Sulfated CXCR3 Peptide Trap Use as a Promising Therapeutic Approach for Age-Related Macular Degeneration.

BACKGROUND AND OBJECTIVES: Chemokines have various biological functions and potential roles in the development or progression of neuroinflammatory diseases. However, the specific pathogenic roles of chemokines in the major cause for vision loss among the elderly, the leading cause of blindness in older individuals, remain elusive. Chemokines interact with their receptors expressed in the endothelium and on leukocytes. The sulfation of tyrosine residues in chemokine receptors increases the strength of ligand-receptor interaction and modulates signaling. Therefore, in the present study, we aimed to construct a human recombinant sulfated CXCR3 peptide trap (hCXCR3-S2) and mouse recombinant sulfated CXCR3 peptide trap (mCXCR3-S2) to demonstrate in vivo effects in preventing choroidal neovascularization (CNV) and chemotaxis. MATERIALS AND METHODS: We generated expression vectors for mCXCR3-S2 and hCXCR3-S2 with GST domains and their respective cDNA sequences. Following overexpression in E. coli BL21 (DE3), we purified the fusion proteins from cell lysates using affinity chromatography. First, the impact of hCXCR3-S2 was validated in vitro. Subsequently, the in vivo efficacy of mCXCR3-S2 was investigated using a laser-induced CNV mouse model, a mouse model of neovascular age-related macular degeneration (AMD). RESULTS: hCXCR3-S2 inhibited the migration and invasion of two human cancer cell lines. Intravitreal injection of mCXCR3-S2 attenuated CNV and macrophage recruitment in neovascular lesions of mouse models. These in vitro and in vivo effects were significantly stronger with CXCR3-S2 than with wild-type CXCR3 peptides. CONCLUSION: These findings demonstrate that the sulfated form of the CXCR3 peptide trap is a valuable tool that could be supplemented with antivascular endothelial growth factors in AMD treatment.

Methylsulfonylmethane ameliorates metabolic-associated fatty liver disease by restoring autophagy flux via AMPK/mTOR/ULK1 signaling pathway.

Introduction: Metabolism-associated fatty liver disease (MAFLD) is a global health concern because of its association with obesity, insulin resistance, and other metabolic abnormalities. Methylsulfonylmethane (MSM), an organic sulfur compound found in various plants and animals, exerts antioxidant and anti-inflammatory effects. Here, we aimed to assess the anti-obesity activity and autophagy-related mechanisms of Methylsulfonylmethane. Method: Human hepatoma (HepG2) cells treated with palmitic acid (PA) were used to examine the effects of MSM on autophagic clearance. To evaluate the anti-obesity effect of MSM, male C57/BL6 mice were fed a high-fat diet (HFD; 60% calories) and administered an oral dose of MSM (200 or 400 mg/kg/day). Moreover, we investigated the AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin complex 1 (mTORC1)/UNC-51-like autophagy-activating kinase 1 (ULK1) signaling pathway to further determine the underlying action mechanism of MSM. Results: Methylsulfonylmethane treatment significantly mitigated PA-induced protein aggregation in human hepatoma HepG2 cells. Additionally, Methylsulfonylmethane treatment reversed the PA-induced impairment of autophagic flux. Methylsulfonylmethane also enhanced the insulin sensitivity and significantly suppressed the HFD-induced obesity and hepatic steatosis in mice. Western blotting revealed that Methylsulfonylmethane improved ubiquitinated protein clearance in HFD-induced fatty liver. Remarkably, Methylsulfonylmethane promoted the activation of AMPK and ULK1 and inhibited mTOR activity. Conclusion: Our study suggests that MSM ameliorates hepatic steatosis by enhancing the autophagic flux via an AMPK/mTOR/ULK1-dependent signaling pathway. These findings highlight the therapeutic potential of MSM for obesity-related MAFLD treatment.

Polycystin-1 Enhances Stemmness Potential of Umbilical Cord Blood-Derived Mesenchymal Stem Cells.

