The Sonic Hedgehog Signaling Pathway Induces Myopic Development by Activating Matrix Metalloproteinase (MMP)-2 in Guinea Pigs

Purpose

To investigate whether the Sonic hedgehog (Shh) signaling induces myopic development by increasing the expression of matrix metalloproteinase (MMP)-2 in guinea pigs.

Methods

A translucent diffuser was glued onto the right eye to induce form-deprivation myopia (FDM) in 10 guinea pigs. Four guinea pigs were served as a control group. The other 100 guinea pigs were subdivided into 5 groups (20 per group) and received a 10 µl intravitreal injection every 2 days for 4 times. Two groups were injected with 20 or 50 µg/ml Shh amino-terminal peptide (Shh-N) into the right eye and 0.1% bovine serum albumin into the other. FDM was induced in the right eyes of the three cyclopamine-treated groups and both eyes were injected with 50, 100, or 200 µg/ml cyclopamine. Retinoscopic refraction and eye dimensions were assessed on Day 14 of treatment. MMP-2 protein expression was determined in both scleras by western blotting.

Results

Both concentrations of Shh-N stimulated myopic development and axial growth as compared with control eyes. Myopia and axial elongation were significantly greater in the 50 µg/ml than in the 20 µg/ml Shh-N group (P<0.001 and P = 0.0019, respectively). All three doses of cyclopamine significantly attenuated myopic development compared with the FDM group (P<0.0001). Cyclopamine at 100 or 200 µg/ml significantly reduced axial elongation compared with the FDM group (P = 0.044 and P = 0.001, respectively). FDM-induced myopia and axial elongation were significantly greater in the 50 µg/ml than in the 200 µg/ml cyclopamine group (P<0.0001 and P = 0.008, respectively). MMP-2 expression was significantly greater in Shh-N–treated eyes than in the control eyes, and was lower in the cyclopamine plus FDM groups than in the FDM group.

Conclusions

The Shh signaling pathway induces myopic development by activating MMP-2 in guinea pigs.     

Selective Modulation of Wnt Ligands and Their Receptors in Adipose Tissue by Chronic Hyperadiponectinemia

Background

Adiponectin-transgenic mice had many small adipocytes in both subcutaneous and visceral adipose tissues, and showed higher sensitivity to insulin, longer life span, and reduced chronic inflammation. We hypothesized that adiponectin regulates Wnt signaling in adipocytes and thereby modulates adipocyte proliferation and chronic inflammation in adipose tissue.

Materials and Methods

We examined the expression of all Wnt ligands and their receptors and the activity of Wnt signaling pathways in visceral adipose tissue from wild-type mice and two lines of adiponectin-transgenic mice. The effects of adiponectin were also investigated in cultured 3T3-L1 cells.

Results

The Wnt5b, Wnt6, Frizzled 6 (Fzd6), and Fzd9 genes were up-regulated in both lines of transgenic mice, whereas Wnt1, Wnt2, Wnt5a, Wnt9b, Wnt10b, Wnt11, Fzd1, Fzd2, Fzd4, Fzd7, and the Fzd coreceptor low-density-lipoprotein receptor-related protein 6 (Lrp6) were reduced. There was no difference in total β-catenin levels in whole-cell extracts, non-phospho-β-catenin levels in nuclear extracts, or mRNA levels of β-catenin target genes, indicating that hyperadiponectinemia did not affect canonical Wnt signaling. In contrast, phosphorylated calcium/calmodulin-dependent kinase II (p-CaMKII) and phosphorylated Jun N-terminal kinase (p-JNK) were markedly reduced in adipose tissue from the transgenic mice. The adipose tissue of the transgenic mice consisted of many small cells and had increased expression of adiponectin, whereas cyclooxygenase-2 expression was reduced. Wnt5b expression was elevated in preadipocytes of the transgenic mice and decreased in diet-induced obese mice, suggesting a role in adipocyte differentiation. Some Wnt genes, Fzd genes, and p-CaMKII protein were down-regulated in 3T3-L1 cells cultured with a high concentration of adiponectin.

Conclusion

Chronic hyperadiponectinemia selectively modulated the expression of Wnt ligands, Fzd receptors and LRP coreceptors accompanied by the inhibition of the Wnt/Ca²⁺ and JNK signaling pathways, which may be involved in the altered adipocyte cellularity, endogenous adiponectin production, and anti-inflammatory action induced by hyperadiponectinemia.     

