Discovery of Molecular Markers to Discriminate Corneal Endothelial Cells in the Human Body

The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant number of CECs results in corneal edema called bullous keratopathy which can lead to severe visual loss. Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.     

Recovery of Corneal Endothelial Cells from Periphery after Injury

Background

Wound healing of the endothelium occurs through cell enlargement and migration. However, the peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium in endothelial injury.

Aim

To investigate the recovery process of corneal endothelial cells (CECs) from corneal endothelial injury.

Methods

Three patients with unilateral chemical eye injuries, and 15 rabbit eyes with corneal endothelial chemical injuries were studied. Slit lamp examination, specular microscopy, and ultrasound pachymetry were performed immediately after chemical injury and 1, 3, 6, and 9 months later. The anterior chambers of eyes from New Zealand white rabbits were injected with 0.1 mL of 0.05 N NaOH for 10 min (NaOH group). Corneal edema was evaluated at day 1, 7, and 14. Vital staining was performed using alizarin red and trypan blue.

Results

Specular microscopy did not reveal any corneal endothelial cells immediately after injury. Corneal edema subsided from the periphery to the center, CEC density increased, and central corneal thickness decreased over time. In the animal study, corneal edema was greater in the NaOH group compared to the control at both day 1 and day 7. At day 1, no CECs were detected at the center and periphery of the corneas in the NaOH group. Two weeks after injury, small, hexagonal CECs were detected in peripheral cornea, while CECs in mid-periphery were large and non-hexagonal.

Conclusions

CECs migrated from the periphery to the center of the cornea after endothelial injury. The peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium.     

Interleukin-1β-Induced Wnt5a Enhances Human Corneal Endothelial Cell Migration through Regulation of Cdc42 and RhoA

Wnt5a can activate β-catenin-independent pathways for regulation of various cellular functions, such as migration, that play critical roles in wound repair. Investigation of Wnt5a signaling may help identify therapeutic targets for enhancing corneal endothelial wound healing that could provide an alternative to corneal transplantation in patients with blindness from endothelial dysfunction. However, Wnt5a signaling in corneal endothelial cells (CECs) has not been well characterized. In this study, we show transient induction of Wnt5a by interleukin-1β (IL-1β) stimulation proceeds through NF-κB in human CECs. This leads to binding of Fzd5 to Ror2, resulting in activation of disheveled protein (Dvl) and subsequently disheveled-associated activator of morphogenesis 1 (DAAM1). This leads to activation of Cdc42 and subsequent inhibition of RhoA. Inhibition of RhoA leads to parallel dephosphorylation and inactivation of LIM domain kinase 2 along with dephosphorylation and activation of slingshot 1, resulting in dephosphorylation and activation of cofilin and leading to enhanced cell migration. These findings suggest that Wnt5a enhances cell migration through activation of Cdc42 and inactivation of RhoA in human CECs.     

角膜内皮细胞的检测、受损因素及治疗新进展

摘要：角膜内皮细胞(corneal endothelial cells,CECs)通过其屏障作用及离子泵功能维持角膜透明,但年龄、疾病及创伤等因素会使其功能有所降低,导致角膜失代偿、角膜水肿、大泡性角膜病变,甚至视力的丧失。因此,CECs可谓是角膜的"生命"。近年来,CECs的检测方法逐渐新增,并且不同检测指标具有不同意义;导致CECs受损的因素也有所增加,同时CECs损伤后的治疗也逐渐成为近年来的研究热点。早期发现CECs受损、明确病因并进行干预或治疗会减少CECs的损伤,对指导临床工作有重要的意义。本文主要对CECs检测方法及指标、损伤因素及其治疗的最新进展进行了综述。     

