Wnt-C59 inhibits proinflammatory cytokine expression by reducing the interaction between β-catenin and NF-κB in LPS-stimulated epithelial and macrophage cells

Dysregulation of the Wnt pathway causes various diseases including cancer, Parkinson’s disease, Alzheimer’s disease, schizophrenia, osteoporosis, obesity and chronic kidney diseases. The modulation of dysregulated Wnt pathway is absolutely necessary. In the present study, we evaluated the anti-inflammatory effect and the mechanism of action of Wnt-C59, a Wnt signaling inhibitor, in lipopolysaccharide (LPS)-stimulated epithelial cells and macrophage cells. Wnt-C59 showed a dose-dependent anti-inflammatory effect by suppressing the expression of proinflammatory cytokines including IL6, CCL2, IL1A, IL1B, and TNF in LPS-stimulated cells. The dysregulation of the Wnt/β-catenin pathway in LPS stimulated cells was suppressed by Wnt-C59 treatment. The level of β-catenin, the executor protein of Wnt/β-catenin pathway, was elevated by LPS and suppressed by Wnt-C59. Overexpression of β-catenin rescued the suppressive effect of Wnt-C59 on proinflammatory cytokine expression and nuclear factor-kappa B (NF-κB) activity. We found that the interaction between β-catenin and NF-κB, measured by co-immunoprecipitation assay, was elevated by LPS and suppressed by Wnt-C59 treatment. Both NF-κB activity for its target DNA binding and the reporter activity of NF-κB-responsive promoter showed identical patterns with the interaction between β-catenin and NF-κB. Altogether, our findings suggest that the anti-inflammatory effect of Wnt-C59 is mediated by the reduction of the cellular level of β-catenin and the interaction between β-catenin and NF-κB, which results in the suppressions of the NF-κB activity and proinflammatory cytokine expression.     

The effect of various factors on variability of retinal nerve fiber layer thickness measurements using optical coherence tomography

Purpose

To evaluate the effects of various factors on the variability of retinal nerve fiber layer (RNFL) thickness measurements using the Stratus optical coherence tomography (OCT) in normal and glaucomatous eyes.

Methods

Four hundred seventy-four subjects (103 normal eyes and 371 glaucomatous eyes) were scanned to determine the RNFL thickness measurements using the Stratus OCT. Measurements were obtained twice during the same day. The standard deviation (SD) was used to compare the variability in RNFL thickness measurements of the normal subjects to that of the glaucomatous patients. Multivariate regression analysis was used to evaluate which covariates were independent predictors of SD in overall mean RNFL thickness.

Results

The mean SD of all RNFL thickness measurements was larger in the glaucoma group except in one sector. In the multivariate regression analysis, the average signal strength (SS) and the relative SS change (difference in SS between initial and repeat scans, divided by initial SS) were independent predictors of the SD in the RNFL thickness measurements (partial R² = 0.018, 0.013; p = 0.016, 0.040, respectively).

Conclusions

Glaucomatous eyes tend to be more variable than normal eyes in RNFL thickness measurement using the Straus OCT. The average SS and the relative SS changes appear to correlate with the variability in RNFL thickness measurement. Therefore, the results of the RNFL analysis should not be interpreted independently of these factors.     

Effect of Antifreeze Protein on Mouse Ovarian Tissue Cryopreservation and Transplantation

Purpose

To investigate the effect of antifreeze protein (AFP) supplementation on ovarian vitrification and transplantation.

Materials and Methods

In this experimental study, we researched a total of 182 ovaries from 4-week-old ICR mice. The equilibration solution included 20% ethylene glycol (EG), and the vitrification solution included 40% EG, 18% Ficoll, and 0.3 M sucrose. Intact ovaries were first suspended in 1 mL of equilibration solution for 10 min, and then mixed with 0.5 mL of vitrification solution for 5 min. Ovaries were randomly assigned to 3 groups and 0, 5, or 20 mg/mL of type III AFP was added into the vitrification solution (control, AFP5, and AFP20 groups, respectively). The vitrified ovaries were evaluated after warming and 2 weeks after autotransplantation. The main outcome measurements are follicular morphology and apoptosis assessed by histology and the TUNEL assay.

Results

A significantly higher intact follicle ratio was shown in the AFP treated groups (control, 28.9%; AFP5, 42.3%; and AFP20, 44.7%). The rate of apoptotic follicles was significantly lower in the AFP treated groups (control, 26.6%; AFP5, 18.7%; and AFP20, 12.6%). After transplantation of the vitrified-warmed ovaries, a significantly higher intact follicle ratio was shown in the AFP20 group. The rate of apoptotic follicles was similar among the groups.