A complex interaction between Wnt signaling and TNF-α in nucleus pulposus cells

Introduction

Increased expression of the proinflammatory cytokine TNF-α in intervertebral discs (IVDs) leads to inflammation, which results in progressive IVD degeneration. We have previously reported that activation of Wnt-β-catenin (hereafter called Wnt) signaling suppresses the proliferation of nucleus pulposus cells and induces cell senescence, suggesting that Wnt signaling triggers the process of degeneration of the IVD. However, it is not known whether cross talk between TNF-α and Wnt signaling plays a role in the regulation of nucleus pulposus cells. The goal of the present study was to examine the effect of the interaction between Wnt signaling and the proinflammatory cytokine TNF-α in nucleus pulposus cells.

Methods

Cells isolated from rat nucleus pulposus regions of IVDs were cultured in monolayers, and the expression and promoter activity of Wnt signaling and TNF-α were evaluated. We also examined whether the inhibition of Wnt signaling using cotransfection with Dickkopf (DKK) isoforms and Sclerostin (SOST) could block the effects of pathological TNF-α expression in nucleus pulposus cells.

Results

TNF-α stimulated the expression and promoter activity of Wnt signaling in nucleus pulposus cells. In addition, the activation of Wnt signaling by 6-bromoindirubin-3′-oxime (BIO), which is a selective inhibitor of glycogen synthase kinase 3 (GSK-3) activity that activates Wnt signaling, increased TNF-α expression and promoter activity. Conversely, the suppression of TNF-α promoter activity using a β-catenin small interfering RNA was evident. Moreover, transfection with DKK-3, DKK-4, or SOST, which are inhibitors of Wnt signaling, blocked Wnt signaling-mediated TNF-α activation; these effects were not observed for DKK-1 or DKK-2.

Conclusions

Here, we have demonstrated that Wnt signaling regulates TNF-α and that Wnt signaling and TNF-α form a positive-feedback loop in nucleus pulposus cells. The results of the present study provide in vitro evidence that activation of Wnt signaling upregulates the TNF-α expression and might cause the degeneration of nucleus pulposus cells. We speculate that blocking this pathway might protect nucleus pulposus cells against degeneration.     

Expression of Wnt signaling components during Xenopus pronephros development

Background

The formation of the vertebrate kidney is tightly regulated and relies on multiple evolutionarily conserved inductive events. These are present in the complex metanephric kidney of higher vertebrates, but also in the more primitive pronephric kidney functional in the larval stages of amphibians and fish. Wnts have long been viewed as central in this process. Canonical β-Catenin-dependent Wnt signaling establishes kidney progenitors and non-canonical β-Catenin-independent Wnt signaling participate in the morphogenetic processes that form the highly sophisticated nephron structure. While some individual Wnt signaling components have been studied extensively in the kidney, the overall pathway has not yet been analyzed in depth.

Methodology/Principal Findings

Here we report a detailed expression analysis of all Wnt ligands, receptors and several downstream Wnt effectors during pronephros development in Xenopus laevis using in situ hybridization. Out of 19 Wnt ligands, only three, Wnt4, Wnt9a and Wnt11, are specifically expressed in the pronephros. Others such as Wnt8a are present, but in a broader domain comprising adjacent tissues in addition to the kidney. The same paradigm is observed for the Wnt receptors and its downstream signaling components. Fzd1, Fzd4, Fzd6, Fzd7, Fzd8 as well as Celsr1 and Prickle1 show distinct expression domains in the pronephric kidney, whereas the non-traditional Wnt receptors, Ror2 and Ryk, as well as the majority of the effector molecules are rather ubiquitous. In addition to this spatial regulation, the timing of expression is also tightly regulated. In particular, non-canonical Wnt signaling seems to be restricted to later stages of pronephros development.

Conclusion/Significance

Together these data suggest a complex cross talk between canonical and non-canonical Wnt signaling is required to establish a functional pronephric kidney.     