Raf kinase inhibitor protein mediated signaling inhibits invasion and metastasis of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the most common type of liver cancer with high mortality and poor prognosis. Mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways have been implicated in promoting tumor cell proliferation and invasion of HCC cells.MethodsAs a potential inhibitor of tumor metastasis, the role of Raf kinase inhibitor protein (RKIP) in HCC development and the functional relevance with MAPK and NF-κB signaling pathways were investigated. The levels of RKIP expression were examined in human HCC tissues and correlated with tumor stages and metastatic status. Function of RKIP in cellular proliferation, migration, invasion and apoptosis was investigated in HCC cell lines by either overexpressing or knocking down RKIP expression. Mouse xenograft model was established to assess the effect of RKIP expression on tumor growth.ResultsOur results demonstrated decreased RKIP expression in HCC tissues and a strong correlation with tumor grade and distant metastasis. Manipulation of RKIP expression in HCCLM3 and HepG2 cells indicated that RKIP functioned to inhibit HCC cell motility and invasiveness, and contributed to tumor growth inhibition in vivo. Mechanistic studies showed that the function of RKIP was mediated through MAPK and NF-κB signaling pathways. However, cell type-dependent RKIP regulation on these two pathways was also suggested, indicating the complex nature of signaling network.ConclusionOur study provides a better understanding on the molecular mechanisms of HCC metastasis and sets the foundation for the development of targeted therapeutics for HCC.     

NLRC3 regulates cellular proliferation and apoptosis to attenuate the development of colorectal cancer

Nucleotide-binding domain, leucine-rich-repeat–containing proteins (NLRs) are intracellular innate immune sensors of pathogen-associated and damage-associated molecular patterns. NLRs regulate diverse biologic processes such as inflammatory responses, cell proliferation and death, and gut microbiota to attenuate tumorigenesis. In a recent publication in Nature, we identified NLRC3 as a negative regulator of PI3K–mTOR signaling and characterized its potential tumor suppressor function. Enterocytes lacking NLRC3 cannot control cellular proliferation because they are unable to suppress activation of PI3K–mTOR signaling pathways. In this Extra-View, we explore possible mechanisms through which NLRC3 regulates cellular proliferation and cell death. Besides interacting with PI3K, NLRC3 associates with TRAF6 and mTOR, confirming our recent finding that NLRC3 negatively regulates the PI3K–mTOR axis. Herein, we show that NLRC3 suppresses c-Myc expression and activation of PI3K–AKT targets FoxO3a and FoxO1 in the colon of Nlrc3^?/? mice, suggesting that additional signaling pathways contribute to increased cellular proliferation. Moreover, NLRC3 suppresses colorectal tumorigenesis by promoting cellular apoptosis. Genes encoding intestinal stem cell markers BMI1 and OLFM4 are upregulated in the colon of Nlrc3^?/? mice. Herein, we discuss recent findings and explore mechanisms through which NLRC3 regulates PI3K–mTOR signaling. Our studies highlight the therapeutic potential of modulating NLRC3 to prevent and treat cancer.     

Cell-by-Cell Alignment of Repeated Specular Microscopy Images from the Same Eye

Purpose

Modern specular microscopes (SM) robustly depict the same central area of the corneal endothelium at different time points through a built-in fixation light. However, repeated image acquisitions slightly shift and rotate because of minute changes in head position in the chin and forehead rest. This prevents the manual retrieval of individual corneal endothelial cells (CECs) in repeated measurements because SM images usually lack obvious landmarks. We devised and validated an image registration algorithm that aligns SM images from the same eye to make corresponding CECs coincide.

Methods

We retrospectively selected 27 image pairs for the presence of significant image overlap. Each image pair had been recorded on the same day and of the same eye. We applied our registration method in each image pair. Two observers independently validated, by means of alternation flicker, that the image pairs had been correctly aligned. We also repeatedly applied our registration method on unrelated image pairs by randomly drawing images and making certain that the images did not originate from the same eye. This was done to assess the specifity of our method.

Results

All automated registrations of the same-day and same-eye image pairs were accurate. However, one single image incorrectly failed to trigger the non-match diagnosis twice in 81 registration attempts between unrelated images. As it turned out, this particular image depicted only 73 CECs. The average number of CECs was 253 (range 73–393).

Conclusion

Repeated non-contact SM images can be automatedly aligned so that the corresponding CECs coincide. Any successful alignment can be considered as proof of the retrieval of identical CECs as soon as at least 100 CEC centroids have been identified. We believe our method is the first to robustly confirm endothelial stability in individual eyes.     

High Throughput Gene Expression Analysis Identifies Reliable Expression Markers of Human Corneal Endothelial Cells

Considerable interest has been generated for the development of suitable corneal endothelial graft alternatives through cell-tissue engineering, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as Descemet’s Stripping Endothelial Keratoplasty (DSEK) and Descemet’s Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types was also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type.     