Polycystic Kidney Disease (PKD) is a disorder that affects the kidneys and other organs, and its major forms are encoded by polycystin-1 (PC1) and polycystin-2 (PC2), as PKD1 and PKD2. It is located sandwiched inside and outside cell membranes and interacts with other cells. This protein is most active in kidney cells before birth, and PC1 and PC2 work together to help regulate cell proliferation, cell migration, and interactions with other cells. The molecular relationship and the function between PKD1 and cancer is well known, such as increased or decreased cell proliferation and promoting or suppressing cell migration depending on the cancer cell type specifically. However, its function in stem cells has not been revealed. Therefore, in this study, we investigated the biological function of PC1 and umbilical cord blood-derived mesenchymal stem cell (UCB-MSC). Furthermore, we assessed how it affects cell migration, proliferation, and the viability of cells when expressed in the PKD1 gene. In addition, we confirmed in an ex vivo artificial tooth model generated by the three-dimension printing technique that the ability to differentiate into osteocytes improved according to the expression level of the stemness markers when PKD1 was expressed. This study is the first report to examine the biological function of PKD1 in UCB-MSC. This gene may be capable of enhancing differentiation ability and maintaining long-term stemness for the therapeutic use of stem cells.

Ryk modulates the niche activity of mesenchymal stromal cells by fine-tuning canonical Wnt signaling.

The importance of modulating the intensity of Wnt signaling has been highlighted in various biological models, but their mechanisms remain unclear. In this study, we found that Ryk-an atypical Wnt receptor with a pseudokinase domain-has a Wnt-modulating effect in bone marrow stromal cells to control hematopoiesis-supporting activities. We first found that Ryk is predominantly expressed in the mesenchymal stromal cells (MSCs) of the bone marrow (BM) compared with hematopoietic cells. Downregulation of Ryk in MSCs decreased their clonogenic activity and ability to support self-renewing expansion of primitive hematopoietic progenitors (HPCs) in response to canonical Wnt ligands. In contrast, under high concentrations of Wnt, Ryk exerted suppressive effects on the transactivation of target genes and HPC-supporting effects in MSCs, thus fine-tuning the signaling intensity of Wnt in BM stromal cells. This ability of Ryk to modulate the HPC-supporting niche activity of MSCs was abrogated by induction of deletion mutants of Ryk lacking the intracellular domain or extracellular domain, indicating that the pseudokinase-containing intracellular domain mediates the Wnt-modulating effects in response to extracellular Wnt ligands. These findings indicate that the ability of the BM microenvironment to respond to extracellular signals and support hematopoiesis may be fine-tuned by Ryk via modulation of Wnt signaling intensity to coordinate hematopoietic activity.

Sestrin2 inhibits YAP activation and negatively regulates corneal epithelial cell proliferation.

Corneal wound healing is essential for the maintenance of corneal integrity and transparency and involves a series of physiological processes that depend on the proliferation of epithelial cells. However, the molecular mechanisms that control corneal epithelial cell proliferation are poorly understood. Here, we show that Sestrin2, a stress-inducible protein, is downregulated in the corneal epithelium during wound healing and that the proliferation of epithelial basal cells is enhanced in Sestrin2-deficient mice. We also show that YAP, a major downstream effector of the Hippo signaling pathway, regulates cell proliferation during corneal epithelial wound repair and that Sestrin2 suppresses its activity. Moreover, increased levels of reactive oxygen species in the Sestrin2-deficient corneal epithelium promote the nuclear localization and dephosphorylation of YAP, activating it to enhance the proliferation of corneal epithelial cells. These results reveal that Sestrin2 is a negative regulator of YAP, which regulates the proliferative capacity of basal epithelial cells, and may serve as a potential therapeutic target for corneal epithelial damage.

Plant-derived angiogenin fusion protein's cytoprotective effect on trabecular meshwork damage induced by Benzalkonium chloride in mice.