Conclusion

The results of the present study suggest that supplementing AFP in the vitrification solution has beneficial effects on the survival of ovarian tissue during cryopreservation and transplantation.     

Polyphenol‐rich apple extract inhibits dexamethasone‐induced sebaceous lipids production by regulating SREBP1 expression

Sebum production and excretion is a primary function of the sebaceous glands, but abnormally increased sebum production is a major cause of acne vulgaris. To identify a new candidate that regulates sebum production, we investigated the possible inhibitory effects of apple polyphenols (APP) purified from unripe apples on primary cultured human sebocytes and in patients with acne vulgaris. Dexamethasone (Dex) increased lipid synthesis and expression of the sterol response element‐binding protein 1 (SREBP 1) and its target enzymes, acetyl‐CoA carboxylase (ACC) and fatty acid synthase (FAS), in the sebocytes. However, APP inhibited Dex‐induced lipid production and expression of SREBP‐1, ACC and FAS. APP also inhibited the increase in the expression and activation of glucocorticoid receptor in the sebocytes. Taken together, these results suggest that APP may be useful to regulate sebum production and may alleviate sebum‐involved skin disease, such as acne vulgaris.     

Coupled Ca2+/H+ transport by cytoplasmic buffers regulates local Ca2+ and H+ ion signaling

Ca²⁺ signaling regulates cell function. This is subject to modulation by H⁺ ions that are universal end-products of metabolism. Due to slow diffusion and common buffers, changes in cytoplasmic [Ca²⁺] ([Ca²⁺]_i) or [H⁺] ([H⁺]_i) can become compartmentalized, leading potentially to complex spatial Ca²⁺/H⁺ coupling. This was studied by fluorescence imaging of cardiac myocytes. An increase in [H⁺]_i, produced by superfusion of acetate (salt of membrane-permeant weak acid), evoked a [Ca²⁺]_i rise, independent of sarcolemmal Ca²⁺ influx or release from mitochondria, sarcoplasmic reticulum, or acidic stores. Photolytic H⁺ uncaging from 2-nitrobenzaldehyde also raised [Ca²⁺]_i, and the yield was reduced following inhibition of glycolysis or mitochondrial respiration. H⁺ uncaging into buffer mixtures in vitro demonstrated that Ca²⁺ unloading from proteins, histidyl dipeptides (HDPs; e.g., carnosine), and ATP can underlie the H⁺-evoked [Ca²⁺]_i rise. Raising [H⁺]_i tonically at one end of a myocyte evoked a local [Ca²⁺]_i rise in the acidic microdomain, which did not dissipate. The result is consistent with uphill Ca²⁺ transport into the acidic zone via Ca²⁺/H⁺ exchange on diffusible HDPs and ATP molecules, energized by the [H⁺]_i gradient. Ca²⁺ recruitment to a localized acid microdomain was greatly reduced during intracellular Mg²⁺ overload or by ATP depletion, maneuvers that reduce the Ca²⁺-carrying capacity of HDPs. Cytoplasmic HDPs and ATP underlie spatial Ca²⁺/H⁺ coupling in the cardiac myocyte by providing ion exchange and transport on common buffer sites. Given the abundance of cellular HDPs and ATP, spatial Ca²⁺/H⁺ coupling is likely to be of general importance in cell signaling.Most cells are exquisitely responsive to calcium (Ca²⁺) (1) and hydrogen (H⁺) ions (i.e., pH) (2). In cardiac myocytes, Ca²⁺ ions trigger contraction and control growth and development (3), whereas H⁺ ions, which are generated or consumed metabolically, are potent modulators of essentially all biological processes (4). By acting on Ca²⁺-handling proteins directly or via other molecules, H⁺ ions exert both inhibitory and excitatory effects on Ca²⁺ signaling. For example, in the ventricular myocyte, H⁺ ions can reduce Ca²⁺ release from sarcoplasmic reticulum (SR) stores, through inhibition of the SR Ca²⁺ ATPase (SERCA) pump and ryanodine receptor (RyR) Ca²⁺ channels (5, 6). In contrast, H⁺ ions can enhance SR Ca²⁺ release by stimulating sarcolemmal Na⁺/H⁺ exchange (NHE), which raises intracellular [Na⁺] and reduces the driving force for Ca²⁺ extrusion on Na⁺/Ca²⁺ exchange (NCX), leading to cellular retention of Ca²⁺ (7, 8). Ca²⁺ signaling is thus subservient to pH.Cytoplasmic Ca²⁺ and H⁺ ions bind avidly to buffer molecules, such that <1% of all Ca²⁺ ions and <0.001% of all H⁺ ions are free. Some of these buffers bind H⁺ and Ca²⁺ ions competitively, and this has been proposed to be one mechanism underlying cytoplasmic Ca²⁺/H⁺ coupling (9). Reversible binding to buffers greatly reduces the effective mobility of Ca²⁺ and H⁺ ions in cytoplasm (10, 11) and can allow for highly compartmentalized ionic microdomains, and hence a spatially heterogeneous regulation of cell function. In cardiac myocytes under resting (diastolic) conditions, the cytoplasm-averaged concentration of free [Ca²⁺] ([Ca²⁺]_i) and [H⁺] ([H⁺]_i) ions is kept near 10⁻⁷ M by membrane transporter proteins. Thus, [H⁺]_i is regulated by the balance of flux among acid-extruding and acid-loading transporter proteins at the sarcolemma [e.g., NHE and Cl⁻/OH⁻ (CHE) exchangers, respectively] (4). Similarly, the activity of SERCA and NCX proteins returns [Ca²⁺]_i to its diastolic level after evoked signaling events (3, 12). Despite these regulatory mechanisms, cytoplasmic gradients of [H⁺]_i and [Ca²⁺]_i do occur in myocytes and are an important part of their physiology. Gradients arise from local differences in transmembrane fluxes that alter [H⁺]_i or [Ca²⁺]_i. For example, spatial [H⁺]_i gradients are produced when NHE transporters, expressed mainly at the intercalated disk region, are activated (4, 13) or when membrane-permeant weak acids, such as CO₂, are presented locally (14). Similarly, release of Ca²⁺ through a cluster of RyR channels in the SR produces [Ca²⁺]_i nonuniformity in the form of Ca²⁺ sparks (15). Given the propensity of cytoplasm to develop ionic gradients, it is important to understand their underlying mechanism and functional role.The present work demonstrates a distinct form of spatial interaction between Ca²⁺ and H⁺ ions. We show that cytoplasmic [H⁺] gradients can produce stable [Ca²⁺]_i gradients, and vice versa, and that this interaction is mediated by low-molecular-weight (mobile) buffers with affinity for both ions. We demonstrate that the diffusive counterflux of H⁺ and Ca²⁺ bound to these buffers comprises a cytoplasmic Ca²⁺/H⁺ exchanger. This acts like a “pump” without a membrane, which can, for instance, recruit Ca²⁺ to acidic cellular microdomains. Cytoplasmic Ca²⁺/H⁺ exchange adds a spatial paradigm to our understanding of Ca²⁺ and H⁺ ion signaling.     

