the CK1δ/CK1ε inhibitor SR3029 caused an increase in Axin1 protein levels and attenuated RSPO1-induced degradation of the Axin1 protein. Moreover, we observed an increase in the number of associations of LRP6 with CK1ε and Axin1 following RSPO1 stimulation. Overexpression of LRP6 further potentiated Axin1 degradation mediated by RSPO1 or CK1ε. In addition, recombinant RSPO1 and Wnt3A proteins synergistically downregulated the protein expression of Axin1 and enhanced the transcriptional activity of the SuperTOPFlash reporter. Taken together, these results uncover the novel mechanism by which RSPOs activate Wnt/ß-catenin signaling through LRP6/CK1ε-mediated degradation of Axin.

Wnt3a/GSK3ß/ß-catenin Signalling Modulates Doxorubicin-associated Memory Deficits in Breast Cancer.

BACKGROUND: Chemobrain is widespread in breast cancer patients receiving chemotherapy. However, the exact mechanism, especially the associated signalling pathway, is not currently clear. This study was to evaluate the behavioural changes in breast cancer mice after chemotherapy and to further explore the role of Wnt3a/glycogen synthase kinase (GSK3ß)/ß-catenin signalling in chemobrain. METHODS: MMTV-PyMT(+) breast cancer mice were injected intraperitoneally with doxorubicin (4 mg/kg) once a week for three weeks to establish a chemobrain model. The Morris water maze (MWM) and novel object recognition (NOR) tests were performed to assess the learning and memory ability. Electron microscopy was used to observe the structural changes in the hippocampal CA1 region. The brain tissue of breast cancer mice after chemotherapy was taken out for mRNA-seq detection. Then, the expression levels and phosphorylation of key proteins in the Wnt3a/GSK3 ß/ß-catenin signalling pathway were evaluated through Western blotting (WB) and immunofluorescence. RESULTS: Doxorubicin-induced spatial and short-term memory impairment was observed in breast cancer mice, and obvious neuronal damage could be seen in the hippocampal CA1 region. Immunofluorescence staining for GSK3ß was increased. Wnt signalling pathway is highly enriched from mRNA-seq analysis, with GSK3ß genes at important nodes. The relative protein levels of p-PI3K, p-AKT, p-GSK3 ß, Wnt3a and TCF-1 were decreased significantly, while the p-ß-catenin level was increased. After injection of the GSK3ß inhibitor sb216763 (1 ng/0.5 µl/side), hippocampal neuronal injury was alleviated to some extent, and the changes in the expression of proteins upstream and downstream of this signalling pathway were reversed. CONCLUSION: Wnt3a/GSK3 ß/ß-catenin signalling is likely involved in doxorubicin-induced memory impairment. This result provides basic evidence for the further study of chemobrain in breast cancer.

RIPK4 downregulation impairs Wnt3A-stimulated invasiveness via Wnt/ß-catenin signaling in melanoma cells and tumor growth in vivo.

PURPOSE: The role of Wnt signaling in oncogenesis and drug resistance is well known. Receptor-interacting protein kinase (RIPK4) contributing to the increased activity of many signaling pathways, including Wnt/ß-catenin, may be an important target for designing new drugs for metastatic melanoma, but its role in melanoma is not fully understood. METHODS: We tested the effect of genetic manipulation of RIPK4 (CRISPR/Cas9) on xenograft growth. In addition, immunohistochemistry was used to detect active ß-catenin, Ki67 and necrosis in xenografts. Wnt signaling pathway activity was examined using Western blot and Top-Flash. The effect of RIPK4 knockout on melanoma cells in vitro stimulated Wnt3A on wound overgrowth, migration and invasion ability was then evaluated. RESULTS: Our study showed that CRISPR/Cas9-mediated RIPK4 knockout (KO) significantly reduced tumor growth in a mouse model of melanoma, particularly of WM266.4 cells. RIPK4 KO tumors exhibited lower percentages of Ki67+ cells as well as reduced necrotic area and decreased levels of active ß-catenin. In addition, we observed that RIPK4 knockout impaired Wnt3A-induced activation of LRP6 and ß-catenin, as manifested by a decrease in the transcriptional activity of ß-catenin in Top-Flash in both tested melanoma cell lines, A375 and WM266.4. Prolonged incubation (48 h) with Wnt3A showed reduced level of MMP9, C-myc, and increased SOX10, proteins whose transcription is also dependent on ß-catenin activity. Moreover, RIPK4 knockout led to the inhibition of scratch overgrowth, migration and invasion of these cells compared to their controls. CONCLUSION: RIPK4 knockdown inhibits melanoma tumor growth and Wnt3A stimulated migration and invasion indicating that RIPK4 might be a potential target for melanoma therapy.