BACKGROUND: Benzalkonium chloride (BAK), commonly used in glaucoma treatment, is an eye drop preservative with dose-dependent toxicity. Previous studies have observed the multi-functional benefits of angiogenin (ANG) against glaucoma. In our study, we evaluated ANG's cytoprotective effect on the trabecular meshwork (TM) damage induced by BAK. Additionally, we developed a plant-derived ANG fusion protein and evaluated its effect on TM structure and function. METHODS: We synthesized plant-derived ANG (ANG-FcK) by fuzing immunoglobulin G's Fc region and KDEL to conventional recombinant human ANG (Rh-ANG) purified from transgenic tobacco plants. We established a mouse model using BAK to look for degenerative changes in the TM, and to evaluate the protective effects of ANG-FcK and Rh-ANG. Intraocular pressure (IOP) was measured for 4 weeks and ultrastructural changes, deposition of fluorescent microbeads, type I and IV collagen, fibronectin, laminin and α-SMA expression were analyzed after the mice were euthanized. RESULTS: TM structural and functional degeneration were induced by 0.1% BAK instillation in mice. ANG co-treatment preserved TM outflow function, which we measured using IOP and a microbead tracer. ANG prevented phenotypic and ultrastructure changes, and that protective effect might be related to the anti-fibrosis mechanism. We observed a similar cytoprotective effect in the BAK-induced degenerative TM mouse model, suggesting that plant-derived ANG-FcK could be a promising glaucoma treatment.

Effects of Ranibizumab, Bevacizumab, and Aflibercept on Senescent Retinal Pigment Epithelial Cells.

PURPOSE: Anti-vascular endothelial growth factor (VEGF) agents have been used for the last 10 years, but their safety profile, including cytotoxicity against various ocular cells such as retinal pigment epithelial (RPE) cells, remains a serious concern. Safety studies of VEGF agents conducted to date have primarily relied on healthy RPE cells. In this study, we assessed the safety of three anti-VEGF agents, namely, ranibizumab, bevacizumab, and aflibercept, on senescent RPE cells. METHODS: Senescent human induced pluripotent stem cell-derived RPE cells were generated by continuous replication and confirmed with senescence biomarkers. The viability, proliferation, protein expression, and phagocytosis of the senescent RPE cells were characterized 3 days after anti-VEGF treatment with clinical doses of ranibizumab, bevacizumab, or aflibercept. RESULTS: Clinical doses of ranibizumab, bevacizumab, or aflibercept did not decrease the viability or alter proliferation of senescent RPE cells. In addition, the anti-VEGF agents did not induce additional senescence, impair the protein expression of zonula occludens-1 and RPE65, or reduce the phagocytosis capacity of senescent RPE cells. CONCLUSIONS: Clinical dosages of ranibizumab, bevacizumab, or aflibercept do not induce significant cytotoxicity in senescent RPE cells.

Ambient fine particulate matters induce cell death and inflammatory response by influencing mitochondria function in human corneal epithelial cells.

Ambient fine particulate matter (AFP) is a main risk factor for the cornea as ultraviolet light. However, the mechanism of corneal damage following exposure to AFP has been poorly understood. In this study, we first confirmed that AFP can penetrate the cornea of mice, considering that two-dimensional cell culture systems are limited in reflecting the situation in vivo. Then, we investigated the toxic mechanism using human corneal epithelial (HCET) cells. At 24h after exposure, AFP located within the autophagosome-like vacuoles, and cell proliferation was clearly inhibited in all the tested concentration. Production of ROS and NO and secretion of pro-inflammatory cytokines were elevated in a dose-dependent manner. Additionally, conversion of LC3B from I-type to II-type and activation of caspase cascade which show autophagic- and apoptotic cell death, respectively, were observed in cells exposed to AFP. Furthermore, AFP decreased mitochondrial volume, inhibited ATP production, and altered the expression of metabolism-related genes. Taken together, we suggest that AFP induces cell death and inflammatory response by influencing mitochondrial function in HCET cells. In addition, we recommend that stringent air quality regulations are needed for eye health.

Nano-sized iron particles may induce multiple pathways of cell death following generation of mistranscripted RNA in human corneal epithelial cells.