Functional Wnt signaling is increased in idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease, characterized by distorted lung architecture and loss of respiratory function. Alveolar epithelial cell injury and hyperplasia, enhanced extracellular matrix deposition, and (myo)fibroblast activation are features of IPF. Wnt/β-catenin signaling has been shown to determine epithelial cell fate during development. As aberrant reactivation of developmental signaling pathways has been suggested to contribute to IPF pathogenesis, we hypothesized that Wnt/β-catenin signaling is activated in epithelial cells in IPF. Thus, we quantified and localized the expression and activity of the Wnt/β-catenin pathway in IPF.

Methodology/Principal Findings

The expression of Wnt1, 3a, 7b, and 10b, the Wnt receptors Fzd1-4, Lrp5-6, as well as the intracellular signal transducers Gsk-3β, β-catenin, Tcf1, 3, 4, and Lef1 was analyzed in IPF and transplant donor lungs by quantitative real-time (q)RT-PCR. Wnt1, 7b and 10b, Fzd2 and 3, β-catenin, and Lef1 expression was significantly increased in IPF. Immunohistochemical analysis localized Wnt1, Wnt3a, β-catenin, and Gsk-3β expression largely to alveolar and bronchial epithelium. This was confirmed by qRT-PCR of primary alveolar epithelial type II (ATII) cells, demonstrating a significant increase of Wnt signaling in ATII cells derived from IPF patients. In addition, Western blot analysis of phospho-Gsk-3β, phospho-Lrp6, and β-catenin, and qRT-PCR of the Wnt target genes cyclin D1, Mmp 7, or Fibronectin 1 demonstrated increased functional Wnt/β-catenin signaling in IPF compared with controls. Functional in vitro studies further revealed that Wnt ligands induced lung epithelial cell proliferation and (myo)fibroblast activation and collagen synthesis.

Conclusions/Significance

Our study demonstrates that the Wnt/β-catenin pathway is expressed and operative in adult lung epithelium. Increased Wnt/β-catenin signaling may be involved in epithelial cell injury and hyperplasia, as well as impaired epithelial-mesenchymal cross-talk in IPF. Thus, modification of Wnt signaling may represent a therapeutic option in IPF.     

Visual acuity measures do not reliably detect childhood refractive error--an epidemiological study

Purpose

To investigate the utility of uncorrected visual acuity measures in screening for refractive error in white school children aged 6-7-years and 12-13-years.

Methods

The Northern Ireland Childhood Errors of Refraction (NICER) study used a stratified random cluster design to recruit children from schools in Northern Ireland. Detailed eye examinations included assessment of logMAR visual acuity and cycloplegic autorefraction. Spherical equivalent refractive data from the right eye were used to classify significant refractive error as myopia of at least 1DS, hyperopia as greater than +3.50DS and astigmatism as greater than 1.50DC, whether it occurred in isolation or in association with myopia or hyperopia.

Results

Results are presented from 661 white 12-13-year-old and 392 white 6-7-year-old school-children. Using a cut-off of uncorrected visual acuity poorer than 0.20 logMAR to detect significant refractive error gave a sensitivity of 50% and specificity of 92% in 6-7-year-olds and 73% and 93% respectively in 12-13-year-olds. In 12-13-year-old children a cut-off of poorer than 0.20 logMAR had a sensitivity of 92% and a specificity of 91% in detecting myopia and a sensitivity of 41% and a specificity of 84% in detecting hyperopia.

Conclusions

Vision screening using logMAR acuity can reliably detect myopia, but not hyperopia or astigmatism in school-age children. Providers of vision screening programs should be cognisant that where detection of uncorrected hyperopic and/or astigmatic refractive error is an aspiration, current UK protocols will not effectively deliver.     

Refractive Errors in 3–6 Year-Old Chinese Children: A Very Low Prevalence of Myopia?

Purpose

To examine the prevalence of refractive errors in children aged 3–6 years in China.

Methods

Children were recruited for a trial of a home-based amblyopia screening kit in Guangzhou preschools, during which cycloplegic refractions were measured in both eyes of 2480 children. Cycloplegic refraction (from 3 to 4 drops of 1% cyclopentolate to ensure abolition of the light reflex) was measured by both autorefraction and retinoscopy. Refractive errors were defined as followed: myopia (at least −0.50 D in the worse eye), hyperopia (at least +2.00 D in the worse eye) and astigmatism (at least 1.50 D in the worse eye). Different definitions, as specified in the text, were also used to facilitate comparison with other studies.