Arthroscopic versus mini-open rotator cuff repair: a comparison of clinical outcomes and patient satisfaction

This study compares the results of arthroscopic and arthroscopically assisted mini-open rotator cuff repair in a series of 84 patients who underwent repair of small, medium, or large tears between March 1997 and September 2001 with at least 2 years of follow-up. There were 42 arthroscopic repairs and 42 mini-open repairs. Of the patients, 81 (96.4%) had good or excellent UCLA (University of California, Los Angeles) scores (40 arthroscopic repairs [95.2%] and 41 mini-open repairs [97.6%]); there were 2 fair results and 1 poor outcome. The ASES (American Shoulder and Elbow Surgeons) scores averaged 91.1 for the arthroscopic group and 90.2 for the mini-open group (P > .05). Six patients required further surgery (three from the arthroscopic group and three from the mini-open group). Of 84 patients, 83 (98.8%) reported being satisfied with the procedure. At greater than 2 years of follow-up, arthroscopic and mini-open rotator cuff repairs produced similar results for small, medium, and large rotator cuff tears with equivalent patient satisfaction rates.     

Effects of Argon Laser Iridotomy on the Corneal Endothelium of Pigmented Rabbit Eyes

Purpose

In Asian countries, laser iridotomy for the treatment of angle-closure glaucoma is a common cause of bullous keratopathy, which may be associated with a shallow anterior chamber and dark iris pigmentation in Asians. Several cases of corneal decompensation after argon laser iridotomy have been reported. In the present study, we evaluated the harmful effects of argon laser iridotomy on the corneal endothelium.