Iron is closely associated with an ambient particulate matters-induced inflammatory response, and the cornea that covers the front of the eye, is among tissues exposed directly to ambient particulate matters. Prior to this study, we confirmed that nano-sized iron particles (FeNPs) can penetrate the cornea. Thus, we identified the toxic mechanism of FeNPs using human corneal epithelial cells. At 24h after exposure, FeNPs located inside autophagosome-like vacuoles or freely within human corneal epithelial cells. Level of inflammatory mediators including nitric oxide, cytokines, and a chemokine was notably elevated accompanied by the increased generation of reactive oxygen species. Additionally, cell proliferation dose-dependently decreased, and level of multiple pathways of cell death-related indicators was clearly altered following exposure to FeNPs. Furthermore, expression of gene encoding DNA binding protein inhibitor (1, 2, and 3), which are correlated to inhibition of the binding of mistranscripted RNA, was significantly down-regulated. More importantly, expression of p-Akt and caspase-3 and conversion to LC3B-II from LC3B-I was enhanced by pretreatment with a caspase-1 inhibitor. Taken together, we suggest that FeNPs may induce multiple pathways of cell death via generation of mistranscripted RNA, and these cell death pathways may influence by cross-talk. Furthermore, we propose the need of further study for the possibility of tumorigenesis following exposure to FeNPs.

Role of TOPK in lipopolysaccharide-induced breast cancer cell migration and invasion.

Inflammation has been known to be linked to invasion or metastasis of breast cancer, which has poor prognosis, although the regulatory mechanism remains to be undiscovered. Here we show that T-LAK cell-originated protein kinase (TOPK) mediates pro-inflammatory endotoxin lipopolysaccharide (LPS)-induced breast cancer cell migration and invasion. The mRNA or protein level of TOPK, toll- like receptor4 (TLR4), interleukin (IL)-6, vascular endothelial growth factor (VEGF) or matrix metalloproteinase9 (MMP9) genes related to TLR4 signaling or tumor progression was induced by LPS treatment in MCF7 breast cancer cells, but the induction was abolished by stable knocking down of TOPK in MCF7 cells. Also, TOPK depletion decreased LPS-induced phosphorylation of p38, but not ERK and JNK among mitogen-activated protein kinases (MAPKs). On the other hand, we revealed that TOPK is essential for transcriptional activity of NF-κB or MMP9 promoter triggered by LPS. The induced promoter activity of NF-κB or MMP9 but not AP-1 was inhibited by knocking down of TOPK. Furthermore, we demonstrated that inhibitor of TOPK or MMP9 as well as MMP9 siRNA efficiently blocked LPS-induced migration or invasion of breast cancer cell lines. Interestingly, both of expression of TOPK and TLR4 were markedly increased in high-grade breast cancer. Collectively, we conclude that TOPK functions as a key mediator of LPS/TLR4-induced breast cancer cell migration and invasion through regulation of MMP9 expression or activity, implying a potential role of TOPK as a therapeutic target linking LPS-induced inflammation to breast cancer development.

Laser Photocoagulation Induces Transduction of Retinal Pigment Epithelial Cells by Intravitreally Administered Adeno-Associated Viral Vectors.

Retinal transduction by intravitreally administered adeno-associated viral (AAV) vector is previously known to be extremely limited to the neural retina except AAV2 capsid type. Recently, we showed that prior laser photocoagulation enhances retinal transduction of intravitreally administered AAV vectors, including the outer retina and retinal pigment epithelium (RPE). Here, by performing short-pulse laser pretreatment on the mouse retina, we demonstrate RPE cells transduced by three different capsid types of AAV vectors, AAV2, AAV5, and AAV8, using RPE wholemounts. For all capsid types, laser pretreatment effectively induced the transduction of RPE cells in and around the laser site.

Over-expression of translationally controlled tumor protein in lens epithelial cells seems to be associated with cataract development.

Inhibition of Na,K-ATPase causes opacification of the lens through abnormal increases in sodium and calcium levels, disturbed osmotic equilibrium, activation of proteolytic enzymes and cell damage. We previously identified Translationally Controlled Tumor Protein (TCTP) as a cytoplasmic repressor of Na,K-ATPase and confirmed that systemic hypertension is induced in transgenic mice over-expressing TCTP through inhibition of vascular Na,K-ATPase and increased intracellular calcium mobilization. In the current study, we confirmed the role of TCTP in causing intracellular calcium mobilization by inhibiting Na,K-ATPase in a human lens epithelial cell line and further showed that some of the TCTP-transgenic mice develop cataracts with an incidence rate of 7.38% compared to 1.47% in controls. We demonstrated that TCTP acts as a cataractogenic factor through the repression of Na,K-ATPase activity and calcium mobilization in lens epithelial cells.