Results

The mean spherical equivalent refractive error was at least +1.22 D for all ages and both genders. The prevalence of myopia for any definition at any age was at most 2.5%, and lower in most cases. In contrast, the prevalence of hyperopia was generally over 20%, and declined slightly with age. The prevalence of astigmatism was between 6% and 11%. There was very little change in refractive error with age over this age range.

Conclusions

Previous reports of less hyperopic mean spherical equivalent refractive error, and more myopia and less hyperopia in children of this age may be due to problems with achieving adequate cycloplegia in children with dark irises. Using up to 4 drops of 1% cyclopentolate may be necessary to accurately measure refractive error in paediatric studies of such children. Our results suggest that children from all ethnic groups may follow a similar pattern of early refractive development, with little myopia and a hyperopic mean spherical equivalent over +1.00 D up to the age of 5–6 yearsin most conditions.     

Dickkopf-1 as a Novel Predictor Is Associated with Risk Stratification by GRACE Risk Scores for Predictive Value in Patients with Acute Coronary Syndrome: A Retrospective Research

Objective

Dickkopf-1 (DKK-1), a major regulator of the Wnt pathway, plays an important role in cardiovascular disease. However, no study has evaluated the association of DKK-1 and acute coronary syndrome (ACS). We investigated this association and whether the Global Registry of Acute Coronary Events (GRACE) hospital-discharge risk score predicting major adverse cardiac events (MACE) can be improved by adding the DKK-1 value.

Methods

We enrolled 291 patients (46 with ST-segment elevation myocardial infarction [STEMI] and 245 with non-ST elevated ACS [NSTE-ACS]) who were divided into groups by tertiles of baseline plasma DKK-1 level measured by ELISA. The GRACE risk score was calculated and predictive value alone and together with DKK-1 and/or high-sensitivity C-reactive protein (hs-CRP) level were assessed, respectively.

Results

Compared with patients with NSTE-ACS, those with STEMI had higher plasma DKK-1 level at baseline (P = 0.006). Plasma DKK-1 level was correlated with hs-CRP level (r = 0.295, P<0.001) and was greater with high than intermediate or low GRACE scores (P = 0.002 and P<0.001, respectively). We found 44 (15.1%) MACEs during a median 2-year follow-up. DKK-1 levels were higher for patients with than without events (P<0.001). The rate of MACE increased with increasing DKK-1 level (P<0.001). The area under the receiver operating characteristic curve for GRACE score with MACE was 0.524 and improved to 0.791 with the addition of hs-CRP level, 0.775 with the addition of DKK-1 level and 0.847 with both values added.

Conclusions

DKK-1 is an independent predictor of long-term MACE of patients with ACS. The long-term predictive ability of post-discharge GRACE score may be enhanced by adding DKK-1 level.     

One-Step Transepithelial Topography-Guided Ablation in the Treatment of Myopic Astigmatism

Purpose

To evaluate one-step topography-guided transepithelial ablation in the treatment of low to moderate myopic astigmatism using a 1KHz excimer laser.

Methods

Retrospective study of 117 consecutive eyes available for evaluation 12 months after surgery. Pre- and post-operative visual and refractive data as well as post-operative pain and haze were analyzed. A novel technique integrating custom refractive- and epithelial- ablation in a single uninterrupted procedure was used.

Results

The mean pre-operative spherical equivalent (SE) and the mean cylinder were: –3.22 diopters (D) ±1.54 (SD) (range –0.63 to –7.25 D) and –0.77 D ±0.65 (range 0 to –4.50 D), respectively. At 12 months after surgery: no eyes lost ≥2 lines of corrected distant visual acuity (CDVA). Safety and efficacy indexes were 1.27 and 1.09, respectively. Uncorrected distant visual acuity (UDVA) was ≥20/20 in 96.6% of the eyes. Manifest refraction spherical equivalent was within ±0.5 D of the desired refraction in 93.2% of the eyes. Average root mean square (RMS) wavefront error measured at central 6 mm, increased from 0.38 pre-operatively to 0.47 µm post-operatively. Refractive stability was achieved and sustained 1 month after surgery. No visually significant haze was registered during the observation period. Post-operative pain was reported in 4.5% of patients.

Conclusions

One-step transepithelial topography-guided treatment for low to moderate myopia and astigmatism performed with a 1 KHz laser, provided safe, effective, predictable and stable results with low pain and no visually significant haze.     