PACAP through EGFR transactivation preserves human corneal endothelial integrity

The corneal endothelium is composed of a single hexagonal-shaped cells layer adherent to the Descemet's membrane. The primary function of these cells is maintaining of tissue clarity by regulating its hydration. Trauma, aging or other pathologies cause their loss, counterbalanced by enlargement of survived cells unable to guarantee an efficient fluid pumping to and from the stroma. Regenerative medicine using human corneal endothelial cells (HCECs) isolated from peripheral corneal-scleral tissue of a donor could be an attractive solution, overcoming transplantation problems. In a previous study, we have demonstrated that HCECs treatment with pituitary adenylate cyclase–activating polypeptide (PACAP) following growth factors deprivation prevents their degeneration. However, the molecular mechanism mediating this effect has not been clarified, yet. Here, we have shown for the first time the expression of PACAP and its receptor (PAC1R) in human corneal endothelium and demonstrated that this peptide, selectively binding to PAC1R, induces epidermal growth factor receptor (EGFR) phosphorylation and the MAPK/ERK1/2 signaling pathway activation. In conclusion, our data have suggested that PACAP could represent an important trophic factor in maintaining human corneal endothelial integrity through EGFR transactivation. Therefore, PACAP, as well as epidermal growth factor and fibroblast growth factor, could co-operate to guarantee tissue physiological functioning by supporting corneal endothelial barrier integrity.     

BMP and Notch Signaling Pathways differentially regulate Cardiomyocyte Proliferation during Ventricle Regeneration

Adult mammalian hearts show limited capacity to proliferate after injury, while zebrafish are capable to completely regenerate injured hearts through the proliferation of spared cardiomyocytes. BMP and Notch signaling pathways have been implicated in cardiomyocyte proliferation during zebrafish heart regeneration. However, the molecular mechanism underneath this process as well as the interaction between these two pathways remains to be further explored. In this study we showed BMP signaling was activated after ventricle ablation and acted epistatic downstream of Notch signaling. Inhibition of both signaling pathways differentially influenced ventricle regeneration and cardiomyocyte proliferation, as revealed by time-lapse analysis using a cardiomyocyte-specific FUCCI (fluorescent ubiquitylation-based cell cycle indicator) system. Further experiments revealed that inhibition of BMP and Notch signaling led to cell-cycle arrest at different phases. Overall, our results shed light on the interaction between BMP and Notch signaling pathways and their functions in cardiomyocyte proliferation during cardiac regeneration.     

Inhibition of TGF-β Signaling Enables Human Corneal Endothelial Cell Expansion In Vitro for Use in Regenerative Medicine

Corneal endothelial dysfunctions occurring in patients with Fuchs'' endothelial corneal dystrophy, pseudoexfoliation syndrome, corneal endotheliitis, and surgically induced corneal endothelial damage cause blindness due to the loss of endothelial function that maintains corneal transparency. Transplantation of cultivated corneal endothelial cells (CECs) has been researched to repair endothelial dysfunction in animal models, though the in vitro expansion of human CECs (HCECs) is a pivotal practical issue. In this study we established an optimum condition for the cultivation of HCECs. When exposed to culture conditions, both primate and human CECs showed two distinct phenotypes: contact-inhibited polygonal monolayer and fibroblastic phenotypes. The use of SB431542, a selective inhibitor of the transforming growth factor-beta (TGF-β) receptor, counteracted the fibroblastic phenotypes to the normal contact-inhibited monolayer, and these polygonal cells maintained endothelial physiological functions. Expression of ZO-1 and Na⁺/K⁺-ATPase maintained their subcellular localization at the plasma membrane. Furthermore, expression of type I collagen and fibronectin was greatly reduced. This present study may prove to be the substantial protocol to provide the efficient in vitro expansion of HCECs with an inhibitor to the TGF-β receptor, and may ultimately provide clinicians with a new therapeutic modality in regenerative medicine for the treatment of corneal endothelial dysfunctions.     