Wnt-3a improves functional recovery after spinal cord injury by regulating the inflammatory and apoptotic response in rats via wnt/ß-catenin signaling pathway.

The specific molecular mechanism of neuroprotective effects of wnt-3a on spinal cord injury (SCI) has not been elucidated. In our study, we evaluated the recovery of motor function after SCI by BBB, observed neuronal apoptosis by western blot and TUNEL, observed the changes of neuronal inflammation by western blot and immunofluorescence staining, and observed the changes of motoneurons and spinal cord area in the anterior horn of the spinal cord via Nissl and HE staining. We found that wnt-3a could significantly promote the recovery of motor function, reduce the loss of motor neurons in the anterior horn of the spinal cord, promote the recovery of injured spinal cord tissue, inhibit neuronal apoptosis and inflammatory response, and ultimately promote neuronal function after SCI. However, when XAV939 inhibits the wnt/ß-catenin signaling pathway, the neuroprotective effects of wnt-3a are also significantly inhibited. The above results together indicated that wnt-3a exerts its neuroprotective effect on after SCI via activating the wnt/ß-catenin signaling pathway.

The selenoprotein P-LRP5/6-WNT3A complex promotes tumorigenesis in sporadic colorectal cancer.

Some studies suggest that the trace element selenium protects against colorectal cancer (CRC). However, the contribution of selenoprotein P (SELENOP), a unique selenocysteine-containing protein, to sporadic colorectal carcinogenesis challenges this paradigm. SELENOP is predominately secreted by the liver but is also expressed in various cells of the small intestine and colon in mice and humans. In this issue of the JCI, Pilat et al. demonstrate that increased SELENOP expression promoted the progression of conventional adenomas to carcinoma. SELENOP functioned as a modulator of canonical WNT signaling activity through interactions with WNT3A and its coreceptor LDL receptor-related protein 5/6 (LRP5/6). Secreted SELENOP formed a concentration gradient along the gut crypt axis, which might amplify WNT signaling activity by binding to LRPL5/6. The mechanism for WNT control via SELENOP may affect colorectal tumorigenesis and provide therapeutic targets for CRC.

hsa­miR­216a­3p regulates cell proliferation in oral cancer via the Wnt3a/ß­catenin pathway.

Oral cancer is one of the leading causes of death worldwide, with a reported 5­year survival rate of ~50% after treatment. The treatment measures for oral cancer are very expensive and affordability is low. Thus, it is necessary to develop more effective therapies to treat oral cancer. A number of studies have found that miRNAs are invasive biomarkers and have therapeutic potential in a variety of cancers. The present study included 30 oral patients and 30 healthy controls. Clinicopathological characteristic and miR­216a­3p/ß­catenin expression level of 30 oral cancer patients were analyzed. In addition, two oral cancer cell lines (HSC­6 and CAL­27) were used for mechanism­of­action study. The expression level of miR­216a­3p was higher in oral cancer patients compared with healthy controls and positively associated with tumor stage. Inhibition of miR­216a­3p potently suppressed cell viability and induced apoptosis of oral cancer cells. It was found that effects of miR­216a­3p on oral cancer were through Wnt3a signaling. It was also found that the expression level of ß­catenin was higher in oral cancer patients compared with healthy controls and positively associated with tumor stage; the effects of miR­216a­3p on oral cancer were through ß­catenin. In conclusion, miR­216a­3p and the Wnt­ß­catenin signaling pathway may be interesting candidates to develop effective therapies for oral cancers.