Transactivation of EGFR mediates insulin-stimulated ERK1/2 activation and enhanced cell migration in human corneal epithelial cells.

PURPOSE: Insulin activates phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK)-1/2 in human corneal epithelial cells. These events have been shown to be involved in wound healing. However, the mechanism of insulin-induced ERK pathway is not clear during corneal wound healing. In this study, the effect of insulin associated with epidermal growth factor receptor (EGFR) on wound healing in transformed human corneal epithelial cells was investigated to determine the signaling mechanism involved. METHODS: SV40-immortalized human corneal epithelial (THCE) cells were cultured on a diluted Matrigel matrix that resembled the basement membrane of the corneal epithelium. A wound was introduced with a micropipette tip, and closure of the scratch wound was photographed 12 h after exposure to insulin. Activation of EGFR was analyzed by immunoprecipitation, and cytoskeletal rearrangements were visualized with rhodamine-conjugated phalloidin. RESULTS: Exposure of corneal epithelial cells to insulin induced phosphorylation of EGFR. Inhibition of EGFR activation by AG1478 or the MMP inhibitor, GM6001, reduced phosphorylation of insulin-induced ERK in the presence of insulin and delayed wound closure. In addition, cells exposed to insulin contained stress fibers and their submembranous cortical actin was depleted. These effects were inhibited by AG1478. CONCLUSIONS: Inhibition of EGFR activity decreases cell migration involved in insulin-induced wound repair, an effect that mimics inhibition of MMP activation. Inhibition of MMP activity leads to decreased EGFR phosphorylation. Our data show that insulin stimulates wound healing in the corneal epithelium by activating EGFR, and point to a novel insulin signaling pathway that acts during corneal wound healing.

Expression of Wnt and MMP in epithelial cells during corneal wound healing.

PURPOSE: To study the expression of Wnt during corneal wound healing and to understand the signaling mechanism involved. METHODS: Rat cornea was demarcated on the central area by 4-mm trepine, and the epithelium within this area was removed by scalpel. The epithelium was scraped and isolated to extract RNA. To determine the proliferation of corneal epithelial cells, corneoscleral rims from human donors were treated with dispase II for 15 minutes, and epithelial cells were isolated. Cells were plated on a 3T3 feeder cells layer. The proliferation of corneal epithelial cells was evaluated by colony-forming efficiency. RESULTS: Wnt 5b and 7a were rapidly induced in wounded cornea, and Wnt 7a promoted proliferation of corneal epithelial cells. In addition, matrix metalloproteinase (MMP)-12 was expressed in wounded rat corneal epithelium. Transcription of MMP-12 was responsive to Wnt/beta-catenin signaling. Function blockade of MMP-12 delayed Wnt 7a-induced cell proliferation. CONCLUSION: These results indicate that Wnt proteins and MMP-12 regulate the proliferation of corneal epithelial cells and that Wnt signaling contributes to the resurfacing of defective areas during corneal wound healing.

Wnt-7a up-regulates matrix metalloproteinase-12 expression and promotes cell proliferation in corneal epithelial cells during wound healing.

Corneal wound repair involves the rapid coverage of a denuded area by residual epithelial cells. During wound healing, there are different cell behaviors in different regions of the epithelium: cell proliferation in the peripheral epithelium and cell migration in the central epithelium. We found that Wnt-7a was rapidly induced in the wounded cornea, promoted the proliferation of corneal epithelial cells, and enhanced wound closure. Matrix metalloproteinase-12 (MMP-12) was detected in the peripheral epithelium, where cell proliferation was enhanced, but was diminished in the migrating central epithelium. Wnt-7a induced the accumulation of beta-catenin and the activation of Rac and beta-catenin, and Rac synergistically induced the transcription of MMP-12. Blocking the function of MMP-12 delayed wound closure induced by Wnt-7a. Our results also suggest that, in addition to the beta-catenin pathway, Wnt-7a might induce a beta-catenin-independent pathway. By regulating the proliferation of corneal epithelial cells, Wnt-7a and MMP-12 appear to contribute to corneal wound healing.