Correlation between Lamina Cribrosa Tilt Angles,Myopia and Glaucoma Using OCT with a Wide Bandwidth Femtosecond Mode-Locked Laser

Purpose

To measure horizontal and vertical lamina cribrosa (LC) tilt angles and investigate associated factors using prototype optical coherence tomography (OCT) with a broad wavelength laser light source.

Design

Cross sectional study.

Methods

Twenty-eight no glaucoma eyes (from 15 subjects) and 25 glaucoma eyes (from 14 patients) were enrolled. A total of 300 optic nerve head B-scans were obtained in 10 µm steps and the inner edge of Bruch''s membrane opening (BMO) was identified as the reference plane. The vertical and horizontal angles between BMO line and approximate the best-fitting line for the surface of the LC were measured and potential associated factors were estimated with univariate and multivariate logistic regression analyses.

Results

The median (interquartile range) horizontal and vertical tilt angles were 7.10 (2.43–11.45) degrees and 4.15 (2.60–6.85) degrees in eyes without glaucoma and 8.50 (4.40–14.10) degrees and 9.30 (6.90–14.15) degrees in glaucoma eyes, respectively. The refractive errors had a statistically significant association with horizontal LC tilt angles (coefficients, −1.53 per diopter) and glaucoma had a significant correlation with vertical tilt angles (coefficients, 6.56) using multiple logistic regression analysis (p<0.001).

Conclusions

OCT allowed evaluation of the internal tilting of the LC compared with the BMO. The horizontal internal LC tilt angle was correlated with refractive errors, corresponding to myopic physiological changes, and vertical internal LC tilt was correlated with glaucoma, corresponding to glaucomatous pathological changes. These parameters have important implications for investigation of the correlation between myopia, glaucoma and LC morphological features.     

Lamina Cribrosa Defects and Optic Disc Morphology in Primary Open Angle Glaucoma with High Myopia

Purpose

To investigate whether lamina cribrosa (LC) defects are associated with optic disc morphology in primary open angle glaucoma (POAG) eyes with high myopia.

Methods

A total of 129 POAG patients and 55 age-matched control subjects with high myopia were evaluated. Three-dimensional scan images obtained by swept source optical coherence tomography were used to detect LC defects. Radial B-scans and infrared images obtained by spectral domain optical coherence tomography were used to measure β-peripapillary atrophy (PPA) lengths with and without Bruch''s membrane (BM) (temporal, nasal, superior, and inferior), tilt angle (vertical and horizontal), and disc diameter (transverse and longitudinal). Peripapillary intrachoroidal cavitations (PICCs), disc area, ovality index, and cyclotorsion of the optic disc were analyzed as well.

Results

LC defects were found in 70 of 129 (54.2%) POAG eyes and 1 of 55 (1.8%) control eyes (P<0.001). Age, sex, spherical equivalent, axial length, intraocular pressure, and central corneal thickness were not significantly different among POAG eyes with LC defects, POAG eyes without LC defects, and control eyes. Temporal PPA lengths without BM in all three groups correlated significantly with vertical and horizontal tilt angles, although no PPA length with BM correlated significantly with any tilt angle. PICCs were detected more frequently in POAG eyes with LC defects than those without LC defects (P = 0.01) and control eyes (P = 0.02). POAG eyes with LC defects showed a smaller ovality index (P = 0.004), longer temporal PPA without BM (P<0.001), and larger vertical/horizontal tilt angles (vertical, P<0.001; horizontal, P = 0.01), and transverse diameter (P = 0.01). In multivariate analysis for the presence of LC defects, presence of POAG (P<0.001) and vertical tilt angle (P<0.001) were identified as significant.

Conclusions

The presence of LC defects was associated with myopic optic disc morphology in POAG eyes with high myopia.     

Thyroid Hormone-Regulated Wnt5a/Ror2 Signaling Is Essential for Dedifferentiation of Larval Epithelial Cells into Adult Stem Cells in the Xenopus laevis Intestine

Background and Aims

Amphibian intestinal remodeling, where thyroid hormone (T3) induces some larval epithelial cells to become adult stem cells analogous to the mammalian intestinal ones, serves as a unique model for studying how the adult stem cells are formed. To clarify its molecular mechanisms, we here investigated roles of non-canonical Wnt signaling in the larval-to-adult intestinal remodeling during Xenopus laevis metamorphosis.