PDLIM1 inhibits cell migration and invasion in diabetic retinopathy via negatively regulating Wnt3a.

The injury of vascular endothelial cells is a crucial factor in the development of diabetic retinopathy (DR). PDLIM1 (a member of the PDZ and LIM protein family) has been reported to exert an essential function in vascular diseases. This study aimed to elucidate the role of PDLIM1 on retinal vascular endothelial cells in DR. Immunofluorescence staining was used to localize the expression of PDLIM1 in the mouse retina. In some tumor diseases, PDLIM1 has been reported to play a key role in regulating the Wnt pathway. However, no in-depth reports have been found in DR. Retinal capillary endothelial cells (RCECs) were treated with high-glucose and high-lipid (HG/HL) culture medium, and siRNA transfection to investigate the role of PDLIM1 in DR. PDLIM1 and Wnt3a expression was confirmed by qRT-PCR and western blotting. Flow cytometry, Transwell assay, and scratch assay were used to test the ability of cell apoptosis, migration, and invasion. PDLIM1 was mainly expressed in the retinal pigment epithelium (RPE), ganglion cell layer (GCL), inner plexus layer (IPL), and outer plexus layer (OPL). HG/HL increased Wnt3a levels and promoted cell's ability of apoptosis, migration, and invasion, which were reversed by the knockdown of PDLIM1. PDLIM1 was found to play a protective role in diabetic retinopathy by counter-regulating Wnt3a. PDLIM1 ameliorates cell apoptosis, migration, and invasion by negatively regulating Wnt3a in RCECs of DR, which suggests that PDLIM1 might be a promising therapeutic target for DR treatment.

Frizzled receptors facilitate Tiki inhibition of Wnt signaling at the cell surface.

The membrane-tethered protease Tiki antagonizes Wnt3a signaling by cleaving and inactivating Wnt3a in Wnt-producing cells. Tiki also functions in Wnt-receiving cells to antagonize Wnt signaling by an unknown mechanism. Here, we demonstrate that Tiki inhibition of Wnt signaling at the cell surface requires Frizzled (FZD) receptors. Tiki associates with the Wnt-FZD complex and cleaves the N-terminus of Wnt3a or Wnt5a, preventing the Wnt-FZD complex from recruiting and activating the coreceptor LRP6 or ROR1/2 without affecting Wnt-FZD complex stability. Intriguingly, we demonstrate that the N-terminus of Wnt3a is required for Wnt3a binding to LRP6 and activating ß-catenin signaling, while the N-terminus of Wnt5a is dispensable for recruiting and phosphorylating ROR1/2. Both Tiki enzymatic activity and its association with the Wnt-FZD complex contribute to its inhibitory function on Wnt5a. Our study uncovers the mechanism by which Tiki antagonizes Wnt signaling at the cell surface and reveals a negative role of FZDs in Wnt signaling by acting as Tiki cofactors. Our findings also reveal an unexpected role of the Wnt3a N-terminus in the engagement of the coreceptor LRP6.

WNT3a Signaling Inhibits Aromatase Expression in Breast Adipose Fibroblasts-A Possible Mechanism Supporting the Loss of Estrogen Responsiveness of Triple-Negative Breast Cancers.