Redox signaling mediated by the gut microbiota

Abstract

The microbiota that occupies the mammalian intestine can modulate a range of physiological functions, including control over immune responses, epithelial barrier function, and cellular proliferation. While commensal prokaryotic organisms are well known to stimulate inflammatory signaling networks, less is known about control over homeostatic pathways. Recent work has shown that gut epithelia contacted by enteric commensal bacteria rapidly generate reactive oxygen species (ROS). While the induced production of ROS in professional phagocytes via stimulation of formyl peptide receptors (FPRs) and activation of NADPH oxidase 2 (Nox2) is a well-studied process, ROS are also similarly elicited in other cell types, including intestinal epithelia, in response to microbial signals via FPRs and the epithelial NADPH oxidase 1 (Nox1). ROS generated by Nox enzymes have been shown to function as critical second messengers in multiple signal transduction pathways via the rapid and transient oxidative inactivation of a distinct class of sensor proteins bearing oxidant-sensitive thiol groups. These redox-sensitive proteins include tyrosine phosphatases that serve as regulators of MAP kinase pathways, focal adhesion kinase, as well as components involved in NF-κB activation. As microbe-elicited ROS has been shown to stimulate cellular proliferation and motility, and to modulate innate immune signaling, we hypothesize that many of the established effects of the normal microbiota on intestinal physiology may be at least partially mediated by this ROS-dependent mechanism.     

BMP‐2 modulates β‐catenin signaling through stimulation of Lrp5 expression and inhibition of β‐TrCP expression in osteoblasts

Canonical BMP and Wnt signaling pathways play critical roles in regulation of osteoblast function and bone formation. Recent studies demonstrate that BMP‐2 acts synergistically with β‐catenin to promote osteoblast differentiation. To determine the molecular mechanisms of the signaling cross‐talk between canonical BMP and Wnt signaling pathways, we have used primary osteoblasts and osteoblast precursor cell lines 2T3 and MC3T3‐E1 cells to investigate the effect of BMP‐2 on β‐catenin signaling. We found that BMP‐2 stimulates Lrp5 expression and inhibits the expression of β‐TrCP, the F‐box E3 ligase responsible for β‐catenin degradation and subsequently increases β‐catenin protein levels in osteoblasts. In vitro deletion of the β‐catenin gene inhibits osteoblast proliferation and alters osteoblast differentiation and reduces the responsiveness of osteoblasts to the BMP‐2 treatment. These findings suggest that BMP‐2 may regulate osteoblast function in part through modulation of the β‐catenin signaling. J. Cell. Biochem. 108: 896–905, 2009. © 2009 Wiley‐Liss, Inc.     

Sodium current abnormalities and deregulation of Wnt/β-catenin signaling in iPSC-derived cardiomyocytes generated from patient with arrhythmogenic cardiomyopathy harboring compound genetic variants in plakophilin 2 gene

BackgroundMutations in desmosomal genes linked to arrhythmogenic cardiomyopathy are commonly associated with Wnt/β-catenin signaling abnormalities and reduction of the sodium current density. Inhibitors of GSK3B were reported to restore sodium current and improve heart function in various arrhythmogenic cardiomyopathy models, but mechanisms underlying this effect remain unclear. We hypothesized that there is a crosstalk between desmosomal proteins, signaling pathways, and cardiac sodium channels.Methods and resultsTo reveal molecular mechanisms of arrhythmogenic cardiomyopathy, we established human iPSC-based model of this pathology. iPSC-derived cardiomyocytes from patient carrying two genetic variants in PKP2 gene demonstrated that PKP2 haploinsufficiency due to frameshift variant, in combination with the missense variant expressed from the second allele, was associated with decreased Wnt/β-catenin activity and reduced sodium current. Different approaches were tested to restore impaired cardiomyocytes functions, including wild type PKP2 transduction, GSK3B inhibition and Wnt/β-catenin signaling modulation. Inhibition of GSK3B led to the restoration of both Wnt/β-catenin signaling activity and sodium current density in patient-specific cardiomyocytes while GSK3B activation led to the reduction of sodium current density. Moreover, we found that upon inhibition GSK3B sodium current was restored through Wnt/β-catenin-independent mechanism.ConclusionWe propose that alterations in GSK3B-Wnt/β-catenin signaling pathways lead to regulation of sodium current implying its role in molecular pathogenesis of arrhythmogenic cardiomyopathy.     