Methods/Findings

Our quantitative RT-PCR (qRT-PCR) and immunohistochemical analyses indicated that the expressions of Wnt5a and its receptors, frizzled 2 (Fzd2) and receptor tyrosine kinase-like orphan receptor 2 (Ror2) are up-regulated by T3 and are spatiotemporally correlated with adult epithelial development in the X. laevis intestine. Notably, changes in morphology of larval absorptive epithelial cells expressing Ror2 coincide well with formation of the adult stem cells during metamorphosis. In addition, by using organ cultures of the tadpole intestine, we have experimentally shown that addition of exogenous Wnt5a protein to the culture medium causes morphological changes in the larval epithelium expressing Ror2 even in the absence of T3. In contrast, in the presence of T3 where the adult stem cells are formed in vitro, inhibition of endogenous Wnt5a by an anti-Wnt5a antibody suppressed the epithelial morphological changes, leading to the failure of stem cell formation.

Significance

Our findings strongly suggest that the adult stem cells originate from the larval absorptive cells expressing Ror2, which require Wnt5a/Ror2 signaling for their dedifferentiation accompanied by changes in cell morphology.     

Endogenous Wnt/β-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells

Background

Wnt/β-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4.

Methodology/Principal Findings

We tested the hypothesis that Wnt/β-catenin signals control both early mesoderm induction and later cardiac differentiation in this system. Addition of exogenous Wnt3a at the time of induction enhanced cardiac differentiation, while early inhibition of endogenous Wnt/β-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for β-myosin heavy chain (β-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. Measurement of Brachyury expression showed that addition of Wnt3a enhances mesoderm induction, whereas blockade of endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/β-catenin signaling is required for Smad1 activation by BMP4.

Conclusions/Significance

Our data indicate that induction of mesoderm and subsequent cardiac differentiation from human ES cells requires fine-tuned cross talk between activin A/BMP4 and Wnt/β-catenin pathways. Controlling these pathways permits efficient generation of cardiomyocytes for basic studies or cardiac repair applications.     

Bili Inhibits Wnt/β-Catenin Signaling by Regulating the Recruitment of Axin to LRP6

Background

Insights into how the Frizzled/LRP6 receptor complex receives, transduces and terminates Wnt signals will enhance our understanding of the control of the Wnt/ß-catenin pathway.

Methodology/Principal Findings

In pursuit of such insights, we performed a genome-wide RNAi screen in Drosophila cells expressing an activated form of LRP6 and a β-catenin-responsive reporter. This screen resulted in the identification of Bili, a Band4.1-domain containing protein, as a negative regulator of Wnt/β-catenin signaling. We found that the expression of Bili in Drosophila embryos and larval imaginal discs significantly overlaps with the expression of Wingless (Wg), the Drosophila Wnt ortholog, which is consistent with a potential function for Bili in the Wg pathway. We then tested the functions of Bili in both invertebrate and vertebrate animal model systems. Loss-of-function studies in Drosophila and zebrafish embryos, as well as human cultured cells, demonstrate that Bili is an evolutionarily conserved antagonist of Wnt/β-catenin signaling. Mechanistically, we found that Bili exerts its antagonistic effects by inhibiting the recruitment of AXIN to LRP6 required during pathway activation.

Conclusions

These studies identify Bili as an evolutionarily conserved negative regulator of the Wnt/β-catenin pathway.     

The Role of Wnt/β-Catenin Signaling in Enterocyte Turnover during Methotrexate-Induced Intestinal Mucositis in a Rat

Background/Aims

Intestinal mucositis is a common side-effect in patients who receive aggressive chemotherapy. The Wnt signaling pathway is critical for establishing and maintaining the proliferative compartment of the intestine. In the present study, we tested whether Wnt/β-catenin signaling is involved in methotrexate (MTX)-induced intestinal damage in a rat model.

Methods

Non-pretreated and pretreated with MTX Caco-2 cells were evaluated for cell proliferation and apoptosis using FACS analysis. Adult rats were divided into three experimental groups: Control rats; MTX-2 animals were treated with a single dose of MTX given IP and were sacrificed on day 2, and MTX-4 rats were treated with MTX similar to group B and were sacrificed on day 4. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation, and enterocyte apoptosis were measured at sacrifice. Real Time PCR and Western blot was used to determine the level of Wnt/β-catenin related genes and protein expression.