Estrogen-dependent breast cancers rely on a constant supply of estrogens and expression of estrogen receptors. Local biosynthesis, by aromatase in breast adipose fibroblasts (BAFs), is their most important source for estrogens. Triple-negative breast cancers (TNBC) rely on other growth-promoting signals, including those from the Wnt pathway. In this study, we explored the hypothesis that Wnt signaling alters the proliferation of BAFs, and is involved in regulation of aromatase expression in BAFs. Conditioned medium (CM) from TNBC cells and WNT3a consistently increased BAF growth, and reduced aromatase activity up to 90%, by suppression of the aromatase promoter I.3/II region. Database searches identified three putative Wnt-responsive elements (WREs) in the aromatase promoter I.3/II. In luciferase reporter gene assays, promoter I.3/II activity was inhibited by overexpression of full-length T-cell factor (TCF)-4 in 3T3-L1 preadipocytes, which served as a model for BAFs. Full-length lymphoid enhancer-binding factor (LEF)-1 increased the transcriptional activity. However, TCF-4 binding to WRE1 in the aromatase promoter, was lost after WNT3a stimulation in immunoprecipitation-based in vitro DNA-binding assays, and in chromatin immunoprecipitation (ChIP). In vitro DNA-binding assays, ChIP, and Western blotting revealed a WNT3a-dependent switch of nuclear LEF-1 isoforms towards a truncated variant, whereas ß-catenin levels remained unchanged. This LEF-1 variant revealed dominant negative properties, and most likely recruited enzymes involved in heterochromatin formation. In addition, WNT3a induced the replacement of TCF-4 by the truncated LEF-1 variant, on WRE1 of the aromatase promoter I.3/II. The mechanism described here may be responsible for the loss of aromatase expression predominantly associated with TNBC. Tumors with (strong) expression of Wnt ligands actively suppress aromatase expression in BAFs. Consequently a reduced estrogen supply could favor the growth of estrogen-independent tumor cells, which consequently would make estrogen receptors dispensable. In summary, canonical Wnt signaling within (cancerous) breast tissue may be a major factor controlling local estrogen synthesis and action.

Klotho ameliorates angiotension-II-induced endothelial senescence via restoration of autophagy by inhibiting Wnt3a/GSK-3ß/mTOR signaling: A potential mechanism involved in prognostic performance of Klotho in coronary atherosclerotic disease.

OBJECTIVE: We aimed to evaluate the prognostic performance of circulating Klotho in coronary atherosclerotic disease (CAD), and to further explore the effect of Klotho on stress-mediated endothelial senescence and underlying mechanism. METHODS: A cohort of 295 patients had a 12-month follow-up for major adverse cardiovascular events (MACE). Serum Klotho was detected by enzyme linked immunosorbent assay. Cell viability, SA-ß-Gal staining, the expression of P53 and P16 were analyzed for endothelial senescence. Oxidative stress was evaluated by measurement of reactive oxygen species, superoxide dismutase and malondialdehyde. LC3, P62, Wnt3a, GSK-3ß and mTOR were analyzed by western blotting. Autophagosome formation was detected by adenovirus transfection. RESULTS: In epidemiological analysis, low Klotho (≤295.9 pg/ml) was significantly associated with MACE risk (HR=2.266, 95 %CI 1.229-4.176). In experimental analysis, Klotho alleviated endothelial senescence and oxidative stress caused by Ang-II exposure; Klotho restored impaired autophagic flux to ameliorate Ang-II induced endothelial senescence; Ang-II activated Wnt3a/GSK-3ß/mTOR signaling to inhibit autophagy, whereas Klotho restored autophagy through blockade of Wnt3a/GSK-3ß/mTOR signaling; Klotho ameliorated endothelial senescence by suppressing Wnt3a/GSK-3ß/mTOR pathway under Ang-II exposure. CONCLUSIONS: Prognostic significance of Klotho in CAD is potentially ascribed to its anti-endothelial senescence effect via autophagic flux restoration by inhibiting Wnt3a/ GSK-3ß/mTOR signaling.

Wnt3a is a promising target in colorectal cancer.

Most colorectal cancers (CRC) are associated with activated Wnt signaling, making it the fourth most prevalent type of cancer globally. To function properly, the Wnt signaling pathway requires secreted glycoproteins known as Wnt ligands (Wnts). Humans have 19 Wnts, which suggest a complicated signaling and biological process, and we still know little about their functions in developing CRC. This review aims to describe the canonical Wnt signaling in CRC, particularly the Wnt3a expression pattern, and their association with the angiogenesis and progression of CRC. This review also sheds light on the inhibition of Wnt3a signaling in CRC. Despite some obstacles, a thorough understanding of Wnts is essential for effectively managing CRC.

Wnt3a promotes odonto/osteogenic differentiation in vitro and tertiary dentin formation in a rat model.