Input–output behavior of ErbB signaling pathways as revealed by a mass action model trained against dynamic data

The ErbB signaling pathways, which regulate diverse physiological responses such as cell survival, proliferation and motility, have been subjected to extensive molecular analysis. Nonetheless, it remains poorly understood how different ligands induce different responses and how this is affected by oncogenic mutations. To quantify signal flow through ErbB‐activated pathways we have constructed, trained and analyzed a mass action model of immediate‐early signaling involving ErbB1–4 receptors (EGFR, HER2/Neu2, ErbB3 and ErbB4), and the MAPK and PI3K/Akt cascades. We find that parameter sensitivity is strongly dependent on the feature (e.g. ERK or Akt activation) or condition (e.g. EGF or heregulin stimulation) under examination and that this context dependence is informative with respect to mechanisms of signal propagation. Modeling predicts log‐linear amplification so that significant ERK and Akt activation is observed at ligand concentrations far below the K_d for receptor binding. However, MAPK and Akt modules isolated from the ErbB model continue to exhibit switch‐like responses. Thus, key system‐wide features of ErbB signaling arise from nonlinear interaction among signaling elements, the properties of which appear quite different in context and in isolation.     

NEK2 overexpression aggravates IL-22-induced keratinocyte proliferation and cytokine level increases and IMQ-induced psoriasis-like dermatitis

BackgroundPsoriasis is a common inflammatory skin disease characterized by the excessive proliferation and abnormal differentiation of keratinocytes. Protein kinases could act on intracellular signaling pathways associated with cell proliferation.ObjectiveIdentifying more hub protein kinases affecting cellular and molecular processes in psoriasis, and exploring the dynamic effects of baicalin and NEK2 on the IL-22-induced cellular inflammation and IMQ-induced psoriasis-like mice.Methods and resultsIn this study, differentially expressed protein kinases playing a hub role in psoriasis initiation and development were identified using integrative bioinformatics analyses, and NEK2 has been chosen. NEK2 was significantly up-regulated in psoriatic samples according to online datasets and experimental analyses. In IL-22-induced cellular inflammation model in HaCaT cells, NEK2 overexpression promoted, whereas NEK2 knockdown partially abolished IL-22-induced alterations in cell viability, DNA synthesis, cytokine levels, as well as STAT3 phosphorylation and p-RB, cyclin D1, CDK4, and CDK6 protein contents. Baicalin treatment partially suppressed IL-22-induced HaCaT cell viability, DNA synthesis, and increases in cytokine levels, whereas NEK2 overexpression significantly abolished Baicalin-induced protection against cellular inflammation. In IMQ-induced psoriasis-like skin inflammation model in mice, baicalin markedly ameliorated IMQ-induced psoriasis-like symptoms and local skin inflammation, whereas NEK2 overexpression partially eliminated the therapeutic effects of baicalin.ConclusionNEK2, up-regulated in psoriatic lesion skin, could aggravate IMQ-induced psoriasis-like dermatitis and attenuate the therapeutic efficiency of baicalin through promoting keratinocyte proliferation and cytokine levels. The STAT3 signaling might be involved.     

EA15, MIR22, LINC00472 as diagnostic markers for diabetic kidney disease

This study aimed to investigate the molecular mechanisms of diabetic kidney disease (DKD) and to explore new potential therapeutic strategies and biomarkers for DKD. First we analyzed the differentially expressed changes between patients with DKD and the control group using the chip data in Gene Expression Omnibus (GEO) database. Then the gene chip was subjected to be annotated again, so as to screen long noncoding RNAs (lncRNAs) and study expression differences of these lncRNAs in DKD and controlled samples. At last, the function of the differential lncRNAs was analyzed. A total of 252 lncRNAs were identified, and 14 were differentially expressed. In addition, there were 1,629 differentially expressed messenger RNAs (mRNAs) genes, and proliferation and apoptosis adapter protein 15 (PEA15), MIR22, and long intergenic nonprotein coding RNA 472 ( LINC00472) were significantly differentially expressed in DKD samples. Through functional analysis of the encoding genes coexpressed by the three lncRNAs, we found these genes were mainly enriched in type 1 diabetes and autoimmune thyroid disease pathways, whereas in Gene Ontology (GO) function classification, they were also mainly enriched in the immune response, type I interferon signaling pathways, interferon-γ mediated signaling pathways, and so forth. To summary, we identified EA15, MIR22, and LINC00472 may serve as the potential diagnostic markers of DKD.