Wnt signaling enhances FGF2-triggered lens fiber cell differentiation.

Wnt signaling is implicated in many developmental processes, including cell fate changes. Several members of the Wnt family, as well as other molecules involved in Wnt signaling, including Frizzled receptors, LDL-related protein co-receptors, members of the Dishevelled and Dickkopf families, are known to be expressed in the lens during embryonic or postembryonic development. However, the function of Wnt signaling in lens fiber differentiation remains unknown. Here, we show that GSK-3beta kinase is inactivated and that beta-catenin accumulates during the early stages of lens fiber cell differentiation. In an explant culture system, Wnt conditioned medium (CM) induced the accumulation of beta-crystallin, a marker of fiber cell differentiation, without changing cell shape. In contrast, epithelial cells stimulated with Wnt after priming with FGF elongated, accumulated beta-crystallin, aquaporin-0, p57kip2, and altered their expression of cadherins. Treatment with lithium, which stabilizes beta-catenin, induced the accumulation of beta-crystallin, but explants treated with lithium after FGF priming did not elongate as they did after Wnt application. These results show that Wnts promote the morphological aspects of fiber cell differentiation in a process that requires FGF signaling, but is independent of beta-catenin. Wnt signaling may play an important role in lens epithelial-to-fiber differentiation.

Alteration of cadherin in dexamethasone-induced cataract organ-cultured rat lens.

PURPOSE: A side effect associated with long-term treatment of various diseases with steroids is a high incidence of posterior subcapsular cataracts (PSC). To understand the mechanism underlying steroid-induced cataract, the cultured lens model was developed, and the expression of potential candidate proteins during opacity formation was examined. METHOD: Rat lenses were carefully dissected from the surrounding ocular tissue and incubated in medium 199. Dexamethasone was then added to the medium. The lenses were cultured for 7 days and photographed daily to record the development of opacity. Differential expression of candidate proteins was examined by Western blot analysis. RESULT: Various degrees of opacity were observed on the posterior subcapsular region as early as 5 days after incubation with dexamethasone. The expression of E-cadherin and N-cadherin decreased in the cultured rat lenses during the development of opacity. CONCLUSIONS: The pattern of opacity that developed in cultured rat lenses closely resembled that observed in patients with PSC. The results suggest that the decrease in E-cadherin plays a role in the formation of steroid-induced cataract.

Ectopic expression of Axin blocks neuronal differentiation of embryonic carcinoma P19 cells.

Axin regulates Wnt signaling through down-regulation of beta-catenin. To test the role of Wnt signaling in neuronal differentiation, embryonal carcinoma P19 cells (P19 EC), which can be stimulated to differentiate into a neuron-like phenotype in response to retinoic acid (RA), were used. Reverse transcription-PCR and Western blot analysis showed that Axin is expressed in undifferentiated cells, whereas the level is clearly reduced during RA-induced neuronal differentiation. Interestingly, Axin levels were not reduced during endodermal differentiation of P19 EC cells and F9 EC cells by RA, suggesting that the reduction of the Axin level is a specific property of neuronal differentiation. Western analysis showed that the cytoplasmic level of beta-catenin increased during neuronal differentiation of P19 EC cells. Indirect immunofluorescence with beta-catenin antibody showed that the localization of beta-catenin was changed from membrane in undifferentiated cells to nuclei in neuronal P19 EC cells. Induced expression of Axin during endodermal and early neuronal differentiation, using the Tet-On system, did not block normal differentiation. However, maintenance of the Axin level blocked neuronal differentiation and inhibited expression of a neuron-specific marker protein, beta III-tubulin. Also, ectopic induction of a beta-catenin signaling inhibitor, ICAT, inhibited expression of beta III-tubulin. In contrast, addition of Wnt-3A-conditioned medium during the neuronal differentiation period enhanced the expression of beta III-tubulin. Overall, our data show that Wnt-3a/canonical beta-catenin signaling through the down-regulation of Axin may play an important role in neuronal differentiation.