AIM: To investigate the effect of Wnt3a on odonto/osteogenic differentiation of stem cells isolated from human exfoliated deciduous teeth (SHEDs) and reparative dentine formation in a rat model. METHODOLOGY: Stem cells isolated from human exfoliated deciduous teeth were cultured in media with Wnt3a (50-200 ng/ml). Wnt activation was confirmed by ß-catenin immunocytochemistry. Colony-forming unit assay (normalized percentage area), osteogenic gene expression analysis by real-time polymerase chain reaction and mineralization assays measured by the absorption at 540 nm were performed. Tertiary dentine formation in vivo was evaluated using 8-week-old, male Wistar rats. Cavities with pinpoint pulp exposure by a sharp instrument were prepared at the mesial surface of the first molars. Teeth were divided into (n = 6): (1) distilled water (negative control), (2) phosphate-buffered saline (PBS), (3) lithium chloride in DI (20 µM), and (4) Wnt3a in PBS (200 ng/ml). Collagen sponge was used as a scaffold. The cavity was sealed with glass ionomer restoration. Four weeks later, animals were euthanized by sodium pentobarbital (120 mg/kg body weight). Hard tissue formation was evaluated using micro-computerized tomography. Sixty consecutive slides from the initial plane were analysed and calculated as bone/dentine volume per total tissue volume. Paraffin sections (2 µm) were stained with haematoxylin and eosin and Masson's trichrome for morphological evaluation. Data are presented as the mean ± standard error. Mann-Whitney U test was used for two-group comparison. Kruskal Wallis followed by pairwise comparison was employed for three or more group comparisons. Statistical analysis was performed using GraphPad Prism 7. Differences were considered significant at p < .05. RESULTS: Wnt3a decreased SHEDs colony formation and increased OSX, BMP2, and DMP1 expression, corresponding to an increase in mineralization. Additionally, a significant increase in dentine/bone volume per total tissue volume was observed in Wnt3a treated defects. Dentine bridge formation at the exposure sites treated with Wnt3a demonstrated, while fibrous tissues were observed in the control. CONCLUSIONS: Wnt3a suppressed proliferation, increased osteogenic differentiation of SHEDs and promotes tertiary dentine formation. Wnt3a could be utilized as biological molecule for vital pulp therapy.

LINC00936 exacerbated myocardial infarction progression via miR-4795-3p/Wnt3a signaling pathway based on biological and imaging methods.

OBJECTIVE: LncRNAs show great potential in diagnosing and treating myocardial infarction (MI). Clarifying the mechanism of lncRNAs on MI is of great significance for the application of MI biomarkers. Therefore, this report intended to determine the role and mechanism of LINC00936 on MI by biological and imaging methods. METHODS: Hypoxia H9C2 model was established by hypoxia treatment. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay detected the apoptosis of H9C2. H2DCFDA staining and enzyme-linked immunosorbent assay (ELISA) was used to detect the reactive oxygen species (ROS) accumulation and Lactate dehydrogenase (LDH) contents, respectively. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect LINC00936, Wnt3a and miR-4795-3p levels. Western blot detected Wnt3a protein expression. Dual luciferase reporter assays detected the relationship of miR-4795-3p to LINC00936 or Wnt3a. Echocardiography analysis detected cardiac function. 2,3,5-Triphenyltetrazolium chloride (TTC) detected the infarct size. Masson staining detected the pathological changes. RESULTS: LINC00936 level was elevated in the MI patients compared with the controls. Overexpression of LINC00936 promoted apoptosis and ROS accumulation in hypoxia H9C2 model and exacerbated MI progression in vivo. miR-4795-3p bound with LINC00936 in H9C2 cells and miR-4795-3p mimics inhibited apoptosis and ROS accumulation in hypoxia H9C2 model regulated by LINC00936. Wnt3a was targeted by miR-4795-3p and Wnt3a elevation promoted apoptosis and ROS accumulation in hypoxia H9C2 model. CONCLUSION: In this report, we illustrated that LINC00936 exacerbated MI progression via the miR-4795-3p/Wnt3a signaling pathway based on biological and imaging methods. These findings might provide potential molecular target for the diagnosis and treatment of MI.