Methods

Argon laser iridotomy was performed on the right eyes of pigmented rabbits. Changes in corneal thickness and endothelial cell density after laser iridotomy were evaluated. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed for assessment of corneal endothelial cell apoptosis. Combined staining with alizarin red and trypan blue, as well as a live/dead cell assay, were performed for evaluation of damage to the corneal endothelium induced by laser iridotomy.

Results

Corneal thickness did not change immediately after laser iridotomy; however, a significant increase was observed 24 hours after iridotomy (p = 0.001). The endothelial cell density of laser-treated eyes four days after laser iridotomy was significantly decreased compared with control eyes (p < 0.001). TUNEL staining showed many TUNEL-positive cells in the corneal endothelium and corneal stroma. No endothelial trypan blue-stained cell nuclei were observed after laser iridotomy; however, several large endothelial cells with damaged membrane integrity were observed. The live/dead cell assay clearly showed a large number of dead cells stained red in several areas throughout the entire corneal button 24 hours after iridotomy.

Conclusions

Argon laser iridotomy induces corneal endothelial cell apoptosis in pigmented rabbit eyes, resulting in decreased endothelial cell density.     

K6PC-5, a direct activator of sphingosine kinase 1, promotes epidermal differentiation through intracellular Ca2+ signaling

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, regulates multiple cellular responses such as Ca(2+) signaling, growth, survival, and differentiation. Because sphingosine kinase (SphK) is the enzyme directly responsible for production of S1P, many factors have been identified that regulate its activity and subsequent S1P levels. Here we synthesized a previously unidentified SphK activator, K6PC-5, and have studied its effects on intracellular Ca(2+) signaling in HaCaT cells and epidermal differentiation in murine skin. K6PC-5, a hydrophobic compound chemically named N-(1,3-dihydroxyisopropyl)-2-hexyl-3-oxo-decanamide, activated SphK (obtained from C57BL/6 murine blood and F9-12 cell lysates) in a dose-dependent manner. K6PC-5 induced both intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in HaCaT cells and Ca(2+) mobilization in hairless mouse epidermis. Both dimethylsphingosine (DMS) and dihydroxysphingosine (DHS), SphK inhibitors, and transfection of SphK1-siRNA blocked K6PC-5-induced increases in [Ca(2+)](i). The K6PC-5-induced [Ca(2+)](i) oscillations were dependent on thapsigargin-sensitive Ca(2+) stores and Ca(2+) entry, but independent of the classical phospholipase C-mediated pathway. In addition, K6PC-5 enhanced the expression of involucrin and filaggrin, specific differentiation-associated marker proteins in HaCaT cells, whereas transfection of SphK1-siRNA blocked the increase of involucrin. Topical K6PC-5 also enhanced the expression of involucrin, loricrin, filaggrin, and keratin 5 in intact murine epidermis. Finally, topical K6PC-5 inhibited epidermal hyperplasia by exerting antiproliferative effects on keratinocytes in murine epidermis. These results suggest that K6PC-5 acts to regulate both differentiation and proliferation of keratinocytes via [Ca(2+)](i) responses through S1P production. Thus, regulation of S1P levels may represent a novel approach for treatment of skin disorders characterized by abnormal differentiation and proliferation, such as atopic dermatitis and psoriasis.     

Psychological stress regulates antimicrobial peptide expression by both glucocorticoid and β-adrenergic mechanisms

Psychological stress (PS) exerts well-known negative consequences for permeability barrier function in humans and mice, and deterioration of barrier function appears to be attributable largely to excess production of endogenous glucocorticoids (GC). More recently, PS has been shown to compromise antimicrobial defense, also by GC-dependent mechanisms. We assessed here changes in a third antimicrobial peptide (AMP); i.e., the neuropeptide, catestatin (Cst), which also is expressed in the outer epidermis, and previously shown to be regulated by changes in permeability barrier status. In these studies, PS again provoked a decline in both mouse cathelicidin (CAMP) and mouse β-defensin 3 (mBD3) expression, in a GC-dependent fashion. In contrast, Cst immunostaining instead increased after short-term PS, but then began to decline with more sustained PS. In cultured keratinocytes, we showed further that GC downregulate Cst expression, but β-adrenergic blockade increased immunostaining for Cst in the face of long-term PS. Furthermore, β-adrenergic blockade also upregulated CAMP and mBD3 expression. Together, these results suggest that both endogenous GC and β-adrenergic signaling regulate AMP expression.