Results

In the vitro experiment, treatment with MTX resulted in marked decrease in early cell proliferation rates following by a 17-fold increase in late cell proliferation rates compared to early proliferation. Treatment with MTX resulted in a significant increase in early and late apoptosis compared to Caco-2 untreated cells. In the vivo experiment, MTX-2 and MTX-4 rats demonstrated intestinal mucosal hypoplasia. MTX-2 rats demonstrated a significant decrease in FRZ-2, Wnt 3A Wnt 5A, β-catenin, c-myc mRNA expression and a significant decrease in β-catenin and Akt protein levels compared to control animals. Four days following MTX administration, rats demonstrated a trend toward a restoration of Wnt/β-catenin signaling especially in ileum.

Conclusions

Wnt/β-catenin signaling is involved in enterocyte turnover during MTX-induced intestinal mucositis in a rat.     

Defects of the Lamina Cribrosa in High Myopia and Glaucoma

Purpose

We evaluated the prevalence and characteristics of the defects of the lamina cribrosa (LC) in high myopia and glaucoma, and compared them with control eyes using swept-source optical coherence tomography (SS-OCT).

Methods

One hundred fifty-nine eyes of 108 participants were divided into four subgroups; high myopia with glaucoma (MG, 67 eyes of 46 subjects), glaucoma without high myopia (G, 22 eyes of 13 subjects), high myopia without glaucoma (M, 35 eyes of 29 subjects), and a control group with neither glaucoma nor high myopia (C, 35 eyes of 20 subjects). The LC defects were identified and located using a standardized protocol in serial horizontal OCT scans. The prevalence rates of the defects were compared among the groups. Demographic and ocular factors were compared between eyes with and without defects.

Results

LC defects were observed in one eye (0.03%) in the C group, 8 eyes (22.9%) in the M group, 11 eyes (50%) in the G group, and 28 eyes (41.8%) in the MG group. The prevalence rates of the defects differed significantly among the groups (P = 0.0009). Most eyes with defects in the G and MG groups (79.5%) had damage in the corresponding visual hemifields. Other factors such as visual acuity, intraocular pressure, axial length, refractive error, disc ovality, or parapapillary atrophy area did not differ significantly between eyes with and without LC defects.

Conclusions

High myopia and glaucoma significantly increased the risk of LC damage. The LC damage in non-glaucomatous highly myopic eyes may at least partly explain the increased risk of developing glaucoma in myopic eyes.     

The effects of oxygen tension and antiaging factor Klotho on Wnt signaling in nucleus pulposus cells

Introduction

The goals of this study were to examine the oxemic regulation of Wnt signaling to explore whether Wnt signaling accelerates the age-related degeneration of nucleus pulposus cells, and if so, to define the mechanism underlying this effect. We investigated the expression of Klotho, a newly identified antiaging gene, and whether its regulation is attributable to the suppression of Wnt signaling.

Methods

Rat nucleus pulposus cells were cultured under normoxic (21% O₂) or hypoxic (2% O₂) conditions, and the expression and promoter activity of Wnt signaling and Klotho were evaluated. The effect of Klotho protein was examined with transfection experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, senescence-associated β-galactosidase staining, and cell-cycle analysis. To determine the methylation status of the Klotho promoter region, bisulfite genomic sequencing analysis was performed. Its relation with the activation of Wnt signaling was assessed. We also examined whether the expression of Klotho could block the effects of pathological Wnt expression in nucleus pulposus cells.

Results

Nucleus pulposus cells exhibited increased β-catenin mRNA and protein under the hypoxic condition. Klotho protein was expressed in vivo, and protein and messenger RNA expression decreased under the hypoxic condition. Klotho treatment decreased cell proliferation and induced the quiescence of nucleus pulposus cells. In addition, Klotho treatment inhibited expression of β-catenin gene and protein compared with untreated control cells.

Conclusions

These data indicate that Wnt signaling and Klotho form a negative-feedback loop in nucleus pulposus cells. These results suggest that the expression of Klotho is regulated by the balance between upregulation and downregulation of Wnt signaling.